Transformation of Biomass Into Chemical Commodities
Transformation of Biomass Into Chemical Commodities
Transformation of Biomass Into Chemical Commodities
document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in
Chemical Reviews, copyright © American Chemical Society, after peer review. To access the final edited and published
work see Chemical Reviews, volume 114 (2014) pages 1871‐1908 : http://dx.doi.org/10.1021/cr400309c.
1.1. Bio-based and biochemical production ..............2 5.2.1. Ethylene glycol ..................................... 15
3.7. Terminal long chain alkenes .............................9 5.4.3. Sorbitol ................................................ 19
5.1. Aliphatic monoalcohols .................................. 11 6.3. Other ethers ................................................. 20
5.1.5. 1-Butanol .............................................. 13 7.4. Butanal and isobutyraldehyde ........................ 21
Glycerol-3-P Fructose-1,6-P
Ribose-5-P
Sedoheptulose-7-P
-
2 e Glycerone-P Glyceraldehyde-3-P
(= dihydroxyacetone phosphate)
2 e-
1,3-Diphosphoglycerate
Figure 1. Metabolic pathways of some major biomass monomers up to pyruvate. Glycolysis is the direct route from glucose to py-
ruvate. Formation and consumption of ADP, ATP, phosphate, and water have not been indicated. Electrons (e-) are taken up by
NAD+ or NADP+. Components in boldface are treated explicitly in the text.
Table 2 includes all biomass components that are highly by fermentation of syngas on pilot scale and are moving
relevant for renewable chemicals synthesis, except lignin, toward commercialization.29 Syngas fermentation per‐
because lignin is not considered to be enzymatically hy‐ formance does not yet seem to be at the level of carbohy‐
drolysable. Lignin biosynthesis occurs by radical polymer‐ drate fermentation performance, but has the advantage
ization of guaiacyl, syringyl, and p‐hydroxyphenyl units that it also uses the noncarbohydrate (lignin) portion of
from the precursors coniferyl, sinapyl, and p‐coumaryl lignocellulosic biomass.
alcohol, respectively, all containing phenylpropenoid
building blocks.19 The obtained network of aromatic 2. ALKANES
groups can be degraded by some fungi and bacteria using 2.1. Methane
enzymes such as peroxidases and laccase. In the presence
Methane from fossil sources is an important raw material
of O2, these enzymes generate oxidative radicals, leading
for the industrial production of acetylene, synthesis gas,
to substituted aromatic compounds that can be further
H2, methanol, and many other compounds. Biological
metabolized.19 In the presence of H2O2, lipases have been
methane production is also well‐known, and has been
used to form peroxycarboxylic acids in situ that cause
applied since 1881.30
lignin degradation.24 The lignin catabolism of the proteo‐
bacterium Sphingobium sp. SYK6, was recently proposed Methane can be produced from acetic acid by methano‐
to include a glutathione‐dependent cleavage of a β‐O‐4 genic bacteria (Figure 2). This occurs spontaneously dur‐
aryl ether linkage bond by an etherase.25 With the aid of ing biomethanation, a type of anaerobic digestion of bio‐
enzymes, some components useful as fine chemicals have mass. Mixed cultures of microorganisms first hydrolyze
been obtained from lignin, and a quest has begun to iden‐ complex organic molecules into simple sugars, amino
tify metabolic routes from lignin to commodity chemi‐ acids, and fatty acids. In the subsequent acidogenesis
cals.26 stage, these are converted into carboxylic acids such as
acetic, propionic, butyric, and valeric acid, along with
Chemical degradation of the lignin fraction of biomass is
ammonia, carbon dioxide, and hydrogen sulfide. In the
more straightforward, and the degradation products may
third stage, acetogenesis, products from acidogenesis are
be further converted using biochemical methods. In this
further digested to produce carbon dioxide, hydrogen,
context, gasification of lignin (and other biomass) to syn‐
and organic acids, mainly acetic acid. Finally, acetic acid
gas, such as mentioned in Table 1, is particularly relevant.
is converted into methane and carbon dioxide during
This bio‐syngas, a mixture of CO and H2, like syngas of
methanogenesis. This final conversion involves several
fossil origin, can be converted into commodity chemicals
intermediates with enzyme‐catalyzed methyl transfers to
not only by using chemical methods but also by some
coenzymes M and B.31 The maximum yield of methane per
microorganisms.27 Amongst different syngas converting
glucose equivalent is 0.27 g/g, if the rest becomes CO2.
pathways, the Wood‐Ljungdahl pathway can be used to
However, in practice the feedstock is heterogeneous,
produce acetyl‐CoA from syngas.28,29 In short, one CO is
leading to a different yield. Usually a part of the CO2 will
reduced to a methyl group that is linked to another CO.
dissolve in the liquid effluent, leading to biogas contain‐
Subsequently, all kinds of metabolic routes can lead from
ing relatively much methane.
acetyl‐CoA to commodity chemicals. The companies
LanzaTech, Coskata, and INEOS have produced ethanol
5
Advantages of anaerobic digestion for converting biomass Many organisms are able to produce long chain alkanes.
include the absence of requirement to work under sterile This requires biosynthesis of fatty aldehydes, which is
conditions, and automatic product recovery by gas for‐ described in section 5.1.10. The key reaction is conversion
mation. This leads to relatively simple equipment and of fatty aldehydes into alkanes (Figure 3). Decarbonylases,
operations. A disadvantage of the process is the low which release CO, have been shown in vertebrates, in‐
productivity. Typical values are below 0.03 g/(L h).32 sects, plants, and algae.33 Ferritin‐like nonheme dimetal‐
CO2 O2 2 e- H2O carboxylate enzymes catalyze alkane formation from al‐
Acetate Methane Methanol
dehyde in many cyanobacteria. Their coproduct was also
thought to be carbon monoxide, but O2 consumption and
see Figures formate coproduction have recently been observed, sug‐
1 and 4 2 e-
AcCoA 2 e- gesting they are aldehyde‐deformylating oxygenases.34,35
Pyruvate Formate Formaldehyde Incorporating an alkane biosynthesis pathway from cya‐
nobacteria in Escherichia coli has led to long chain al‐
CO2 kanes.36 The pathway coexpresses genes for acyl‐ACP
Hydrogen
(acyl carrier protein) reductase and an enzyme from the
cyanobacterium Synechococcus elongatus converting al‐
Figure 2. Metabolic pathways to H2 and some C1 compounds. dehyde to alkane. This has led to the secretion of 0.3 g/L
C13 to C17 mixtures. The process is currently under optimi‐
2.2. Long chain alkanes
zation, and pilot‐plant fermentations (1000 L scale) have
C10‐C15 alkanes obtained from fossil carbon sources are already been performed.37
used on large scale as main components of diesel fuel, but
they are also used in other fuels, as lubricants, and they
are catalytically dehydrogenated to alkenes, which serve
as intermediate to various other compounds.
O O
see Figure 4 CO2 ATP
Acetyl-CoA HO S-CoA
Malonyl-CoA
ACP
(m)ethanol glycerol
CoA
O O Fatty acid
Triglycerides (m)ethyl esters
R S-ACP O O
ATP CoA
n
el
ACP
on
H2O H2O2 2 e-
tio
OH O O ATP
n
2 e-
2 e-
R S-ACP R S-ACP CO2 + 2 H2O
CoA
-
3-Hydroxyacyl-ACP Acyl-ACP 2e
ACP Fatty aldehydes Terminal alkenes
O2 2 e- CO2 H2O
2 e- 2 e-
O
H2O O2 CO
R S-ACP 4 e-
formate
Enoyl-ACP Fatty alcohols H2O Long-chain alkanes
Figure 3. Pathways to fatty acid and derivatives. Compounds in boldface are treated in the text.
6
3. ALKENES 3.2. Propene
3.1. Ethene Propene (propylene) is one of the most important chemi‐
cals. In 2002, about 53 million t was obtained from petro‐
Ethene (ethylene) is a chemical intermediate used to pro‐
chemical processes for conversion into polymers and nu‐
duce many different products, such as polyethylene, eth‐
merous derivatives.50
ylene oxide, vinyl chloride, and styrene. In 2009 its pro‐
duction capacity has been estimated at around 115 million Biological formation of traces of propene has been ob‐
t/a, mainly (>98%) through petrochemical routes.38 served in aerobic cultures of many different types of or‐
ganisms, in particular Rhizopus strains, but the source
Due to the importance of ethene as bulk chemical, the
was not elucidated.51,52 In other studies, rabbit cytochrome
possibilities to produce it from biomass have received
P‐450‐catalyzed formation of propene from isobutyralde‐
considerable interest.
hyde has been demonstrated, with an activity of 98
Ethene is secreted in small amounts by plants for signal‐ mmol/min of product per mol of P‐450.53 It is assumed
ing functions such as stimulation of fruit‐ripening.39 The that by mono‐oxygenase action, O2 is activated using
best‐known biosynthesis relies on the enzyme 1‐ NADPH as an electron source. One oxygen atom is then
aminocyclopropane‐1‐carboxylate oxygenase, which also reduced to water and the other atom is transferred to the
oxidizes L‐ascorbate and forms cyanide.40 The co‐ isobutyraldehyde, leading to propene and formic acid.54
produced L‐dehydroascorbate can easily be recycled into Using such a reaction for bio‐based propene production
the consumed L‐ascorbate. However, the 1‐ does not seem to be pursued, although the required iso‐
aminocyclopropane‐1‐carboxylate synthesis consumes S‐ butyraldehyde can be produce from glucose (see section
adenosyl‐L‐methionine (SAM). This is a coenzyme used 7.4). It will be easier to use a route involving acid‐
for transmethylation, transsulfuration, and aminopropyla‐ catalyzed dehydration of isopropanol (see section 0) into
tion. For ethene biosynthesis, SAM must be recycled from propene.55 Besides, patent applications claim that this
its side product 5'‐methylthioadenosine by a large meta‐ dehydration can be enzyme‐catalyzed.48,49
bolic effort.
Commercial bio‐based propene production is pursued by
In a second pathway, occurring in an engineered E. coli Braskem, using fermentative ethanol production, fol‐
strain, L‐methionine is deaminated by a transaminase to lowed by chemical conversion into ethene, dimerization
form (S)‐2‐oxo‐4‐thiomethylbutyric acid. This is decom‐ and metathesis. 56
posed into ethene, methanethiol and CO2, in the presence
of an NADH‐Fe(III) oxidoreductase, which activates O2.41
In a third pathway, ethene is produced from 2‐
oxoglutarate by an enzyme that occurs in Penicillium digi‐
tatum and Pseudomonas syringae. The overall enzymatic
reaction is a combination of two ethene‐forming reac‐
tions and one succinate‐forming reaction:42
3 oxoglutarate + L‐arginine + 3 O2 2 ethene + succinate
+ guanidine + L‐Δ1‐pyrroline‐5‐carboxylate + 7 CO2
This ethene forming enzyme has been expressed in vari‐
ous other organisms such as E. coli,43 Saccharomyces cere‐
visiae44 and Pseudomonas putida.45 However, the three
aforementioned pathways will lead to yields of ethene on
glucose below 0.12 g/g,44 which is unattractive for large‐
scale ethene synthesis.
Enzymatic decarboxylation of acrylic acid to ethene has
been suggested to occur in some organisms due to a side
activity of pyruvate decarboxylase,46 but the proof for this
is considered to be weak.47
Currently, the key option to approach a theoretical yield
of ethene on glucose of 2 mol/mol (0.31 g/g) is by using
acid‐catalyzed dehydration of fermentation‐derived etha‐
nol to ethene. This is commercially applied as bio‐based
route.38 It would be attractive if enzymatic dehydration
would be possible, but such activity is only described in
patent applications.48,49
7
Figure 4. Pathways from pyruvate to a range of compounds, indicated in boldface.
3.3. Isobutene mediate in the production of some important chemicals.
Currently, more than 10 million t/a isobutene (2‐ Production and of butadiene in 2009 was about 9.2 mil‐
methylpropene) is produced via petrochemical routes. lion t.64
Therefore, production from biomass via biocatalytic Several metabolic pathways to 1,3‐butadiene have been
pathways is of considerable interest.57 claimed in a patent application of Genomatica,65 but
Biological isobutene formation is known since the 1970s. without any information on actual formation of the com‐
The highest production rate, merely 0.45 mg/(L h), was pound. The final reaction should be an elimination that
found for a strain of the yeast Rhodotorula minuta.58 Iso‐ resembles the final reaction of isoprene pathways:
butene was formed by reductive decarboxylation of isova‐ 2‐butenyl‐4‐diphosphate 1,3‐butadiene + diphosphate
lerate, which is produced in the catabolic pathway of L‐ However, this reaction is hypothetical. It will be very
leucine.59,60 The reaction seems to be a side reaction of a challenging to achieve commercially viable results regard‐
cytochrome P450 monooxygenase that is involved in hy‐ ing biosynthetic 1,3‐butadiene production.
droxylating benzoate.61 Besides the low rate of isobutene
Other patent applications claim butadiene formation by
formation, the used pathway has no obvious potential to
dehydration reactions from but‐3‐en‐1‐ol and but‐3‐en‐2‐
approach a theoretical stoichiometry of 1 isobutene + 2
ol using homologues of known hydratases.48,49 Further‐
CO2 + 2 H2O per mol of glucose.
more, E. coli bacteria are engineered to produce these
Two recent developments may enable much higher yields, precursors from glucose (in a way not explained) and sub‐
although they do not describe reasonable isobutene pro‐ sequently engineered to convert the butenol isomers into
duction levels yet. It has been shown that 3‐ butadiene. A range of enzymatic butadiene formation
hydroxyisovalerate can be converted to isobutene as a reactions, including the aforementioned ones and path‐
side‐activity of mevalonate diphosphate decarboxylase ways to the required precursors, has been described in
(Figure 4).62‐63 Also, patent applications describe isobuta‐ another patent application.66 However, data on actual
nol dehydration as a side activity of engineered oleate butadiene production are not yet available.
hydratase and other hydratases.48‐49 Fermentative produc‐
tion of isobutanol is described in section 0. If a one‐pot
biochemical conversion of carbohydrate to isobutene 3.5. Isoprene
would become possible, isobutene gas would be emitted Isoprene, or 2‐methyl‐1,3‐butadiene, is suitable for addi‐
from a fermentor, and isobutanol recovery and its chemo‐ tion polymerization. The polymer has traditionally been
catalytic dehydration could be bypassed.57 extracted from rubber trees, but nowadays more than
800,000 t/a isoprene is produced via petrochemistry for
polymerization to synthetic rubber and elastomer.67,68
3.4. Butadiene
In nature, isoprene is formed by various microbial, plant,
1,3‐Butadiene is a very important monomer for synthetic
and animal species. Plants emit it in massive amounts,
rubbers but also for some plastics. Besides, it is an inter‐
8
estimated at 600 million t/a, into the atmosphere.69 The published for amorpha‐4,11‐diene, a precursor of the an‐
known metabolic pathways are the mevalonate (MEV) timalarial agent artemisinin: up to 41 g/L was produced at
and the methylerythritol phosphate (MEP) pathway. In a productivity of 0.35 g/(L h) using engineered S. cere‐
plants, both pathways exist. The MEV pathway, shown in visiae in aerobic fermentation with ethanol feeding.74
Figure 5, is used by archaea, some bacteria and most eu‐
karyotes (including the yeast S. cerevisiae), while the MEP
pathway is used in most bacteria (including E. coli) and
green algae. In either pathway, isoprene is formed by
elimination of pyrophosphate from 3,3‐dimethylallyl py‐
rophosphate by the key enzyme isoprene synthase. Since
the yield of isoprene from naturally‐occurring organisms
is commercially unattractive, Genencor (now DuPont)
and Goodyear use genetically engineered E. coli for the
production of isoprene through fermentation of glu‐
cose.70,68 Although the MEP pathway might yield up to
0.30 g/g isoprene on glucose, they focused on the better
known MEV pathway, which has a maximum yield of 0.25
g/g according to the theoretical net overall reaction:68
1.5 Glucose + 2 O2 Isoprene + 4 CO2 + 5 H2O
This work resulted in the emission of isoprene with the
fermentor off‐gas, from which it was collected with high
purity; the boiling point of isoprene is 34 oC and it is hard‐
ly water soluble. The isoprene was recovered from the off‐
gas and polymerized to rubber. The collected amount of
isoprene corresponded to 60 g/L in the fermentor broth.
The productivity was 2 g/(h L) and the yield on glucose
0.11 g/g.68 This has been used in the production of proto‐
type tires.
3.6. Farnesene
Isoprenoids are hydrocarbons consisting of several iso‐
prene units. Their biosynthesis resembles that of iso‐ Figure 5. Mevalonate pathway to isoprene and farnesene.
prene. The C15 hydrocarbon farnesene is the most relevant
isoprenoid with respect to large scale bio‐based produc‐
tion.
3.7. Terminal long chain alkenes
Farnesene is the name of a group of natural sesquiterpene
isomers, including β‐farnesene (7,11‐dimethyl‐3‐ Linear C6‐C20 hydrocarbons with a terminal unsaturation
methylene‐1,6,10‐dodecatriene). Their catalytic hydro‐ (‐olefins) are very useful as precursor for various sur‐
genation leads to farnesane, which is being evaluated as face‐active agents. Petrochemical production of terminal
diesel fuel, using farnesene produced by Amyris in a long chain alkenes amounted to 2.15 million t in 1994.75
demonstration plant.71 Heterologous E. coli and S. cere‐ Natural linear alkene production such as occurring in
visiae strains have been developed. In S. cerevisiae, the many bacteria proceeds by Claisen condensation and
mevalonate pathway enzymes, converting acetyl‐CoA into yields nonterminal alkenes.76,77 However, in some eukary‐
farnesyl diphosphate, are overexpressed, and the latter otes and some bacteria, Jeotgalicoccus for example, fatty
intermediate is converted into (E)‐β‐farnesene and di‐ acids are converted into terminal alkenes and CO2. The
phosphate (see Figure 5). This final reaction is catalyzed responsible enzyme, OleT, is a cytochrome P450, which is
by a farnesene synthase, due to expression of the corre‐ assumed to consume H2O2 and to form two equivalents of
sponding gene sequence from Artesemia annua. Upon H2O when abstracting hydrogens from the and β posi‐
improvement of the S. cerevisiae strain and the fermenta‐ tions of the fatty acid.78
tion conditions, farnesene has been produced at a yield In insects, terminal alkenes are formed from fatty alde‐
on glucose of 0.12 g/g. The product has low water solubili‐ hydes using cytochrome P450 enzymes that consume
ty which should facilitate separation from the culture NADPH and O2, and release NADP+, CO2 and water.79
broth.72 Production of up to 1.1 g/L farnesene at a produc‐
tivity of 0.01 g/(L h) has been disclosed.73
The potential of engineering the mevalonate pathway for
sesquiterpene biosynthesis is more evident from results
9
4. AROMATIC HYDROCARBONS 4.2. Styrene
4.1. Toluene Styrene, also known as phenylethene, is petrochemically
produced at about 26 million t/a, especially as a monomer
Toluene is one of the best known petrochemicals, con‐
for the synthesis of many useful polymers.83 It has been
sumed in amounts of about 20 million t/a for use in fuel
demonstrated that styrene can also be formed by micro‐
and for conversion into benzene and other chemicals.80
organisms from renewable substrates such as glucose.84
However, low concentrations of biogenic toluene have The conversion was achieved by the co‐expression of
been detected in freshwater lakes. This toluene originates phenylalanine ammonia lyase from Arabidopsis thaliana
from anaerobic degradation of phenylalanine by bacteria and trans‐cinnamate decarboxylase from S. cerevisiae in
such as Tolumonas auensis.81 The pathway is assumed to an L‐phenylalanine over‐producing E. coli host (Figure 6).
involve oxidation of phenylalanine to phenylacetate, This led to the accumulation of up to 0.26 g/L in shake
which is then decarboxylated (cf. Figure 6).82 The respon‐ flask cultures. This is close to the styrene toxicity thresh‐
sible enzymes are not known, and there are no publica‐ old (determined as 0.3 g/L). While genetic engineering
tions on production of toluene from glucose using such approaches will be required to obtain commercially at‐
enzymes. tractive productivities and yields, other approaches will
be required to address the toxicity threshold. Organic‐
solvent resistant bacteria such as Pseudomonas putida S12
could be used as host, like in the biosynthesis of p‐
hydroxybenzoate85 and p‐hydroxystyrene,86 and combined
with in‐situ extraction of the product.86
Figure 6. Pathways to toluene, phenol, and styrene. Only the key compounds are shown.
10
4.3. Naphthalene days.97 Only small amounts of methanol were produced,
Naphthalene was produced at about one million t in 1987, and not much recent work has been done in this field.
for use as precursor of various chemicals.87 The current
production level might be very different but will still be at
commodity scale level. Besides this fossil carbon based 5.1.2. Ethanol
production, biosynthesis might be possible. Naphthalene Ethanol fermentation is known since ancient times, and
has been found, for example, in termite nests, where it is currently it is the most important industrial fermentation
used by termites as fumigant.88 The endophytic fungus process. For several decades, petrochemical production of
Muscodor vitigenus emits traces of naphthalene when ethanol from ethene has been important, but nowadays
grown of agar plates with glucose.89 The origin of this the reverse reaction, ethene production by dehydration of
naphthalene is not clear yet. Similarly, there is some evi‐ fermentative ethanol, is gaining importance.38 Ethanol
dence for biogenic emission of traces of many other vola‐ production has been estimated at 99 million t/a in 2010.98
tile organic compounds, including benzene, toluene, and It is mostly used as fuel,99 but also as solvent, as chemical
o‐ and m‐xylene, by endophytic fungi90,91 and plants.92 The intermediate, and in beverages.
lack of identification of any biosynthetic pathway pre‐ During anaerobic fermentation, ethanol is produced from
cludes that they might be engineered into microorgan‐ glucose or other carbohydrates via pyruvate (Figure 4).
isms for bio‐based production of these aromatic com‐ Pyruvate decarboxylase converts pyruvate to acetalde‐
pounds. On the other hand, it also becomes very obvious hyde, and alcohol dehydrogenase reduces the acetalde‐
that nature’s biocatalytic potential could be explored hyde to ethanol, consuming the NADH that had been
much further. For example, the number of different spe‐ produced during pyruvate formation. This simple path‐
cies of fungal endophytes has been estimated at at least way is commonly exploited in the yeasts such as S. cere‐
one million, and their biosynthetic capability is still a field visiae, which is the default organism, but also in bacteria
very open for exploration.93 such as Zymomonas mobilis or recombinant E. coli. The
overall reaction with glucose is:
C6H12O6 2 C2H6O + 2 CO2
5. ALCOHOLS
This reaction also generates ATP, and cells will multiply if
5.1. Aliphatic monoalcohols ATP, glucose, and nutrients are available in sufficient
amounts. Cell formation is usually accompanied by for‐
5.1.1. Methanol
mation of small amounts of side product such as glycerol.
Methanol has historically been obtained by distillation of This reduces the ethanol yield, but can be minimized by
wood. Currently, it is produced from fossil carbon sources glucose feeding, and retaining cells in the fermentor using
such as methane gas by chemical processes, at a scale of centrifugation, flocculation, membranes, or immobiliza‐
46 million t/a in 2010.94 About 85% of the methanol pro‐ tion.100 The resulting high cell concentrations favor high
duced is used as a starting material or solvent for synthe‐ productivities. When also removing ethanol in‐situ using
sis. The remainder is used in the fuel and energy sector; vacuum, 82 g/(L h) has been achieved using S.
this use is increasing. cerevisiae.101 At high levels of inoculation and nutrient
Bio‐based methanol can be obtained from bio‐based me‐ supply, up to 21.5 % (v/v) ethanol (~170 g/L) has been
thane using chemical catalysis, but biocatalysis is an al‐ produced.102 Ethanol yields on glucose often approach the
ternative. In microbial ecology, oxidation of methane to theoretical maximum of 0.51 g/g.
methanol is widely known as part of the global carbon The current focus of ethanol research is on fermenting all
cycle.95 Many bacteria perform such oxidation, using in kinds of nonfood carbohydrates103,104 and even bio‐based
some cases soluble methane monooxygenase containing a syngas.29,27 The cells are selected for or adapted to condi‐
di‐iron center but in most cases a membrane‐bound en‐ tions of high stress due to high ethanol
zyme believed to contain copper and iron. The soluble concentration,105,106 high temperature,107,108 and presence
enzyme has been studied best. It consists of three com‐ of toxic components in the carbohydrate feed.109,110
ponents: a hydroxylase that houses the active site, a re‐
ductase that shuttles electrons from NADH to the active 5.1.3. 1-Propanol
site, and a regulatory protein.96 Industrially, 1‐propanol is obtained via propanal from
The reaction stoichiometry is: ethene, to be used as solvent or chemical intermediate.111
Methane + NAD(P)H + O2 methanol + NAD(P)+ + H2O Small quantities of 1‐propanol are formed during tradi‐
So despite the oxidation, NAD(P)H has to be added as tional ethanolic fermentation if the starch source contains
reducing agent (see Figure 2) and this requires a regen‐ protein. The biodegradation of threonine leads to this 1‐
eration reaction. Formate and formate dehydrogenase propanol.112 The metabolic pathway runs via 2‐
have been used in case of immobilized cells of Methylosi‐ oxobutyrate (Figure 7).
nus trichosporium that displayed methane monooxygen‐
ase activity in batch and continuous reactors during some
11
O O O O NH2 O
O NH2 NH2
O- - O- O- P O- OH
CO2 O HO ATP HO O -
2e Pi O
O O O OH O O
see Figure 1 Pyruvate Oxaloacetate Aspartate Aspartyl-4-phosphate Aspartate semialdehyde
AcCoA 2 e-
glutamate 2-oxoglutarate CO2
CoA
O O
NH2
- O-
O H2N OH OH
HO HO
O
-Alanine O
Citramalate
Homoserine
H2O ATP
NH3 Pi H2O
O O- O OH O NH2 O NH2
O -
-
O O OH P OH
- -
O HO O
O H2O CO2 2 e - OH
O O OH O O
Citraconate 3-Methylmalate 2-Oxobutyrate Threonine Phosphohomoserine
CO2 2 e-
O HO
Propanal 1-Propanol
Figure 7. Pathways to 1-propanol, propanal and some amino acids.
Upon introduction of the promiscuous 2‐oxoacid decar‐ dehydratase from K. oxytoca. Dehydrogenases native to E.
boxylase from Lactococcus lactis and alcohol dehydro‐ coli will reduce propanal into 1‐propanol. After 48 h fer‐
genase 2 from S. cerevisiae into E. coli, 2‐oxobutyrate was mentation using 20 g/L glucose, 1‐propanol was produced
converted via propanal into 1‐propanol.113‐114 This approach at 0.25 g/L, with 0.46 g/L 1,2‐propanediol remaining un‐
was improved when starting form a threonine hyper‐ converted and with L‐lactate as major product.117
producing E. coli strain.115 In aerobic fed‐batch culture,
glucose was converted into 10.8 g/L of 1‐propanol with 0.11
g/g yield and 0.14 g/(L h) productivity. Using glycerol, 10.3 5.1.4. Isopropanol
g/L of 1‐propanol was obtained with 0.26 g/g yield and Isopropanol, also called isopropyl alcohol or 2‐propanol,
0.083 g/(L h) productivity. is industrially produced from propylene, at a scale of
The most direct route to 2‐oxobutyrate does not involve more than 2.3 million t/a in 2003.111 It is used primarily as
transamination followed by deamination of threonine, a solvent in inks and surfactants, but aso as chemical in‐
but runs via (R)‐citramalate, a C5 dicarboxylate that can termediate.
be formed from pyruvate and acetyl‐CoA by citramalate Isopropanol is a fermentation product of many species of
synthase. Citramalate is dehydrated to citraconate, which Clostridium beijerinckii. A screen of 52 strains of C. bei‐
is rehydrated to (2R,3S)‐3‐methylmalate. An oxidative jerinckii showed a maximum production of isopropanol of
decarboxylation yields 2‐oxobutyrate (Figure 7). These 1.8 g/L.118 Although such isopropanol production has been
last three steps are performed by native enzymes involved improved using immobilized cells in continuous systems,
in leucine biosynthesis, which can run via this pathway. the formation of acetone and 1‐butanol via related path‐
To take advantage of the pathway via citramalate, the ways (see sections 8.1 and 5.1.5) complicates the process.119
specific activity of (R)‐citramalate synthase from Meth‐ To prevent such co‐production, the isopropanol pathway
anococcus jannaschii was improved using directed evolu‐ (Figure 4) has been introduced in E. coli.120,121 Two mole‐
tion and the improved enzyme was expressed in E. cules of acetyl‐CoA, obtained via glycolysis, are con‐
coli.113,116 After knocking out competing pathways and evo‐ densed by acetyl‐CoA acetyltransferase to one molecule of
lutionary engineering, the best strain produced more than acetoacetyl‐CoA. Acetoacetyl‐CoA transferase transfers
3.5 g/L 1‐propanol in 92 h with a yield on glucose of about CoA from acetoacetyl‐CoA to acetate or to butyrate, form‐
0.18 g/g. ing acetoacetate, which is then converted to acetone and
Another approach for the biosynthesis of 1‐propanol ex‐ CO2 by an acetoacetate decarboxylase. Finally, an NADPH
tended the well‐known 1,2‐propanediol pathway, as de‐ dependent alcohol dehydrogenase reduces acetone to
scribed in section 5.2.2. The dehydration of 1,2‐ isopropanol. Isopropanol production of 4.9 g/L has been
propanediol into propanal was achieved upon introducing achieved in 30 h using an E. coli strain with C. acetobu‐
in E. coli also a coenzyme B‐12 dependent propanediol tylicum acetyl‐CoA acetyltransferase, E. coli acetoacetyl‐
12
CoA transferase, C. acetobutylicum acetoacetate decar‐ Metabolic engineering of Clostridia and many other mi‐
boxylase, and C. beijerinckii secondary alcohol dehydro‐ croorganisms is used to adapt the original butanol fer‐
genase.121 mentation in order to:124,125
Using a pH‐controlled fed‐batch culture with the inter‐ ‐ maximize butanol yield on carbohydrate (by minimizing
mittent addition of glucose, a recombinant E. coli strain acetate, butyrate, acetone, and ethanol formation, and
formed 40 g/L isopropanol with a productivity of 0.67 minimizing the need for cell disposal and re‐growth)
g/(L h) and a yield on glucose of 0.24 g/g. To overcome ‐ ferment lignocellulosic sugars and other feedstocks into
the toxicity of isopropanol for E. coli, a gas stripping re‐ butanol
covery method was incorporated into the fed‐batch cul‐
‐ increase the rate of butanol production by the used mi‐
ture system. Using this approach an amount of isopropa‐
croorganism
nol was produced equivalent to 143 g/L in the fermentor,
with only a slightly lower productivity and yield.122 ‐ increase the tolerance of the used microorganism to
butanol.
The theoretical yield of the used pathway is 1 mol propa‐
nol per glucose, equal to 0.33 g/g and indicating room for Using engineered E. coli, achieved yields on glucose are
improvement. up to 0.36 g/g.126 A very high ABE productivity, more 16
g/(L h), has been achieved upon immobilizing C. bei‐
5.1.5. 1-Butanol jerinckii.127 The most challenging issue remains the buta‐
Already in 1861, Pasteur observed 1‐butanol production by nol concentrations achieved in fermentors, which remain
anaerobic fermentation.123 The so‐called ABE (acetone‐ limited to 14‐20 g/L.125,127,128,126,129 The low final butanol con‐
butanol‐ethanol) fermentation using Clostridium bacteria centration leads to relatively short batch fermentations
gives three products in a molar ratio of about 3:6:1 using and relatively long downtimes after each batch, thus inef‐
starch or another glucose source. An industrial process ficient fermentor use and high investments. Moreover, for
was developed in the beginning of the 20th century, both batch and continuous fermentation, the recovery of
which became the main source of 1‐butanol and acetone. butanol from dilute aqueous solution is energy intensive,
These were used as solvent and chemical building which is expensive and also energetically unfavorable, in
block.123 particular if the butanol is to be used as fuel.130 In‐situ
stripping can prolong production and lead to higher bu‐
In the early 1960s, petrochemical production methods led
tanol concentration in the condensed vapor than in the
to the decline of the ABE fermentation industry. The pet‐
fermentor.131,126
rochemical production has been estimated at 2.8 million
t/a in 2008.98 Because of the more difficult fermentation, 1‐butanol does
not yet seem to be competitive with ethanol as biofuel,
Nowadays, there is a renewed attention for ABE fermenta‐
despite it much better properties. Nevertheless, commer‐
tion because of interest in sustainability, and economic
cial fermentative 1‐butanol production has started again,
opportunities due to increasing oil prices. On a mass basis
especially in China. It uses Clostridia and may amount
1‐butanol has a 31 % higher combustion value than etha‐
about 500,000 t/a by now.129 This butanol is probably used
nol, and it can be used as “drop‐in” fuel.
as chemical rather than as fuel.
ABE fermentation is performed by a large variety of Clos‐
An alternative to the Clostridial pathway from glucose to
tridia strains. When only butanol is desired, the ideal
1‐butanol proceeds via citramalate and 2‐oxobutyrate,
stoichiometry from hexose sugars is:
which are shown in Figure 7. 2‐Oxobutyrate can be re‐
duced to 1‐butanol. Upon introducing this pathway in E.
C6H12O6 C4H10O + 2 CO2 + H2O coli, 0.5 g/L 1‐butanol has been obtained.113 Finally, the
potential of the hyperthermophile Pyrococcus furiosus to
This overall reaction can be coupled to the formation of reduce butyric acid (see section 9.3.1) to 1‐butanol at 5 bar
ATP, thus enabling cell growth and maintenance.123 Brief‐ H2 has been demonstrated.132
ly, the pathway from sugars involves formation of acetyl‐
5.1.6. 2-Butanol
CoA via pyruvate. Acetyl‐CoA is dimerized to acetoacetyl‐
CoA, which is reduced to butyryl‐CoA, and then further Presently 2‐butanol is produced at about 5 million t/a by
via butanal to 1‐butanol (Figure 4). The actual biosynthe‐ petrochemical methods.133 It can be used as solvent and as
sis in Clostridia involves some deviations. Initially, acetate precursor of amines, esters, and other valuable deriva‐
and butyrate are released from their CoA derivatives, but tives.
when this leads to a too low pH these acids are assimilat‐ The occurrence of small amounts of 2‐butanol in some
ed again and ABE formation starts. The cells can gain fermented products is well known. Distillates of ferment‐
some more ATP from this conversion, until toxic butanol ed wine residues can contain up to 3 g per liter ethanol.134
levels are reached. The background is the well‐known pathway to 2,3‐
butanediol (see section 5.2.6), which involves decarboxy‐
lative dimerization of pyruvate to 2‐acetolactate, and
13
which is performed by S. cerevisiae, for example. Certain the pathway from acetyl‐CoA to isopropanol but does not
strains of Lactobacillus can dehydrate 2,3‐butanediol to clarify this. A patent application of Genomatica139 de‐
an enol, which isomerizes spontaneously to 2‐butanone. scribes a comparable route but focuses on microbial bu‐
Subsequent enzymatic reduction by NAD‐dependent al‐ tanone formation (see section 8.2).
cohol dehydrogenase yields 2‐butanol (Figure 8).135,136
Patents of DuPont describe E. coli strains which have
5.1.7. Isobutanol
been metabolically engineered for 2‐butanol production The fields of application of isobutanol (2‐methyl‐1‐
according to the aforementioned pathway.133,137 The high‐ propanol) closely resemble those of 1‐butanol. Its indus‐
est reported concentration of 2‐butanol is only 0.034 g/L, trial production from fossil carbon sources has been esti‐
with a yield on glucose below 0.01 g/g and a productivity mated at 500,000 t/a.98
of 1.4 mg/(L h). This demonstrates the concept but also Following 1‐butanol, isobutanol production from biomass
suggests that industrial application is still very remote. using recombinant microorganisms is approaching large
The potential of the pathway, however, is indicated by the scale commercialization. Plans of Gevo to build a 55,000
theoretical stoichiometry of 0.41 g/g, identical to the one t/a isobutanol plant140 were halted shortly later, however.
given for 1‐butanol. A modified pathway involving phos‐ E. coli is the main organism that has been described for
phorylated intermediates instead of 2,3‐butanediol can isobutanol production.113,141,142,143 Many alternatives are cur‐
also be used.133,137 rently being studied because of their robustness or ability
to deal with lignocellulosic sugars, for example S. cere‐
visiae,144,145 C. glutamicum,146,147 B. subtilis,148 and Clostridi‐
um cellulyticum.149 The metabolic pathway generally used
is a modification of the pathway to L‐valine (see Figure 8).
After conversion of carbohydrates to pyruvate, two mole‐
cules of pyruvate are coupled to acetolactate by acetolac‐
tate synthase, releasing CO2. Using NAD(P)H, this is re‐
duced to 2,3‐dihydroxyisovalerate, which is then dehy‐
drated to 2‐oxoisovalerate. A subsequent decarboxylation
yields isobutyraldehyde, which is reduced to isobutanol
using NAD(P)H. Thus, a maximum of 1 mol isobutanol
per mol of glucose (0.41 g/g) can be achieved if competing
reactions are blocked. Another requirement is that the
type of reduced cofactor (NADH or NADPH) formed dur‐
ing pyruvate formation corresponds to the type required
for the subsequent reduction steps. By choosing suitable
enzymes, the pathway’s dependency on NADPH was re‐
moved and the maximum yield was achieved, using an‐
aerobic conditions.142
The highest published isobutanol concentrations are
about 22 g/L, achieved at yield on glucose of about 0.35
g/g and with a productivity of about 0.2 g/(L h).113,143 The
toxicity of isobutanol to E. coli limits the production. Us‐
ing in‐situ stripping with gas at nontoxic isobutanol con‐
centrations, a productivity of 0.69 g/(L h) was maintained
during 72 h.150 This approach also facilitates product re‐
covery.
5.1.8. Pentanols
Besides stereoisomers, there are eight alcohol isomers
with the formula C5H12O. The individual alcohols and also
their mixtures are also known as amyl alcohol and can be
Figure 8. Pathways via acetolactate. Compounds in boldface used as solvent, fuel component, and ester precursor.
are treated in the text. Amyl alcohol production was estimated at 50,000 t/a in
2008.98
A very different route to 2‐butanol has been described in
a patent application of Toyota.138 An E. coli strain in which Amyl alcohol is the main component of fusel alcohol, a
the pathway to isopropanol was introduced (see section byproduct of industrial ethanol fermentation, and is
0), formed up to 0.23 g/L 2‐butanol after 48 h. The patent formed through three enzymatic steps: Amino acids are
application suggests promiscuity of enzymes involved in transaminated; the formed 2‐oxoacids are decarboxylated;
14
and the resulting aldehydes are reduced to alcohols using ated to 1‐hexanol by the hyperthermophile Pyrococcus
NADH‐dependent enzymes.151 furiosus at 5 bar H2.132
By genetically modifying amino acid metabolic pathways
in E. coli, pentanol production has been increased. Broad‐
5.1.10. Fatty alcohols
range 2‐oxoacid decarboxylase from Lactococcus lactis Fatty alcohols are aliphatic alcohols with chain lengths
and alcohol dehydrogenase from S. cerevisiae were intro‐ between C6 and C22. They can be unsaturated. Natural
duced, besides upregulating the pathway for production fatty alcohols are derived from renewable resources such
of the 2‐oxoacid precursor and blocking alternative path‐ as fats, oils, and waxes of plant, animal or microbial
ways. Using the isoleucine pathway, 1.25 g/L 2‐methyl‐1‐ origin, whereas synthetic fatty alcohols are produced from
butanol was obtained at a yield on glucose of 0.17 g/g and petrochemicals such as olefins and paraffins. The produc‐
a productivity of 0.05 g/(L h).152 Using the valine pathway, tion capacity of fatty alcohols was estimated to be 2.8 mil‐
1.28 g/L 3‐methyl‐1‐butanol (isopentanol) was obtained at lion t/a in 2007, nearly equally distributed between petro‐
a yield on glucose of 0.11 g/g.153 Further engineering in chemical and natural feedstocks. The production of natu‐
combination with in‐situ extraction by 50 % (v/v) oleylal‐ ral fatty alcohols proceeds by catalytic hydrogenation of
cohol prolonged the production of 3‐methyl‐1‐butanol fatty acids or their methyl esters.158
while maintaining the yield of 0.11 g/g.154 The aqueous Recently, biochemical alternatives have been shown to be
concentration reached 0.8 g/L but the organic phase con‐ possible (Figure 3). In engineered E. coli, glucose was
centration became 8.8 g/L, so the overall concentration converted to fatty acid, from which the CoA derivative
was 4.8 g/L, and the productivity based on aqueous fer‐ was formed. Subsequently, a fatty acyl‐CoA reductase and
mentation volume was 0.16 g/(L h). a native aldehyde reductase led to 0.06 g/L fatty
2‐Oxohexanoate, the 2‐oxoacid that would be converted alcohol.159 The intermediate fatty aldehydes can also be
to 1‐pentanol, is not normally found in microorganisms. formed from fatty acids using a broad specificity carbox‐
However, an enzyme involved in 2‐oxoisovalerate 3‐step ylic acid reductase.160 Such an enzyme was expressed in E.
chain elongation to 2‐oxoisocaproate was engineered and coli using a putative carboxylic acid reductase sequence
became sufficiently promiscuous to also elongate 2‐ that had been seen in the genome of Mycobacterium
oxopentanoate to 2‐oxohexanoate.155 Besides, the enzyme marinum. The enzyme consumes ATP and NADPH and
for subsequent decarboxylation was engineered. This al‐ has as prosthetic group covalently linked 4′‐
lowed production of 0.75 g/L of 1‐pentanol by E. coli at a phosphopantetheine. In this case fatty alcohols were
yield on glucose of 0.04 g/g and a productivity of 0.02 formed from fatty aldehydes using aldehyde reductases
g/(L h). such as obtained from the cyanobacterium Synechocystis
species PCC 680.161 The achieved fatty alcohol concentra‐
The aforementioned production levels have not been op‐
tion was 0.35 g/L. Since fatty acyl CoA reductase from
timized and are not yet commercially interesting, but
Marinobacter aquaeolei has been found to catalyze
they demonstrate the principle.
NADPH‐dependent four‐electron reduction of fatty acyl‐
5.1.9. 1-Hexanol CoA to fatty alcohol, the intermediate free fatty aldehyde
might be bypassed.162
1‐Hexanol is used as a solvent, as a basic material for the
perfume industry, and for the production of plasticizers.
It is prepared at modest scale from ethene or from natural 5.2. Aliphatic diols and triols
products.98
Biosynthesis of small amounts of 1‐hexanol from glucose 5.2.1. Ethylene glycol
has been achieved in E. coli,113,155 for example by extending Ethylene glycol or 1,2‐ethanediol is used mainly as anti‐
the 2‐oxoacid pathway that was previously described for 1‐ freeze agent and as a raw material for the manufacture of
butanol production.113 In this pathway, one acetyl‐CoA polyester. Global demand was estimated at ca. 18 million
was used to extend the chain length by one carbon unit. t/a.163 It is produced via thermal hydrolysis of ethylene
An alternative pathway should be able to achieve a higher oxide.164 Biocatalytic hydrolysis is possible as well when
carbon yield.156 As discussed before, Clostridial 1‐butanol using a suitable epoxide hydrolase,165 but this would pose
biosynthesis proceeds via coupling of two acetyl‐CoA to no clear advantage and is not pursued.
acetoacetyl‐CoA, which is reduced to butyryl‐CoA (see Besides using ethylene oxide such as described in Section
Figure 4). Addition of a third acetyl‐CoA can lead to hex‐ 6.1, bio‐based routes to ethylene glycol might use direct
anoyl‐CoA and subsequently to 1‐hexanol, using enzymes fermentation of carbohydrates. Figure 9 shows that eth‐
with the proper selectivity. The feasibility of 1‐hexanol ylene glycol formation is linked to the central metabolism
synthesis using E. coli has been demonstrated by incorpo‐ via glyoxylate, which could be reduced in three steps to
rating such a pathway. Several optimizations including ethylene glycol. The final reduction is catalyzed by lactal‐
directed evolution by random mutagenesis improved 1‐ dehyde reductase. Its natural reaction is the oxidation of
hexanol production to almost 0.5 g/L.156‐157 Interestingly, (S)‐propane‐1,2‐diol with NAD+ to (S)‐lactaldehyde. The
hexanoic acid (see section 9.4.2) is completely hydrogen‐ E. coli enzyme will at almost the same rate also convert
15
ethylene glycol to glycolaldehyde and the other way can potentially lead to a higher yield, but achieved yields
around.166 Recently, it has been claimed that mutants of are still modest.168
the NADP dependent alcohol dehydrogenase YqhD can
also be used for ethylene glycol production.167 Also, a
metabolic route to ethylene glycol has been assembled in
E. coli.163 It relies on D‐xylose, which is oxidized to D‐
xylonate by D‐xylonate dehydrogenase; then a dehydra‐
tase forms 2‐dehydro‐3‐deoxy‐D‐xylonate, which is split
by an aldolase into pyruvate and glycolaldehyde (Figure
9). While glycolaldehyde is reduced to ethylene glycol,
pyruvate is converted into side products, which limits the
maximum yield on xylose of the pathway to 0.41 g/g. The
authors of this first attempt achieved 0.29 g/g, and a final
ethylene glycol concentration of 12 g/L at a productivity of
0.24 g/(L h). These promising results should stimulate
follow up work.
O
O
see Figure 13
O- Figure 10. Pathways to glycerol, propanediols and 3-
Glyoxylate hydroxypropionate. If glycerol is used as starting compound,
OH
reactions in reverse direction can be used.
2 e-
O OH
OH OH 5.2.3. 1,3-Propanediol
D-Xylose The main use of 1,3‐propanediol is as monomer, for ex‐
O
ample to obtain a polyester with terephthalic acid. It has
HO
O- traditionally been produced via petrochemical routes, at a
Glycolate scale much smaller than for 1,2‐propanediol, but recently
O OH 2 e- 1,3‐propanediol production by fermentation of glucose
- and glycerol has been developed.
O OH
OH OH Glycerol fermentation to 1,3‐propanediol is performed by
HO many natural organisms. The pathway involves merely
D-Xylonate O
Glycolaldehyde two steps, and requires anaerobic conditions (Figure 10).
Firstly, coenzyme B12‐dependent glycerol dehydratase is
H2O 2 e-
O used to obtain 3‐hydroxypropionaldehyde, which is then
Pyruvate reduced to 1,3‐propanediol by an NADH‐dependent oxi‐
-
O OH doreductase. This pathway is used, for example, by Clos‐
HO
O OH OH tridium butyricum AKR102a.174 In fed‐batch fermentations
2-Dehydro-3-deoxy-D-xylonate Ethylene glycol on 1‐L and 200‐L scale the strain reached 94 g/L 1,3‐
Figure 9. Pathways to ethylene glycol and glycolate. propanediol with an overall productivity of 3.3 g/(L h).
The yield was 0.52 g/g, because the requirement for
5.2.2. 1,2-Propanediol NADH implies that part of glycerol is oxidized to side
1,2‐Propanediol is a commodity chemical with global de‐ products such as carboxylic acids and CO2. Using a meta‐
mand estimated around 1.4 million t/a. It is produced via bolically engineered C. acetobutylicum strain, 0.54 g/g has
petrochemistry and has a major role in applications such been achieved.175
as antifreeze and heat‐transfer fluids, plasticizers and Glucose fermentation to 1,3‐propanediol requires a path‐
thermoset plastics, and cosmetics.168 way that directs glucose towards glycerol, which is then
In wild‐type Clostridium thermosaccharolyticum, glyc‐ further converted as mentioned before. For this, the gly‐
erone phosphate (dihydroxyacetone phosphate) is con‐ colysis is performed up to glycerone phosphate (dihy‐
verted to methylglyoxal, which is then reduced to (R)‐1,2‐ droxyacetone phosphate), which is then reduced and
propanediol via hydroxyacetone (Figure 10). A fraction of dephosphorylated to glycerol (Figure 10).176 Such a path‐
the methylglyoxal is also converted to D‐lactate. In an way has been introduced in E. coli. However, E. coli lacks
anaerobic batch fermentation at 60 oC this led to 9.05 g/L coenzyme B12, which is required by glycerol dehydratase.
(R)‐1,2‐propanediol, with a yield on glucose of 0.20 g/g Therefore, the pathway to synthesize coenzyme B12 was
and a productivity of 0.36 g/L.169 More recently, pathways introduced in E. coli, including a reactivation enzyme.176,177
from glucose to (R)‐1,2‐propanediol have been introduced In a patent of DuPont,177 the highest final 1,3‐propanediol
in E. coli170,171 and S. cerevisiae,172,173 but so far not with im‐ concentration is 141 g/L, with a yield on glucose of 0.44
proved results. Also, glycerol has been used as starting g/g. A higher yield, 0.51 g/g, is mentioned for a fermenta‐
compound. Glycerol is more reduced than glucose and
16
tion that led to 135 g/L 1,3‐propanediol and a productivity see Figure 13 Succinyl-CoA
of 3.5 g/(L h).176 2 e-
The fermentative production of 1,3‐propanediol is per‐ O O
CO2 CoA
formed at 45,000 t/a scale by a joint venture of DuPont - O
O O-
and Tate & Lyle. Metabolic Explorer is currently building O
O O-
a plant for 1,3‐propanediol based on glycerol. 2-Oxoglutarate Succinate semialdehyde
18
technologicial production has been pursued. This involves before reduction to mannitol 1‐phosphate and
NAD(P)H dependent xylose reductase. For example, xyli‐ dephosphorylation. Consequently, the yield on glucose
tol has been produced in the presence of significant can be 1 mol/mol (1.01 g/g) if a reducing agent would be
amounts of yeast extract during cell recycling of Candida available. Wild‐type lactic acid bacteria use 1/3 of the glu‐
tropicalis ATCC 13803. The yield of xylitol on xylose was cose for NAD(P)H generation, limiting the yield to 0.67
0.83 g/g, at a productivity of 4.0 g/(L h) and final xylitol g/g.201 Theoretically, 0.93 g/g might be obtained if the
concentration of 244 g/L.193 Using recombinant S. cere‐ glucose carbons used for NAD(P)H generation would all
visiae, almost quantitative yields on xylose have been ob‐ end up as CO2. Such approaches have been studied with
tained while using glucose as co‐substrate.194,195 yeasts, for example. Using classical mutagenesis, mutants
of the yeast Candida magnoliae NCIM 3470 were generat‐
5.4.3. Sorbitol ed that produced in a two‐stage fermentation mannitol
Sorbitol is a C6‐sugar alcohol obtained by reduction of up to 240 g/L, at a yield of 0.81 g/g and a productivity of 4
the aldehyde group of L‐sorbose or D‐glucose, and is g/(L h) without formation of any by‐product.201 The first
therefore also called D‐glucitol. The reduction of glucose stage was aerobic growth and the second stage anaerobic
can efficiently be done with H2 using nickel catalysts. This conversion of fructose rather than glucose.
has led to industrial production amounting to 500,00o t/a Similar as for sorbitol, an elegant enzymatic cofactor re‐
in 1994, for use of sorbitol in food products, as polymeric generation system has been developed to produce manni‐
building block, and in the classical vitamin C synthesis.196 tol simultaneously with gluconic acid (a compound treat‐
Recently, the production has been estimated at 700,000 ed in section 9.4.6) at 50‐60 g/L.204,205 This starts with a
t/a.197 glucose/fructose mixture (1:1) such as obtained from glu‐
Nevertheless, biochemical production has also received cose using glucose isomerase or sucrose using invertase. A
considerable interest. Most research has focused on using glucose dehydrogenase converts glucose to gluconic acid,
the potential of the bacterium Zymomonas mobilis to co‐ using NAD+ and generating NADH. Mannitol dehydro‐
produce sorbitol and D‐gluconic acid from sucrose or 1:1 genase uses NADH to convert fructose to mannitol and
glucose‐fructose mixtures. A single enzyme, glucose‐ regenerates NAD+.
fructose oxidoreductase, a tetrameric protein with tightly
coupled NADP, is responsible for both the reduction of
5.5. Phenol
fructose to sorbitol and the dehydrogenation of glucose to
glucono‐δ‐lactone. Additionally, glucono‐δ‐lactonase is Production of phenol from fossil resources amounts to
present to speed up the (otherwise spontaneous) hydroly‐ about 9 million t/a, mostly for the production of polycar‐
sis of glucono‐δ‐lactone to gluconic acid.198,199 bonates and resins.206
Although systems with isolated enzymes and with immo‐ Using a solvent‐tolerant Pseudomonas putida S12 strain,
bilized cells have been developed,196 the best results seem glucose has been converted into phenol with a yield of
to have been obtained with free, untreated, Z. mobilis 0.035 g/g. The pathway (Figure 6) follows formation of L‐
ATCC 29191 in a batch system. The use of up to 650 g/L of tyrosine, and its conversion with water into phenol, py‐
an equimolar mixture of glucose and fructose resulted in ruvate and ammonia, using a tyrosine–phenol lyase en‐
an almost complete conversion to sorbitol and gluconic coded by a gene from Pantoea agglomerans. The P. putida
acid, with final concentrations of 300 and 320 g/L, respec‐ strain produced 0.14 g/L of phenol in 24 h in a shake flask
tively, without ethanol formation. Yields were 0.91 g/g for using mineral medium supplemented with glucose and
both products, in 8 h of operation.200 However, commer‐ some salicylate. In a fed‐batch culture, 0.47 g/L phenol
cial operation was not achieved.196 was achieved. To overcome the product toxicity, 1‐octanol
was used in‐situ as extractant. The phenol concentration
5.4.4. D-Mannitol in the octanol phase reached 5.5 g/L and the productivity
Mannitol is a C6‐sugar alcohol naturally produced by was doubled as compared to the single phase fed‐batch.207
many organisms to protect cells and proteins against heat Further improvements will be required to achieve com‐
and osmotic changes. It is used as low‐caloric and low‐ mercial significance.
cariogenic sweetener in food for diabetic patients, and is
widely used as filler in pharmaceutical applications.201
Industrial production of mannitol has been performed by 6. ETHERS
reduction of glucose–fructose mixtures with hydrogen 6.1. Epoxyethane
using nickel as a catalyst. However, this mainly leads to
Epoxyethane, also known as ethylene oxide and as
sorbitol.202
oxirane, is a simple cyclic ether. World production of
Therefore biochemical alternatives have been epoxyethane was ca. 15 million t/a in 2000. The most im‐
developed.203 These rely on NAD(P)H dependent reduc‐ portant application is production of ethylene glycol.208
tion of fructose, obtained by isomerization of glucose.
Bio‐based epoxyethane will require bio‐based ethene (see
Alternatively, glucose is phosphorylated to glucose 1‐
section 3.1). Although epoxidation of ethene is conven‐
phosphate, which is isomerized to fructose 1‐phosphate
19
iently carried out on industrial scale using chemical catal‐ enhanced mutant strain of the methylotrophic yeast Can‐
ysis, biocatalytic epoxidation has been studied as well.209 dida boidinii, 35 g/L formaldehyde has been produced at
The reason for this was probably that production of epox‐ aerobic conditions. The productivity was high, about 5
yethane was a model system for production of other epox‐ g/(L h), but the yield on methanol was only 0.36 g/g,
ides, such as styrene oxide, where the biocatalysis can be probably due to further oxidation. Under appropriate
used to obtain enantiopure products. Epoxidations have culture conditions, alcohol oxidase comprised nearly 50%
been performed using immobilized Mycobacterium Py1. of the cells’ total soluble proteins. 214,215
An NAD(P)H‐consuming monooxygenase used O2 for the
epoxidation, thus allowing gas phase supply of reactants if
7.2. Acetaldehyde
this gas phase also contained propanal as cosubstrate for
NAD(P)H regeneration.209 Acetaldehyde is produced from fossil resources at a scale
of about 1 million t/a, although Brazilian production is
The biocatalytic epoxidation of ethene is a very slow reac‐
bio‐based, using catalytic bioethanol oxidation.216 Acetal‐
tion compared to the chemocatalytic epoxidation and
dehyde is an important intermediate in the production of
does not offer a clear advantage. Introducing the enzyme
many chemicals.
for epoxidation in a hypothetical ethene‐emitting micro‐
organism would require aerobic conditions for epoxida‐ Acetaldehyde is the precursor of ethanol in fermentations
tion activity. This might easily conflict with the condi‐ (see Figure 4), and usually its formation is minimized. S.
tions required for biochemical ethene formation. cerevisiae strains that were used for wine production
formed 0.01‐0.09 g/L of acetaldehyde, although certain
wines contain 0.3 g/L.217 The toxicity of acetaldehyde to
6.2. Epoxypropane the producing microorganisms, in combination with the
Epoxypropane, also called propylene oxide or ease of petrochemical production, has limited the devel‐
methyloxirane, is produced at about 9 million t/a. It is the opment of dedicated fermentation processes for acetalde‐
starting material for a broad spectrum of polymers (poly‐ hyde. Heat‐treated (nonviable) cells of Candida boidinii,
urethanes, polyesters) and liquid chemicals (propylene however, have produced 75 g/L acetaldehyde by aerobic
glycol, polyglycols, propylene glycol ethers).210 oxidation of ethanol.215 The yield on ethanol was almost
The chemistry and biochemistry of epoxypropane resem‐ quantitative, 0.98 mol/mol, corresponding to 0.94 g/g,
ble those of epoxyethane. An important difference is that whereas the productivity was 4.2 g/(L h).
in case of propene epoxidation, enantioselectivity may Engineered E. coli has been used for the following overall
play a role. The biocatalytic approach is usually R‐ reaction:218
selective.211 Bio‐based (R)‐epoxypropane production will glucose 2 acetaldehyde + 2 H2 + 2 CO2
require bio‐based propene (see section 3.2), which is not
This was done by deleting some native pathways, and
yet available.
introducing an exogenous acetyl‐CoA reductase, which
converts acetyl‐CoA with NADH into acetaldehyde. The
6.3. Other ethers achieved molar yield was 86%, corresponding to a mass
For other ethers that are currently produced at significant yield of acetaldehyde on glucose of 0.42 g/g, with a
scale by petrochemistry, such as methyl‐tert‐butyl ether, productivity of 0.03 g/(L h). In addition, the process is
ethyl‐tert‐butyl ether, and tetrahydrofuran, biocatalytic anaerobic and produces H2 as a valuable coproduct. The
routes have not yet been proposed. Biosynthetic ether achieved acetaldehyde concentration was only 0.7 g/L,
formation is not a focal area in bio‐based production. because it is toxic to the cells, but its high volatility will
However, an analysis of the occurrence of ethers in natu‐ enable in‐situ removal by stripping.
ral products showed that there are many types of enzy‐
matic ether formation known, so this is actually a rich 7.3. Propanal
field of biochemistry.212
Propanal, often called propionaldehyde, is used as inter‐
mediate in chemical synthesis of a variety of compounds.
World production was estimated at 154000 t/a in 1988.219
7. ALDEHYDES Production uses fossil resources.
7.1. Formaldehyde Fermentative 1‐propanol production proceeds via pro‐
Formaldehyde has numerous applications, in particular panal (see section 5.1.3 and Figure 7). On the other hand,
for producing resins. In 1996, the production capacity of propanal has also been produced from 1‐propanol using
formaldehyde was about 9 million t/a.213 Besides this fos‐ heat‐treated (nonviable) cells of Candida boidinii contain‐
sil‐carbon based production, biochemical production has ing alcohol oxidase.215 In some cases the yield was quanti‐
received some interest. It is based on oxidation of metha‐ tative, but it dropped to 0.82 g/g at conditions leading to
nol, so it would require bio‐based methanol to become the highest propanol concentration (16 g/L). The produc‐
bio‐based (cf. Figure 2). Using an alcohol oxidase‐ tivity was 0.9 – 3.2 g/(L h).
20
acidogenic phase.123 However, additional uptake mecha‐
7.4. Butanal and isobutyraldehyde nisms recently found for butyric acid complicate this pic‐
ture.225
These C4 aldehydes are jointly produced by catalytic con‐
version of propene, at a scale of about 6 and 1 million t/a, It has been shown that nonenzymatic decarboxylation of
respectively. They are used for production of a range of acetoacetate occurs in vitro, under conditions similar to in
other chemicals, such as their alcohols.220 vivo ABE fermentation.226 This explains why knocking out
the acetoacetate decarboxylase does not eliminate ace‐
Also in metabolic routes, formation of butanal and isobu‐
tone formation.
tyraldehyde precedes the formation of the corresponding
alcohols, as shown in Figure 4 and Figure 8, respectively. Currently, commercial acetone production using the ABE
Small amounts are excreted by several microorganisms.221 process is mostly in China.125 The acetone capacity will be
A mutant of C. acetobutylicum was found to produce up about 150,000 t/a. Continuous fermentation is performed
to 1.7 g/L butanal during ABE fermentation, and it was using C. acetobutylicum mutants obtained via chemi‐
mentioned that due to its low boiling point (75 oC) recov‐ cal/physical mutagenesis and selection during the fer‐
ery should be easier than for 1‐butanol (b.p 117 oC).222 mentation process. According to data from Chinese ABE
fermentation industry,227 the isolated yield of acetone
A recent study focused on maximizing aldehyde produc‐
produced from starch is 0.11 g/g. The achieved acetone
tion. Deleting all genes expected to be involved in isobu‐
concentration in the fermentation is about 2 g/L. Proba‐
tyraldehyde reduction to isobutanol from a isobutanol
bly this could be improved if desired. In the 1990s, over‐
producing E. coli strain led to formation on mainly isobu‐
expressing the acetone‐forming enzymes in a Clostridium
tyraldehyde from glucose, with a yield of 0.18 g/g.223 The
acetobutylicum strain led to 8.7 g/L,228 and expressing an
productivity was about 0.29 g/L, and this was maintained
acetone pathway in E. coli led to 8.9 g/L,229 but recent
during 120 h using in‐situ product removal by gas strip‐
metabolic engineering studies focus on maximizing 1‐
ping. Although the dissolved concentration was kept at
butanol formation.
about 1 g/L, the amount produced in this way corre‐
sponded to about 35 g/L in the fermentor, which other‐
wise would have been toxic to the cells. 8.2. Butanone
Butanone, also known as 2‐butanone and as methyl ethyl
8. KETONES ketone, is produced via petrochemical routes at a scale of
8.1. Acetone about 1 million t/a.139 It is mainly used as solvent for coat‐
Acetone is a chemical intermediate and an excellent sol‐ ings, adhesives, and inks, as well as a chemical building
vent for a wide range of industrial materials. Data collect‐ block.
ed from some major countries indicate a world produc‐ Butanone is an intermediate in the pathway to 2‐butanol
tion capacity in excess of 3 million t/a.224 As co‐product of described in section 5.1.6 and Figure 8. Using the studied
the more valuable phenol, acetone is not under high de‐ recombinant E. coli strains, the formation of up 0.032 g/L
mand. Replacement of petrochemical by bio‐based pro‐ of butanone from glucose has been claimed. 133,137
duction is economically challenging. Technically, howev‐ Coupling of acetyl‐CoA with propionyl‐CoA can give, de‐
er, it would be relatively easy. During the first part of the pending on the enzyme used, 3‐oxopentanoyl‐CoA or 3‐
20th century, large scale industrial fermentation using oxo‐2‐methyl‐butanoyl‐CoA. Subsequent release of the
Clostridium strains was used to produce acetone together CoA group and decarboxylation have been suggested by
with 1‐butanol and some ethanol from carbohydrates.123 researchers from Genomatica as pathways to butanone.139
This acetone‐butanol‐ethanol (ABE) fermentation was The highest concentration of butanone, 0.14 g/L, was ob‐
developed during the First World War because of a high tained using in an E. coli strain a route which combined
need for acetone, which was solvent in the manufacture genes of succinyl‐CoA transferase from Heliobacter pylori,
of gun powder. of thiolase from an Acinetobacter strain, and of decarbox‐
As explained for 1‐butanol in section 5.1.5, acetone is ylase from C. acetobutylicum. The strain had been over‐
formed during the solventogenic phase of the ABE fer‐ produced for succinate production to improve the carbon
mentation. Acetic and butyric acid, which are formed flux toward propionyl‐CoA.
during the acidogenic phase, are taken up again in the Bio‐based levulinic acid230 (4‐oxopentanoic acid; see Fig‐
solventogenic phase by the bacteria and converted into ure 12) is used for another route to butanone. Levulinic
acetyl‐CoA or butyryl‐CoA. This involves simultaneous acid decarboxylation by a side activity of acetoacetate
conversion of acetoacetyl‐CoA into acetoacetic acid decarboxylase has been found in Clostridium acetobutyli‐
(Figure 4). The resulting acetoacetic acid is converted into cum and expressed in E. coli.231 The molar yield of buta‐
acetone by acetoacetate decarboxylase, so that the pH of none on initial levulinic acid was 90%, reaching 0.32 g/L
the solution moves toward more neutral levels and the butanone without byproduct at a productivity of 0.1 g/L.
bacteria can continue to grow. According to this route, It is obvious that this interesting route should be devel‐
the molar amount of acetone produced is limited by the oped further.
molar amount of butyric and acetic acid at the end of the
21
8.3. Cyclohexanone
Cyclohexanone is produced together with cyclohexanol
from fossil carbon sources, as precursor for adipic acid
and caprolactam.
A patent application describes metabolic pathway from
glucose to cyclohexanone.232 It involves hypothetical en‐
zymatic reactions. No actual biosynthesis of cyclohexa‐
none has been shown, though.
Figure 12. Pathways via 5-hydroxymethylfurfural. Com-
pounds in boldface are treated in the text. Dashed arrows are
chemical reactions.
9. CARBOXYLIC ACIDS
As shown subsequently, many carboxylic acids can be
produced using biochemical methods. The applications
usually require the undissociated acid form, whereas the
production methods usually are performed using con‐
comitant titration with base to keep neutral pH, because
the pKa values of the carboxylic acids are typically 3‐5. The
result is usually a carboxylate solution. To obtain the un‐
dissociated acid in reasonable concentration, a pH below
about 3 is required. To simplify the overall production
process, and in particular the product recovery, acid‐
tolerant microorganisms such as the yeast S. cerevisiae are
often developed for carboxylic acid production,233,234 as
alternative to carboxylate‐producing bacteria such as E.
coli.
22
Figure 13. The citrate cycle and some related pathways. The compounds indicted in boldface have individual entries in the text.
23
industrial Acetobacter strain in an aerated repeated fed‐ Alcaligenes eutrophus, productivities of 16 μg/(h.L) have
batch system, optimization of several operation variables been obtained from bicarbonate without optimization.248
using fuzzy control techniques led to up to 203 g/L acetic No attempt, however, seems to be made to produce bio‐
acid at productivities up to 1.6 g/(L h).243 Using immobi‐ based glycolic acid in this way.
lized Acetobacter in continuous fluidized bed reactors, However, it is known that ethylene glycol can be efficient‐
productivities of 9 g/(L h) could be maintained, albeit at ly oxidized via glycolaldehyde to glycolic acid using Glu‐
40 g/L acetic acid. The conversion was also done at pilot conobacter oxidans (cf. Figure 9).250 Recent patent appli‐
scale, and aeration at this high productivity required pure cations of Roquette Freres describe engineering of E. coli
O2.241 to obtain a metabolic route from glucose via pyruvate to
Instead of ethanol fermentation to acetic acid, carbohy‐ glyoxylate and subsequently to glycolic acid.246,251 The
drates can be used. Then, microorganisms will generally highest yield on glucose was 0.36 g/g, with a glycolic acid
produce not only acetic acid. Exceptions are strictly an‐ concentration of 55 g/L and a productivity of 1.23 g/(L h).
aerobic homoacetogens such as Moorella thermoacetica.
Via glycolysis, these organisms convert glucose into two
molecules of pyruvate, which are decarboxylated into two
9.2. C3 carboxylic acids
acetyl‐CoA and two CO2. Acetyl‐CoA is converted into 9.2.1. Propionic acid
two acetate equivalents. The aforementioned reactions
produce eight reducing equivalents, which are utilized to The production in 2006 of propionic acid (propanoic ac‐
reduce the produced two molecules of CO2 to an addi‐ id) was 377,000 t. Food and feed preservation accounted
tional acetate equivalent via the Wood–Ljungdahl path‐ for ca. 78% of the propionic acid consumption in 2009.252
way. Thus, the net product formation reaction would be: Commercial production of propionic acid is entirely by
petrochemical routes, although a number of pilot plants
glucose 3 acetic acid has been designed and built to produce it by fermenta‐
In this way yields on glucose of ~0.8 g/g have been tion.253
achieved, leading to acetate concentrations up to 100 g/L In the 19th century, studies of the anaerobic propionic acid
at productivities of 0.8 g/(L h).244 However, such concen‐ fermentation already resulted in the formulation of the
trations can only be achieved at neutral pH, whereas the so‐called Fitz equation:253
ethanol oxidation proceeds at uncontrolled pH (reaching
~2) and yields the desired acetic acid rather than an ace‐ 3 lactic acid
tate salt. 2 propionic acid + 1 acetic acid + 1 CO2 + 1 H2O
Acetic acid is also formed by the anaerobic digestion of However, dependent on the bacterial species, one out of
biomass. The four stages of anaerobic fermentation are several different pathways (see Figure 14) is used to
explained when treating methane in section 2.1. The achieve this stoichiometry. In the dicarboxylate pathway,
fourth stage of methane formation can be prevented, for glucose is converted into pyruvate or phosphoenolpy‐
example by adding inhibitors. Then, acetic acid, hydro‐ ruvate, which are carboxylated with CO2 to oxaloacetate.
gen, and carbon dioxide are produced. This production of This is reduced and rearranged in a number of steps to
acetic acid has been advocated as a way to access various (S)‐methylmalonate‐CoA, which is then decarboxylated
commodity chemicals via anaerobic digestion of waste.245 to propionyl‐CoA, to lead to propionate. Figure 14 shows
Besides all aforementioned microbial conversions, acetate also the methylcitrate cycle for conversion of pyruvate to
can also be directly liberated from acetylated biomass propionate. The acrylate pathway is similar until py‐
components such as hemicellulose, by the action of en‐ ruvate. After reduction to lactate, 2‐phospholactoyl‐CoA
zymes such as acetyl xylan esterase.21 However, this will is formed to enable dehydration to acrylyl‐CoA. Finally
probably lead to relatively low concentrations of acetate this is reduced to propionyl‐CoA, which is then hydro‐
and does not seem to be considered as acetic acid produc‐ lyzed.253 In each pathway, acetate production leads to
tion method. NADH required for the reduction in the propionate
branch. Obviously, the acetate production constrains the
9.1.3. Glycolic acid achievable propionic acid yield. According to the given
equation, the theoretical propionic acid yield on lactic
Glycolic acid, also known as 2‐hydroxyacetic acid, is cur‐
acid (or glucose) is 0.55 g/g. Elimination of the acetate‐
rently not an important compound despite its simple
forming pathway has been studied,254 but an alternative
structure. Annual consumption in the USA of 15000 t has
NADH supply route will be required instead.
been mentioned246 but also worldwide production of
about 2000 t/a.247 Some higher plants, eukaryotic algae, Another problem is the toxicity of propionic acid to the
and phototrophic bacteria, but also chemolithoauto‐ bacteria. However, a high propionic acid‐tolerant strain
trophs can synthesize it.248,249 Oxygenolytic cleavage of D‐ has been developed by adapting a metabolically engi‐
ribulose 1,5‐diphosphate (RuDP) produces phosphoglyco‐ neered Propionibacterium acidipropionici with gradually
late which is then hydrolyzed to glycolate. The oxygena‐ increased propionic acid concentration in the fermenta‐
tion reaction is catalyzed by RuDP carboxylase. Using tion broth via fed‐batch fermentation. The adapted mu‐
24
tant grew faster than its parental strain and produced 97
and 104 g/L propionic acid from glucose and lactate, re‐
spectively.255 Productivities were 0.07 and 0.12 g/(L h).
The average propionic acid yield on glucose was 0.53 g/g,
close to the maximum. Using glycerol, 106 g/L propionic
acid has been achieved, but at the expense of yield and
productivity.256
Figure 14. Pathways to lactate, propionate, and acrylate, starting from pyruvate, which is obtained according to Figure 1.
9.2.2. Acrylic acid duction, because cultivation of R. erythropolis LG12 with
Acrylic acid, also known as 2‐propenoic acid, is produced 40 g/L of acrylic acid resulted in 44% conversion into 3‐
at about 4.2 million t/a by petrochemistry. Its major utili‐ hydroxypropionate.262
zation is in polymers.257 The metabolic pathway from lactate to propionate as
In nature, free acrylate only seems to play a role in the catalyzed by C. propionicum proceeds via acrylyl‐CoA
degradation of dimethylsulfoniopropanoate (DMSP). (Figure 14) and has been reversed for acrylate production.
DMSP is an abundant osmolyte in marine environments. Resting cells of C. propionicum converted up to 18.5% of
It is made by many single‐celled marine phytoplankton, propionate into acrylate when methylene blue was used
marine macroalgae, and a few angiosperms. DMSP is a as an electron acceptor.263 The acrylate concentration
potential energy and carbon source for marine bacteria, reached up to 2.2 g/L. To obtain renewable propionate
and for several bacteria the first step in the degradation of from sugars, these authors had fermented lactose into a
DMSP is its cleavage into dimethylsulfide and acrylate by mixture of propionate, acetate and lactate, using a cocul‐
the enzyme DMSP lyase.258‐259 This pathway is not of in‐ ture of Lactobacillus bulgaricus and Propionibacterium
terest for commercial production of acrylic acid from bi‐ shermanii. This route suffers from some of the same prob‐
omass.257 More promising routes involve propionic, lactic, lems as the aforementioned route from lactate, namely
and 3‐hydroxypropionic acid. The routes to these com‐ the requirement of an external electron acceptor and the
pounds are explained elsewhere in section 9.2. Enzymatic formation of several other fermentation products from
dehydration of lactic acid or its CoA derivative suffers the sugar.
from a poor equilibrium position at ambient conditions. Thus, so far it is not clear how to obtain a high yield of
The equilibrium ratio [acrylyl‐CoA]/[lactoyl‐CoA] is only acrylate on sugars using enzymes.
about 0.005.260 The molar ratio [acrylate]/[lactate] was
found to be approximately 0.03 at equilibrium.261 9.2.3. Lactic acid
Equilibrium limitations will also play a role if 3‐ Lactic acid (2‐hydroxypropanoic acid) and its salts have
hydroxypropionate is used for enzymatic acrylic acid pro‐ traditionally been used widely in food, cosmetic, pharma‐
25
ceutical, and leather industries. The main current applica‐ acid has an existing market. This implies that biocatalytic
tion of lactic acid is as building block for polylactic acid conversion of acrylic acid to 3‐hydroxypropionic acid272
(PLA). PLA is considered to be one of the most promising has no clear commercial production value. Dehydration
biodegradable polymers that can be applied to textiles, of 3‐hydroxypropionic acid may be used to obtain acrylic
packaging materials, and films, for example. PLA was tra‐ acid (see section 9.2.2).
ditionally produced from optically pure L‐lactic acid, and 3‐Hydroxypropionate has several roles in microbial me‐
has desirable properties such as good processability, bio‐ tabolism. This has allowed the formulation of a range of
compatibility, and biodegradability. Its application is lim‐ pathways, which could be potentially constructed for 3‐
ited by its low melting temperature (180 °C), but poly‐L‐ hydroxypropionic acid production from glucose or glyc‐
lactic acid and poly‐D‐lactic acid can form a racemic erol.273 The pathway from glycerol is simple, involving
complex with a melting point of 230 °C.264 Therefore, ste‐ only two enzymatic steps (Figure 10). So far, the highest
reocomplexation improves the mechanical performance, published level of production for this pathway is 39 g/L at
thermal resistance, and hydrolysis resistance of PLA‐ 35 % yield using recombinant E. coli SH‐BGK1.274 This
based materials. strain required (expensive) coenzyme B12 supplementa‐
Fermentative lactic acid production from biomass was tion for activity of glycerol dehydratase, in the first step in
industrially performed already in 1881. There has also the pathway. The dehydration leads to 3‐hydroxypropanal
been petrochemical production, but this has disappeared upon keto‐enol tautomerization, and subsequent reduc‐
again.265 In 2011, the fermentative lactic acid production tion yields 3‐hydroxypropionate. Several enzymes have
has been estimated at 370,000 t/a.233 been used for the reduction, and strains constitutive in
The metabolic pathway from glucose to lactic acid is coenzyme B12 are advocated.275
straightforward: glycolysis to pyruvate, which is reduced For starting from glucose, many pathways have been pro‐
using NADH by D‐ or L‐lactate dehydrogenase (Figure posed, including pathways with hypothetical enzymatic
14). Theoretically, this could overall lead to splitting of 1 conversions.273,276 These pathways have been analyzed
mol glucose into 2 mol lactic acid, corresponding to a with respect to thermodynamic feasibility and achievable
mass yield of 1 g/g, which has indeed been achieved.266 yield. One of the pathways involves isomerization of L‐
Numerous studies has been performed on strain and fer‐ alanine to β‐alanine (2‐aminopropionic acid). Interesting‐
mentation development to achieve efficient processes, not ly, this was a hypothetical conversion at the time of the
only using glucose but also from cheaper carbohydrate conception of the pathway, but later a gene of Bacillus
sources.265,233,267 Here only some of the best results using subtilis lysine 2,3‐aminomutase was mutated to obtain the
glucose are given. desired alanine mutase activity.277
Using immobilized Rhizopus oryzae in a fed‐batch cul‐ Experimental implementation of a metabolic pathway
ture, 280 g/L partly soluble calcium lactate has been from glucose to 3‐hydroxypropionate has been published
achieved, which corresponds to 231 g/L L‐lactic acid.268 for an E. coli strain.278 After glycolysis, pyruvate was con‐
The L‐lactic acid yield on glucose was 0.92 g/g and the verted into acetyl‐CoA. Overexpression of acetyl‐CoA
productivity was 1.83 g/(L h). Fewer studies have been carboxylase led to malonyl‐CoA, which was converted
published on D‐lactic acid production. Using Sporolacto‐ into 0.4 g/L 3‐hydroxypropionate using NADPH‐
bacillus sp. strain CASD, which is a homofermentative D‐ dependent malonyl‐CoA reductase originating from
lactic acid producer, an amount of calcium D‐lactate cor‐ Chloroflexus aurantiacus. This leaves much room for fur‐
responding to 207 g/L lactic acid has been achieved in ther optimization. A recent patent application of
fed‐batch cultivation.269 The productivity was 4.4 g/(L h) OPXBIO279 describes extensive genetic engineering of E.
and the yield on glucose was 0.84 g/g; using different coli. Using the best strain in aerated fed‐batch fermenta‐
feeding the yield was 0.93 g/g, somewhat at the expense tion, a yield on glucose of 0.53 g/g has been achieved with
of achieved concentration and productivity. a final 3‐hydroxypropionate concentration of 48 g/L. Pilot
With respect to optimizing the lactate productivity using scale experiments have already been performed.
cell recycling, an impressive 150 g/(L h) has been
9.2.5. Pyruvic acid
achieved.270
Pyruvic acid, which has the systematic name 2‐
9.2.4. 3-Hydroxypropionic acid oxopropanoic acid, is currently merely a fine‐chemical.
Despite its simple structure, 3‐hydroxypropionic acid has However, it can be produced from glucose very efficiently,
never been a significant petrochemical product. Its devel‐ and might become more important.280,281
opment as a platform chemical took off when Cargill Pyruvate is at important metabolic intermediate between
identified the possibility to produce it efficiently by bio‐ glycolysis and citrate cycle, and a metabolic precursor for
chemical methods from biomass, and its potential as pol‐ ethanol and lactic acid. The pathways to pyruvate are
ymer building block.271 3‐Hydroxypropionic acid might be shown in Figure 1. Many pyruvate‐overproducing strains
used directly in polyesters, but its potential as precursor have been developed, such as a S. cerevisiae strain that
of acrylic acid might be more interesting, because acrylic produced 135 g/L pyruvate with a productivity of 1.4 g/(L
26
h) and an overall yield on glucose of 0.54 g/g.282 Using a biosynthesis of the monomer is relatively simple, and alt‐
recombinant E. coli, 0.87 g/g has been achieved at a hough current industrial production of 3‐hydroxybutyric
productivity of 6.0 g/(L h).283 Prolonged production of acid is only for fine‐chemicals applications, commercial
pyruvate could be achieved by maintaining the concentra‐ production of the polymer is growing, and the monomer
tion in the reactor below 55 g/L. might also become more important.15
Lactic acid is currently available at a much lower price The metabolic pathway from glucose follows acetyl‐CoA
than pyruvic acid, and therefore enzymatic oxidation of formation. Two molecules of acetyl‐CoA are condensed
D‐ and L‐lactic to pyruvic acid has also been pursued. by β‐ketothiolase to acetoacetyl‐CoA. This product is
Efficient production at almost quantitative yields has then reduced to 3‐hydroxybutyryl‐CoA by acetoacetyl‐
been obtained.284,285 Being the metabolic precursor of lac‐ CoA reductase. A synthase catalyzes the polymerization
tic acid, such pyruvic acid production from lactic acid is (Figure 4). Accumulated PHB has been intracellularly
not likely to become the prevailing process if pyruvic acid hydrolyzed by PHB hydrolase leading to excretion of (R)‐
becomes a commodity chemical like lactic acid. 3‐hydroxybutyric acid up to 15 g/L using a Halomonas
strain, for example.291 A much higher concentration, 118
g/L, was achieved using Azohydromonas lata.292 In this
9.3. C4 carboxylic acids case, cells that had accumulated PHB were transferred to
9.3.1. Butyric acid an anaerobic vessel at pH 4, which promoted hydrolysis.
This allowed a high overall productivity of (R)‐3‐
Butyric acid, with the systematic name butanoic acid, is hydroxybutyric acid of 4.9 g/(L h) with a yield on sucrose
produced by petrochemistry at a scale of about 500,000 of about 0.43 g/g.
t/a.286 It is mainly applied in cellulose acetate butyrate
plastics, but also in numerous other products.287 In similar ways, (S)‐3‐hydroxybutyric acid was produced
to a level of 10 g/L, using recombinant E. coli expressing
Butyric acid is a common metabolite produced under an‐ the required (S)‐enzymes.293
aerobic conditions by bacteria from various genera. The
most important strains studied for industrial application 9.3.3. Methacrylic acid / 2-hydroxyisobutyric
are all Clostridia, namely Clostridium butyricum, C. ty‐ acid
robutyricum, and C. thermobutyricum.253‐288
Methacrylic acid (2‐methyl‐2‐propenoic acid) and espe‐
The metabolic pathway from glucose to butyric acid has cially its methyl ester are important precursors for acrylic
been described for 1‐butanol (see section 5.1.5 and Figure polymers. In 2007, production of methyl methacrylate
4). Strains producing butyric acid are not able to reassimi‐ was 2.7 million t, with significant extensions planned.294
late this butyric acid when the medium becomes too acid‐ This makes methacrylic acid by far the most important C4
ic and they do not convert it to 1‐butanol. The pathway up carboxylic acid. Nevertheless, biochemical production has
to butyric acid also leads to production of H2:288 not been published. Only a number of hypothetical path‐
glucose butyric acid + 2 H2 + 2 CO2 + 3 ATP ways has been claimed in a patent application.295 The fo‐
The competing pathway to acetic acid, which produces 4 cus of bio‐based research is on precursors of methacrylic
ATP per glucose but leads to more acidification, has to be acid, such as itaconic acid (see section 9.4.1), isobutene
minimized to maximize butyric acid formation. Since ATP (see section 3.3) and 2‐hydroxyisobutyric acid.296 The lat‐
is required to support cell growth, the best results have ter compound can be obtained from 3‐hydroxybutyryl‐
been obtained using cell retention. Repeated fed‐batch CoA (Figure 4) by the action of a cobalamin dependent 2‐
fermentation of glucose using an immobilized butyric‐ hydroxyisobutyryl‐CoA mutase found in Aquincola ter‐
acid‐tolerant strain C. tyrobutyricum increased its toler‐ tiaricarbonis. This enzyme has been expressed in a re‐
ance to butyric acid in the course of several months, to combinant Cupriavidus necator strain which otherwise
reach finally a fermentation with a concentration of 87 was used for production of PHB (see section 9.3.2). By
g/L.286 The yield on glucose was 0.46 g/g, which is close to deleting the gene for PHB synthase, up to 6.4 g/L 2‐
the maximum for the applied pathway (0.49 g/g for the hydroxyisobutyric acid was obtained from fructose.297 The
equation shown). A reasonable volume‐specific produc‐ productivity was 0.13 g/(L h).
tivity of 1.1 g/(L h) was reached.
9.3.4. Succinic acid
In Sweden, production has been tested at 10 m3 scale us‐
In the past years, succinic acid, also called butanedioic
ing C. tyrobutyricum.289
acid, has been produced from petrochemicals at a scale of
9.3.2. 3-Hydroxybutyric acid about 30,000 t/a.298 Currently, there is a transition toward
a much larger scale using bio‐based production. This suc‐
3‐Hydroxybutyric acid is the monomer of the well‐known
cinic acid will become cheaper and should not only be
bacterial storage polymer polyhydroxybutyrate (PHB).
used as intermediate for various fine chemicals but espe‐
Strains of Cupriavidus necator (at that time called Alcali‐
cially as building block for polymers.
genes eutrophus) produced PHB concentrations up to 180
g/L with productivities up to 3.8 g/(L h).290 Therefore, also
27
Succinic acid occurs in most organisms as intermediate of 9.3.6. Fumaric acid
the citrate cycle (Figure 13). It can be excreted as major Fumaric acid, also known as (E)‐2‐butenedioic acid or
fermentation end‐product by microorganisms like Ac‐ trans‐1,2‐ethylenedicarboxylic acid, is another key inter‐
tinobacillus succinogenes, Anaerobiospirillum suc‐ mediate in the citrate cycle and therefore present in a
ciniciproducens, Mannheimia succiniciproducens and very wide range of organisms. Some fungi, in particular
some recombinant E. coli and Corynebacterium glutami‐ Rhizopus oryzae and arrhizus strains, excrete fumaric acid
cum strains. Much research has been done on maximizing as fermentation product and during the 1940s this has
the yield. Anaerobic conversion is preferred. When using been used at industrial scale to produce fumaric acid.307
only glucose as electron donor, and supplementing addi‐ Later this process was discontinued and replaced by pet‐
tional carbon using CO2 or carbonate salt, the achievable rochemical synthesis. Current production is about 90,000
yield on glucose would be 1.12 g/g according to:298 t/a, mostly for use in polymers and food.308
7/6 glucose + CO2 2 succinic acid + H2O The metabolic pathways to fumaric acid are closely relat‐
To approach this overall reaction stoichiometry, a path‐ ed to those of the aforementioned succinic and L‐malic
way consisting of various parallel portions is required. In acid (Figure 13). The best fumaric acid fermentations have
short, via glycolysis carbohydrates should be converted been obtained using R. arrhizus.309 A concentration of 107
into pyruvate or phosphoenolpyruvate. These C3 interme‐ g/L and a yield on glucose of 0.86 g/g were reached after
diates should be converted into succinate via the oxida‐ 53 h fermentation in a stirred vessel. The fumaric acid was
tive and reductive branch of the citrate cycle, with the present as precipitated calcium salt in mixture with fun‐
glyoxylate shunt in operation to provide the proper bal‐ gal pellets, complicating product recovery. Therefore,
ance of some metabolic intermediates between both heterologous production is being developed using recom‐
branches.299,300 binant E. coli and S. cerevisiae.308
The theoretical yield of ~ 1.1 g/g has been achieved using
pre‐grown engineered E. coli.301 After an aerobic cultiva‐ 9.4. C5 and C6 carboxylic acids
tion stage, anaerobic production occurred up to 99 g/L
succinic acid with a productivity of 1.3 g/(L h). 9.4.1. Itaconic acid
The highest final succinic acid concentration published is Itaconic acid, an unsaturated C5 diacid also known as 2‐
146 g/L, using fed‐batch production using C. glutamicum. methylenebutanedioic acid and as methylenesuccinic
A yield of 0.92 g/g was achieved and the productivity was acid, is used worldwide in the industrial synthesis of res‐
3.2 g/(L h).302 Continuous anaerobic production with An‐ ins and fine‐chemicals.310,311 Since 1955, commercial fer‐
aerobiospirillum succinoproducens that was retained by a mentation is performed and current production is about
membrane led to a very high productivity of 15 g/(L h).303 80,000 t/a.311
In this case, integrated succinate removal by electrodialyis
Itaconic acid is a product of Aspergillus terreus strains
was used to keep the succinate concentration low. The
and some other fungi. The key reaction is a decarboxyla‐
yield was 0.76 g/g because the strain formed side prod‐
tion of aconitate, an intermediate of the citrate cycle, by
ucts such as acetate.
cis‐aconitate decarboxylase (Figure 13). This pathway al‐
Commercial fermentative production of succinic acid has lows a yield of 1 mol/mol glucose, corresponding to a yield
been announced by several companies, using different of 0.72 g/g.312
microorganisms: Myriant, 13,600 t/a using E. coli; Bioam‐
Good results have been published for A. terreus. In aerat‐
ber with Mitsui, 17,000 t/a; BASF with Purac, 10,000 or
ed 15‐L fermentors, an itaconic acid concentration of 86
30,000 t/a using Basfia succiniciproducens; DSM with Ro‐
g/L was achieved with an overall productivity of 0.51 g/(L
quette, 10,000 t/a using S. cerevisiae.304 In the latter case
h) and a yield on glucose of 0.62 g/g.313 The recent discov‐
the fermentation pH is kept low to facilitate product re‐
ery of the A. terreus gene for cis‐aconitase
covery.
decarboxylase314 has led to attempts to achieve itaconic
9.3.5. Malic acid acid production using recombinant strains. For example,
there is a report of engineered Pseudozyma tsukubaensis
Malic acid is the trivial name of 2‐hydroxybutanedioic producing 113 g/L itaconic acid with a yield of 0.45 g/g and
acid. It is widely used in the food industry as an acidulant. a productivity of 0.18 g/(L h).315 In the latter case the pH is
Racemic malic acid is prepared commercially by hydra‐ neutral, whereas A. terreus produces at low pH, which is
tion of maleic anhydride, at 5000 t/a in the USA.247 Bio‐ favorable for itaconic acid recovery.
catalytic hydration of fumaric using fumarase is used in
industry for obtaining (S)‐malic acid.305 Neither process 9.4.2. Hexanoic acid
uses renewable carbon sources, but bio‐based processes
Hexanoic acid (caproic acid) is currently only a fine‐
are being developed. These involve the citrate cycle to
chemical and produced from petrochemicals, but bio‐
produce malate from glucose (Figure 13). Fermentation
based production shows an interesting potential.
using engineered Aspergillus flavus led to 113 g/L malate at
a productivity of 0.59 g/(L h) and a yield of 0.94 g/g.306
28
Hexanoate (caproate) has been found together with some 9.4.4. 2,5-Furandicarboxylic acid
octanoate (caprylate) as elongation product of acetate, Biochemical production of this compound relies on the
the main intermediate of anaerobic digestion under availability of 5‐hydroxymethylfurfural (HMF) by acid‐
methanogenesis‐suppressed conditions (see section 2.1).316 catalyzed dehydration of hexoses.322 Oxidation of both the
Mixed microbial communities produced 8.2 g/L hexano‐ aldehyde and hydroxymethyl group of HMF to carboxylic
ate in a stable batch reactor run from equimolar ethanol acid groups leads to 2,5‐furandicarboxylic acid (Figure 12).
and acetate. The highest hexanoate production rate was This diacid is seen as a bio‐based alternative to petro‐
0.15 g/(L h) with a yield of 0.65 g/g. The ethanol is first chemically produced terephthalic acid, which is used in
converted to acetate, while producing NADH and ATP. formation of polyesters.323 Oxidation of HMF may be done
Acetate is then converted to acetyl‐CoA, which is coupled using heterogeneous or electrochemical catalysis,324,325 but
to butyryl‐CoA.317 Another acetyl‐CoA is required for fur‐ biochemical oxidation has become an alternative. An
ther elongation to hexanoyl‐CoA, which is converted to HMF/furfural oxidoreductase from a Cupriavidus basilen‐
hexanoate. Microbial populations were found to be domi‐ sis strain has been introduced into Pseudomonas putida
nated by relatives of Clostridium kluyveri.316 S12.326 The resulting whole‐cell biocatalyst produced 30
g/L of 2,5‐furandicarboxylic acid from HMF with a yield of
9.4.3. Adipic acid
0.97 mol/mol and a productivity of 0.21 g/(L h) under
Adipic acid, also known as hexanedioic acid, is the most aerobic fed‐batch conditions.327 No residual furan deriva‐
important dicarboxylic acid and currently produced at tives were found, which is highly beneficial for subse‐
about 2.6 million t/a using petrochemical methods.318,234 It quent purification and polymerization.
is applied in the synthesis of polymers such as Nylon 6,6.
To achieve the same oxidation of HMF, the use of chlo‐
To achieve bio‐based adipic acid production, a recombi‐ roperoxidase and C. antarctica lipase B has also been ex‐
nant E. coli strain has been developed that synthesized plored.328,329
from glucose 37 g/L of cis,cis‐muconic acid, an adipic acid
precursor having two C=C bonds in the chain. The yield 9.4.5. Citric acid
was 0.17 g/g after 48 h of culturing under fed‐batch fer‐ Citric acid or 2‐hydroxy‐1,2,3‐propanetricarboxylic acid
mentor conditions. Optimization of this synthesis re‐ was first isolated from lemon juice by Scheele in 1784. In
quired adaptation of the shikimate pathway (see Figure the 19th century this led to natural isolation processes, but
6), which is normally used for biosynthesis or aromatic in the 20th century fermentation of carbohydrates became
amino acids, by expression of three heterologous en‐ the dominant industrial process. Alternative chemical
zymes: 3‐dehydroshikimate dehydratase, protocatechuic routes have been developed, on basis of petroleum con‐
acid decarboxylase, and catechol 1,2‐dioxygenase. Hydro‐ version, and even yeast fermentation of alkanes has been
genation of the resulting cis,cis‐muconic solution with applied, but such processes have only briefly been in op‐
10% Pt on carbon resulted in a 97% conversion (mol/mol) eration.330 Industrial citric acid production in 2007 was
into adipic acid.319 There are no enzymes known yet for estimated at 1.7 million t, and about 70% is used by the
reducing cis,cis‐muconic acid to adipic acid.318 food and beverages industry.331
Alternative pathways to adipic acid have been reviewed The current industrial production relies on the fungus
recently.318 After producing D‐gluconic acid (see section Aspergillus niger. Through the glycolytic pathway, py‐
9.4.6), further oxidation to D‐glucaric acid (a stereoiso‐ ruvate is formed from carbohydrates. Pyruvate is convert‐
mer of 2,3,4,5‐tetrahydroxyhexanedioic acid) can take ed to acetyl‐CoA and CO2, and an equimolar amount of
place, either chemically or biochemically; or glucaric acid pyruvate should be carboxylated with CO2 to oxaloace‐
can be produced via alternative routes from glucose. For tate. Then, citrate synthase condenses acetyl‐CoA with
glucaric acid reduction to adipic acid no enzymes are oxaloacetate to citrate, while releasing CoA (Figure 13).
known yet. Another proposed pathway follows a lysine The pathway consumes O2 to regenerate the NADH that
pathway up to 2‐oxoadipate, and then uses a series of five is formed during glycolysis to NAD+, thus generating ATP.
enzymatic steps up to adipate. Alternatively, hexanoic Besides this ATP, the overall pathway reaction is:
acid (see section 9.4.2) can be ω‐oxidized to adipic acid.
glucose + 1.5 O2 citric acid + 2 H2O
Finally, benzoate (see section 9.6) might be enzymatically
degraded up to cis,cis‐muconate and this might be hy‐ This leads to a maximum yield on glucose of 1.07 g/g, but
drogenated as mentioned before. When feeding benzoate yields up to 0.88 g/g have been reported.332 Final concen‐
to an Arthrobacter strain in an aerated 15 L vessel, 44 g/L trations up to 240 g/L has been achieved in aerated fed‐
cis,cis‐muconate accumulated, with a quantitative yield batch fermentations using A. niger, at a productivity of 1.4
on benzoate.320 The productivity was 0.92 g/(L h), but 5.5 g/(L h).333 In this case no yield was reported, though.
g/(L h) has been achieved in a cell recycling system with a A major advantage of A. niger is that pH 2 can be main‐
P. putida strain.321 tained, where citric acid is mostly undissociated. This
facilitates product recovery.
29
9.4.6. D-Gluconic acid production.344 The annual production has been estimated
Oxidation of the aldehyde group of D‐glucose yields D‐ at 80,000 t/a.345,346
gluconic acid. Gluconic acid and in particular its sodium Two‐stage continuous fermentation using the aforemen‐
salt are produced at a scale of about 60,000‐90,000 t/a for tioned mixed culture gave an overall 2‐KGA productivity
numerous applications. Usually the capacity of gluconic of 2.15 g/(L h) at 113 g/L and a molar yield of 90.1%.343
acid to dissolve multivalent cations such as calcium is
used.334,335,336 Catalytic, electrochemical, enzymatic, and
9.5. Fatty acids
microbial oxidation of D‐glucose can be used to produce
D‐gluconic acid. Large scale production using fungal or Fatty acids are C6‐C24 linear aliphatic carboxylic acids,
bacterial cells is well established. Gluconobacter subox‐ either saturated or unsaturated, occurring is esterified
ydans uses membrane‐bound glucose dehydrogenase with form in fats or oils. World production of fatty acids in
quinoprotein and heme cofactors, and co‐produces H2O 2005‐2006 was estimated at 6.5‐8.1 million t.347
from O2. Aspergillus niger has an FAD‐dependent glucose Fatty acids are produced by hydrolysis of natural fats and
oxidase with co‐produces hydrogen peroxide. A catalase oils. Glycerol is the co‐product of this reaction. To simpli‐
decomposes the peroxide to water and O2. Both routes fy recovery, modern industrial processes operate at 210‐
lead to glucono‐1,5‐lactone, which can spontaneously hy‐ 260 oC and 19‐60 bar without catalyst.347 Thus, immobi‐
drolyze to gluconic acid, but this hydrolysis is accelerated lized or free lipases are not used as catalyst for bulk fatty
by a lactonase.335 acid production, although their mild operation conditions
This simple biotransformation of glucose is among the might save energy costs, and might reduce coloration
most efficient ones described in this review. The highest resulting from degradation of fatty acids at high tempera‐
product concentration, 504 g/L gluconic acid, has been ture. Therefore, lipases are more likely to be useful for
described for fed‐batch fermentation of concentrated glu‐ regioselective hydrolysis of triglycerides to obtain special‐
cose solution using wild‐type Aureobasidium pullulans, a ty fatty acids with heat labile groups, such as polyunsatu‐
yeast‐like fungus.337 The productivity was 4.5 g/(L h) in rated fatty acids.348 Besides, lipases can be used for pro‐
this case, but in continuous fermentations with cell reten‐ ducing related products such as fatty acid methyl esters
tion up to 19 g/(L h) was achieved.338 To achieve such (section 10.1).
rates, pure oxygen rather than air was used. Up to 98% of As alternative to the aforementioned lipid biomass, car‐
the glucose was converted into gluconic acid, correspond‐ bohydrate biomass can be used as source of fatty acids,
ing to a yield of 1.07 g/g. after fermentative conversion (Figure 3). The metabolic
route involves formation of acetyl‐CoA. In E. coli, for ex‐
9.4.7. L-Ascorbic acid / 2-Keto-L-gulonic acid ample, the obtained acetyl groups are used for condensa‐
L‐Ascorbic acid, also known as vitamin C, has always been tion with a growing acyl chain that is bound as thioester
a bio‐based product. The traditional Reichstein‐Grüssner to ACP. After each condensation, the grown chain con‐
synthesis339 has been performed industrially since the tains a β‐oxo group, which is reduced in three enzymatic
1930s. It begins with D‐glucose and includes an enantiose‐ steps to a saturated acyl chain. The process is terminated
lective microbial dehydrogenation of D‐sorbitol to L‐ by thioester hydrolysis to obtain free fatty acid. Many
sorbose. A Gluconobacter oxydans mutant and fermenta‐ recombinant E. coli strains have been developed for such
tion protocol have been developed that gave a theoretical‐ conversions.349 Up to 6.6 g/L fatty acid has been obtained,
ly maximal productivity of 200 g/L of L‐sorbose from 200 using shake flask cultures, at a yield on glucose of 0.28 g/g
g/L of D‐sorbitol in 28 h of fermentation.340 The other and a productivity of 0.11 g/(L h).128
steps in the Reichstein‐Grüssner synthesis are chemical,
and require some protection/deprotection steps. The
9.6. Benzoic acid
overall yield is ~50%.341
Benzoic acid and its derivatives are widely distributed in
To increase the yield and avoid the protec‐
nature. For example, gum benzoin contains 12 – 18% ben‐
tion/deprotection steps, many biocatalytic options have
zoic acid in free and esterified forms.350 Historically, ex‐
been considered, for each individual step and for the
traction processes have been used to produce benzoic
whole synthesis.341‐342 This has led to development of an
acid, but nowadays oxidation of toluene, originating from
innovative process for the production of 2‐keto‐L‐gulonic
fossil carbon sources, is industrially applied. The main
acid (2‐KGA). In a first fermentation stage, L‐sorbose is
destination of benzoic acid is as intermediate for phenol,
produced from D‐sorbitol by batch culture of Acetobacter
followed by use of sodium and potassium benzoate as
melanogenum with a molar yield of about 96%. In a sec‐
food or beverage ingredient.
ond stage, a mixed culture is used in which Ketogulonige‐
nium vulgare is responsible for the conversion of L‐ Recently, the first fermentative formation of benzoic acid
sorbose into 2‐KGA, while Bacillus megaterium turned out was published.351 Through a pathway involving L‐
to be just a supplier of a growth factor.343 The 2‐KGA is phenylalanine, cinnamoyl‐CoA and benzoyl‐CoA, the
chemically converted into L‐ascorbic acid. By now, this aerobic bacterium Streptomyces maritimus produced
process dominates industrial L‐ascorbic acid benzoic acid (see Figure 15). The highest concentration of
30
benzoate obtained using 3% starch as feedstock was 0.46 Thermomyces lanuginose lipase) and more active Novo‐
g/L after 6 days of cultivation, and the estimated yield zym 435 (immobilized Candida antarctica lipase) in mix‐
was 0.02 g/g. tures of rape oil, tert‐butanol, and a slight excess of meth‐
anol, a FAME yield of 95% was obtained at a concentra‐
tion of about 400 g/L and a productivity of about 35 g/(L
h). There was no obvious loss in lipase activity during 200
batches of 12 h, because tert‐butanol eliminated the tox‐
icity of methanol.358 Such a technology has been applied
to achieve industrial biodiesel production in China, at a
scale of 20,000 t/a.356
FAAEs are naturally produced by some eukaryotes, but
this is not well understood.354 E. coli has been engineered
to produce FAEE by esterifying exogenously added oleic
acid with ethanol endogenously produced from glucose.359
This involved incorporating an ethanol formation path‐
way and a low‐specificity acyltransferase. Pilot cultivation
yielded 11 g/L FAEE with a productivity of 0.24 g/(L h). E.
coli has also been engineered to produce FAEE directly
from glucose via fatty acid and ethanol formation path‐
ways. The achieved FAEE concentration was 0.67 g/L, at
9.4% of the theoretical yield.159
Related biochemical routes to FAEE are being developed
for commercialization by the company LS9.360
Figure 15. Proposed pathway to benzoic acid.
10.2. γ-Butyrolactone
γ‐Butyrolactone is the cyclic ester of 4‐hydroxybutyric
10. ESTERS AND LACTONES acid. It is one of the most valuable alternatives to envi‐
Obviously, bio‐based esters can be obtained by esterifica‐ ronmentally harmful chlorinated solvents, and is an im‐
tion of the alcohols and carboxylic acids that have been portant intermediate in fine chemistry.361 It is commer‐
treated earlier in this review. This esterification can be cially produced at a scale of 100,000 t/a via petrochemical
catalyzed by mineral acids or by enzymes such as lipases processes.362
or esterases. Commodity esters that may be enzymatically Enzymatic synthesis of γ‐butyrolactone has been explored
obtained are ethyl acetate352 and dimethyl adipate,353 for using lactonization of 4‐hydroxybutyrate, which itself
example. The latter case was optimized using immobi‐ might obtained by fermentation (Figure 11).363 C. antarcti‐
lized Candida antarctica Lipase B in hexane with excess ca Lipase B showed good lactonization rates, but this re‐
methanol to obtain 98% yield. However, acid‐catalyzed quires a low pH and therefore it was argued that it cannot
esterification is usually cheaper. Potential advantages of be used in‐vivo. However, the next section shows a solu‐
enzymatic esterification are enantio‐ and regioselectivity, tion to this problem for a related compound.
the decreased risk of substrate and product degradation
reactions at the mild reaction conditions used, and the
possibility of including the ester formation reaction in 10.3. γ-Valerolactone
metabolic pathways. Some examples are given subse‐ γ‐Valerolactone is the cyclic ester of 4‐hydroxyvaleric ac‐
quently. id. The chemical synthesis of γ‐valerolactone from bio‐
based levulinic acid230 is receiving increased attention
because of the potential of γ‐valerolactone as transporta‐
10.1. Fatty acid (m)ethyl esters tion fuel and chemical building block.364,365 Alternatively,
Fatty acid methyl esters (FAMEs) and fatty acid ethyl es‐ biotransformation might be used. Pseudomonas putida is
ters (FAEEs) were used as “biodiesel” at a scale of more capable of producing high titers of 4‐hydroxyvalerate
than 12 million t/a in 2008,354 but at about 20 million t/a from levulinic acid.366 This proceeds via the CoA deriva‐
in 2013.355 They are made from fatty acid triesters of glyc‐ tive of 4‐hydroxyvalerate using unknown enzymes (Figure
erol, by transesterification using methanol derived from 12). Lactonization would only be achieved at acidic pH
fossil resources or bioethanol from renewable resources. due to equilibrium restrictions, and this pH would not be
Generally, chemical catalysts are used because enzymes tolerated by the cells. Therefore, human paraoxonase I, a
are too costly. However, many studies have been per‐ lactonizing enzyme, was expressed extracytosolically in P.
formed to improve this situation.356,354,357 With combined putida.367 When using an acidic external pH in a shake
use of more expensive Lipozyme TL IM (immobilized flask experiment, lactonization of 4‐hydroxyvalerate to γ‐
31
valerolactone was enhanced to 2.1 g/L as compared to reaction of ethylene oxide and ammonia. Therefore it
<0.2 g/L when using an intracellular lactonase. Subse‐ might become bio‐based in the future (see section 6.1 on
quently, in a 2‐liter bioreactor fed with levulinate, 27 g/L ethylene oxide).
4‐hydroxyvalerate and 8.2 g/L γ‐valerolactone were ob‐ Another approach is also considered373 (see Figure 16). L‐
tained after 115 h. Yields on levulinate were below 30%, Serine, which may be obtained from sugars by fermenta‐
indicating substantial room for improvement, for example tion,374 can be decarboxylated to 2‐aminoethanol. The
by decreasing the external pH below 6 and by selective in‐ required decarboxylases are pyridoxal 5’‐phosphate de‐
situ removal of the lactone. pendent and have been found in some plants, for exam‐
ple.375‐376 Expression in E. coli has led to small amounts of
product from L‐serine.377 Expression in P. putida also led
11. NITRILES to some formation of 2‐aminoethanol from glucose, but
Several nitriles, are produced at large scale by petrochem‐ this was severely constrained by intracellular availability
ical methods. For acrylonitrile, for example, the produc‐ of serine.378
tion capacity is 5 million t/a, for use in a range of fibers
and resins.368 Bio‐based alternatives including biochemi‐ O O O O
2 e-
cal steps are under development. Glutamic acid can be see Figure 1 P P
HO O O- HO O O-
oxidatively decarboxylated into 3‐cyanopropanoic acid OH OH
OH O
using vanadium chloroperoxidase in the presence of two
3-Phosphoglycerate 3-Phosphonooxypyruvate
equivalents of H2O2 and a catalytic amount of Br‐.369 The
glutamate
obtained yield was 1 mol/mol at a final product concen‐
tration of 0.5 g/L and a productivity of 0.1 g/(L h). Follow‐ 2-oxoglutarate
CO2 O Pi
up chemistry can convert 3‐cyanopropanoic acid into the O O
P
desired acrylonitrile.370 HO OH HO O OH
HO OH
NH2 NH2 NH2
If bio‐based aldehydes become available, they might be
converted into the corresponding nitriles by using an ap‐ Ethanolamine L-Serine 3-Phosphoserine
proach that has been demonstrated for 3‐ Figure 16. Pathway to ethanolamine.
phenylpropionitrile, butyronitrile, and others.371 Alde‐
hydes were chemically converted with hydroxylamine to
oximes. The oximes were not isolated but converted, of‐ 12.2. 1,4-Diaminobutane
ten quantitatively, into nitriles by using E. coli containing
1,4‐Diaminobutane, which bears the trivial name putres‐
heterologous phenylacetaldoxime hydratase. Such aldox‐
cine, is being produced via petrochemistry, especially for
ime dehydratases are widespread in microorganisms.372
conversion into nylon‐4,6. It is a common metabolite,
being the biological degradation product of the amino
acid ornithine. The pathway is shown in Figure 17.
12. AMINES
Overexpression of this pathway in E. coli and deletion of
Many amines are metabolites of living organisms. At competitive pathways has led to a strain producing 24 g/L
physiological pH, they usually will be in the ammonium putrescine from glucose in a fed‐batch culture. The
form, whereas it requires pH values above the pKa (often productivity was 0.75 g/(L h) and the yield on glucose was
pH > 10) to have unprotonated amine as prevalent spe‐ 0.17 g/g.379 Using a similar approach, fed‐batch cultivation
cies. For most applications unprotonated amine is re‐ with C. glutamicum PUT21 was carried out. A putrescine
quired. A high pH and a high free amine concentration titer of 19 g/L at a volumetric productivity of 0.55 g/(L h)
will be toxic for the most common microorganisms; find‐ and a yield of 0.16 g/g glucose were achieved.380
ing stable free enzymes may be easier. Otherwise amine
Assuming the availability of bio‐based arginine (see sec‐
formation should be done under conditions of control of
tion 13.1.3), arginase‐catalyzed hydrolysis to ornithine has
pH at neutral values, leading to ammonium salt for‐
been developed,381 which can be combined with subse‐
mation and significant recovery costs to isolate amines.
quent enzymatic decarboxylation382 to obtain putrescine
This situation is analogous to the biochemical formation
of carboxylic acids as described in the beginning of chap‐ (Figure 17). For B. subtilis arginase, a remarkably good
operational stability (total turnover number, TTN =
ter 9.
1.13∙108) at the pH of arginine free base (pH 11.0) was ob‐
served,383 which should simplify product recovery.
12.1. 2-Aminoethanol
2‐Aminoethanol is also known as (mono)ethanolamine
and is used extensively in gas sweetening, as anticorro‐
sive, detergent and as precursor for the production of
ethylene amines. Currently it is industrially produced by
32
O O O O O fed‐batch fermentation. A higher yield, 0.17 g/g, is de‐
2 e-
- P
O -O O-
H O - scribed using C. glutamicum.389 Under optimized condi‐
O
HN HN tions in a 5 m3 aerated pilot fermentor, BASF has pro‐
duced 72 g/L at a productivity of 0.9 g/(L h) with another
O O C. glutamicum strain. Byproducts levels were significant:
N-Acetylglutamylphosphate N-Acetylglutamate semialdehyde 15 g/L lysine.HCl and 10 g/L acetyl‐diaminopentane.
glutamate
ATP 2-oxoglutarate
O O O
O- NH2 O-
HO
HN HN
O O
N-Acetylglutamate N-Acetylornithine
O O O
HO O- H2N OH
NH2 NH2
Glutamate Ornithine
urea
CO2 H2O CO2
NH O
H2N N OH
H
NH2
Arginine
O
NH2 NH2
- H2N
O Figure 18. Pathways to L-lysine and 1,5-diaminopentane.
-Aminobutyrate 1,4-Diaminobutane
Figure 17. Conversions of glutamate into γ-aminobutyrate and
of glutamate and arginine into 1,4-diaminobutane. Glutamate
formation is shown in Figure 13. Arginine biosynthesis re- 13. AMINO ACIDS
quires ornithine and carbamoyl phosphate, a. o. Amino acids are generally used in nutrition and as pre‐
cursor for a wide range of fine chemicals.
12.3. 1,5-Diaminopentane
13.1. Proteinogenic amino acids
Also 1,5‐diaminopentane, which is often called cadaver‐
ine, can be used in the production of polyamides. In vivo, 13.1.1. L-Threonine
it is formed in the decarboxylation of L‐lysine by lysine
In 2012, the production of L‐threonine has been estimated
decarboxylase (Figure 18). Fermentative lysine production
at 230,000 t/a.17 It is produced using randomly mutagen‐
is an established industrial process (see section 13.1.5), and
ized E. coli strains. In the metabolic pathway (Figure 7),
this has triggered interest in bio‐based 1,5‐
biomass is converted into oxaloacetate, which is further
diaminopentane production.384 Formation of 1,5‐
converted via aspartate and homoserine, a. o. The final
diaminopentane from crude lysine solutions has been
reaction is a conversion of homoserine phosphate into
studied using free or immobilized lysine decarbox‐
threonine by threonine synthase. This PLP‐dependent
ylase.385,386 Using immobilized enzyme was calculated to
enzyme eliminates phosphate and adds water to shift the
be a basis for economically feasible production.386 Howev‐
C‐O bond from the C4 to the C3 position.
er, to avoid having to produce the (immobilized) enzyme,
microbial production from glucose is being pursued using In an optimized fed‐bed fermentation with E. coli, a thre‐
genetically engineered microorganisms such as C. glu‐ onine concentration of 118 g/L was achieved at a yield on
tamicum387,388,389 or E. coli.390 The latter led to a yield on glucose of 0.30 g/g and a productivity of 3.1 g/(L h).391
glucose of 0.12 g/g and a production of 9.6 g/L 1,5‐ Higher yields, 0.39 g/g, have been achieved upon meta‐
diaminopentane with a productivity of 0.32 g (L h) in a bolic engineering of E. coli.392 The achievable theoretical
yield of L‐threonine has been calculated to be 0.81 g/g, if
33
there would be no biomass formation. This shows that hydrolysis can be done using cyanophycinase, an exopep‐
there is still room for improvement. tidase producing β‐Asp‐Arg dipeptide, and a peptidase
hydrolyzing this peptide to its amino acid monomers.398
13.1.2. L-Valine
About 1100 t of L‐valine (2‐amino‐3‐methylbutanoic acid) 13.1.4. L-Glutamic acid
has been produced in 2005,393 making it insignificant in The systematic name of glutamic acid is 2‐
the field of commodity chemicals. However, efficient for‐ aminopentadioic acid. The industrial production of L‐
mation of L‐valine has been achieved using C. glutami‐ glutamate is by fermentation. In 2012 it has been estimat‐
cum, which may be a basis for larger scale production in ed at 2.5 million t/a.17 Most is used as monosodium glu‐
the future. tamate, a flavoring agent and component of some fer‐
L‐Valine is synthesized in C. glutamicum from glucose via mented food. In the mid‐1950s, the bacterium C. glutami‐
pyruvate in a series of reactions that parallel the pathway cum was isolated in Japan. It excreted large quantities of
to isobutanol up to 2‐oxoisovalerate, and is described in L‐glutamate into the culture medium. The key precursor
section 0 and Figure 8. Finally, a reductive amination is is 2‐oxoglutarate, which is formed in the citrate cycle. It is
required to obtain valine. The theoretical yield on glucose converted into L‐glutamate by a reductive amination,
of this pathway is 1 mol/mol, corresponding to 0.65 g/g. catalyzed by NADP‐dependent glutamate dehydrogenase
For the final step to valine, instead of the natural NADPH (Figure 13). Commercial strains have a very low activity
dependent reductive transaminase, an NADH‐dependent for competing pathways.
leucine dehydrogenase has been used, in order to elimi‐ The overall reaction for L‐glutamic acid production from
nate the NADPH dependency of the pathway. In this way glucose is:
a titer of 227 g/L has been achieved with a productivity of C6H12O6 + NH3 + 1.5 O2 C5H9O4N + CO2 + 3 H2O
4.7 g/(L h) with yields up to 0.41 g/g.394 By further elimi‐
The corresponding maximum yield on glucose is 0.82 g/g.
nating side‐reactions, the yield has been increased to 0.57
Sufficient aeration and ammonia addition (also used to
g/g, at a titer of 150 g/L and a productivity 6.3 g/(L h).395
control pH) are critical factors in industrial production.
13.1.3. L-Aspartic acid and L-Arginine The yield of L‐glutamic acid is up to 0.6 g/g of the sugar
supplied, and the final concentration is approximately 100
The production of L‐aspartic acid and L‐arginine have
g/L with a productivity of about 2 g/(L h).399 Using fed‐
been estimated at 15,000 and 3,000 t/a, respectively, in
batch fermentation, 141 g/L glutamate has been obtained
2005.393 Potentially, they could be easily produced in the
with a C. glutamicum strain at a productivity of 3.8 g/(L
future and become more important. Their structures are
h).400
shown in Figure 7 and Figure 17, respectively.
L‐Aspartic acid is currently industrially produced from 13.1.5. L-Lysine
ammonia and fumaric acid using immobilized aspartase Lysine is an amino acid required by all organisms, but not
(L‐aspartate ammonia lyase) from E. coli or suspended all produce it. For animal feed more than 1.4 million t/a of
cells of Brevibacterium bravum.396 To become bio‐based, L‐lysine.HCl is produced by fermentation processes using
renewable rather than fossil‐based fumaric acid would strains of C. glutamicum and E. coli from sugar sources
have to be used. A fermentative route directly from glu‐ such as molasses, sucrose, or glucose.318
cose to aspartic acid cannot simply rely on using aspartase
The metabolic pathway from glucose to lysine follows
with a fumarate‐producing strain, because a favorable
that of aspartate. In two steps, this is converted to aspar‐
equilibrium of the aspartase‐catalyzed reaction might
tate semialdehyde, which is condensed with pyruvate to
require intracellular ammonia and fumaric concentra‐
obtain a C7 compound, L‐2,3‐dihydrodipicolinate. After
tions that are toxic.
two reductions to meso‐diaminopimelate a final decar‐
An interesting source for L‐aspartic acid and also for L‐ boxylation yields lysine (Figure 18). A pathway optimized
arginine is the polypeptide cyanophycin. This consists of a toward redox equivalents is more complicated though,
poly(L‐aspartic acid) backbone with at the β‐carboxy and leads to a theoretical yield of lysine (not its hydro‐
group of each L‐aspartic acid a branch of one L‐arginine, chloride) on glucose of 0.60 g/g.401 Using C. glutamicum,
which is bound at its ‐amino group. It is a nitrogen stor‐ 112 g/L lysine was produced in fed‐batch fermentation
age polymer in cyanobacteria and some other bacteria, with a yield on glucose of 0.44 g/g and a productivity of
but heterologous expression of cyanophycin has been 3.2 g/(L h). This was achieved by implementation of
taken up. Using E. coli DH1 harboring the cyanophycin twelve changes in genes of a wild‐type strain. These
synthetase gene from a Synechocystis sp., production of changes redirected carbon fluxes toward the optimal
cyanophycin at 500 L has been established. Maximum pathway predicted by metabolic modeling. The introduc‐
cyanophycin cell content of up to 0.24 g/g of cellular dry tion of foreign DNA was avoided, so that the obtained
matter was obtained. Synthesis of cyanophycin was found strains are considered nongenetically modified organisms
to be strongly dependent on the presence of complex or‐ by industrial classification.401
ganic nutrients such as protein hydrolysate.397 Subsequent
34
13.2. Nonproteinogenic amino acids To obtain 2‐oxoheptanedionate, it has been suggested to
perform twice C1‐elongation, from the C5 compound 2‐
13.2.1. β-Alanine oxoglutarate, which is an intermediate of the citrate cycle
β‐Alanine is also known as 3‐aminopropanoic acid. It has and can be produced from glucose, via the C6 compound
been envisioned as intermediate in the production of ni‐ 2‐oxoadipate.411 However, direct biochemical formation of
trogen containing base chemicals such as acrylamide and 6‐aminoadipate from glucose was not yet reported.
acrylonitrile.402 Decarboxylation of the ‐carboxylic acid Although there will be enzymes in nature capable of cata‐
group of L‐aspartate leads to β‐alanine (Figure 7). E. coli lyzing the interconversion between 6‐aminohexanoate
L‐aspartate ‐decarboxylase has successfully been used and caprolactam,412 the reaction equilibrium under physi‐
for this.402 However, irreversible deactivation caused by ological conditions will not lead to ring closure, because
transamination of the catalytically essential pyruvoyl 6‐aminohexanoate will be zwitterionic at neutral pH.
group necessitates stabilization by protein engineering Thus, lactam formation will require a separate conversion
before the enzyme can be applied industrially. step.
13.2.2. γ-Aminobutyric acid
This compound is often abbreviated as GABA, and its sys‐ 14. HALOGEN DERIVATIVES
tematic name is 4‐aminobutanoic acid. Currently, it has Enzymatic chlorination and bromination of organic com‐
fine‐chemical applications only. It might be converted to pounds occur widely in nature, and this has inspired nu‐
its lactam, 2‐pyrrolidone, which has important applica‐ merous studies on the mechanism on such halogena‐
tions403 and might also be used to produce Nylon‐4, alt‐ tions.413 Halogenating enzymes have been divided into
hough that is not a polymer of significant current interest. hydrogen peroxide‐requiring haloperoxidases (heme‐
Nevertheless, GABA is a well‐known metabolite,404 and dependent or vanadium‐dependent), O2‐dependent halo‐
decarboxylation of L‐glutamate gives direct access to bio‐ genases (FADH2‐dependent or nonheme iron‐dependent),
based GABA (see Figure 17). Using 40 g/L glucose and 214 and nucleophilic halogenases using chloride, for exam‐
g/L glutamate, a wild‐type Lactococcus brevis strain pro‐ ple.414 Unfortunately, the widely studied haloperoxidases
duced 104 g/L GABA during 72 h fed‐batch catalyze the formation of hypohalous acid (HOCl or
fermentation.405 Immobilized glutamate decarboxylase, HOBr) but have no control on the regioselectivity of the
obtained from recombinant E. coli cell lysate, converted subsequent halogenation, which occurs outside the en‐
glutamate almost quantitatively into 224 g/L GABA in zyme. Chlorination of acetic acid using chloroperoxidase
about 1 h, implying a huge productivity.406 Maintaining led to dichloroacetic rather than monochloroacetic
the activity of the enzyme’s cofactor pyridoxal‐5’‐ acid.415 FADH2‐dependent halogenases also form hypo‐
phosphate (PLP) is an important issue in research on halous acid, but in the best studied cases this does not
GABA production. leave the active site, leading to regioselectivity. The net
reaction involves NADH:
13.2.3. 6-Aminohexanoic acid / Caprolactam
RH + X‐ + NADH + O2 RX + NAD+ + 2 OH‐
6‐Aminohexanoic acid is also known as 6‐aminocaproic
Modest protein engineering of these enzymes has been
acid. Upon ring closure it will lead to caprolactam, which
done, leading to halogenating activity with compounds
is the monomer for Nylon 6. Caprolactam is currently
closely resembling the natural substrate, but not yet with
produced by petrochemical routes at a scale of about
precursors of commodity chemicals.416
500,000 t/a.407 Production of 6‐aminohexanoic acid by
microbial conversion of carbohydrates would lead to a The nonheme iron dependent halogenases consume 2‐
bio‐based route. oxoglutarate:
The first suggested route is based on lysine production RH + X‐ + oxoglutarate + O2 RX + succinate + HCO3‐
from biomass (see section 13.1.5). Lysine can be converted Nucleophic halogenases have been discovered only re‐
into 6‐amino‐2‐hydroxyhexanoate by chemical methods. cently and seem to be more useful as fluorinases than as
6‐Amino‐2‐hydroxyhexanoate can be dehydrated into 6‐ chlorinases.417
aminohex‐2‐enoate. This has been converted into 6‐ Thus, bio‐based commodity chemicals cannot yet be
aminohexanoate using E. coli containing a 3‐enoate re‐ made using the aforementioned enzymes.
ductase gene from C. tyrobutyricum or Moorella thermo‐
acetica.408 However, during the reduction, a severely
competing cyclization to β‐homoproline occurs, and 14.1. Methyl chloride
therefore another pathway is also being patented.409,410 2‐ Methyl chloride (chloromethane) can be produced from
Oxoheptanedioate can be converted into 6‐ HCl and methanol. The order of magnitude of annual
aminohexanoate via (i) decarboxylation to 5‐ production is 1 million t/a. It is mostly used for produc‐
formylpentanoate, which can be transaminated, or (ii) via tion of silicones.418 The used methanol might be bio‐based
transamination to 2‐aminoheptanedioate, which can be (see section 5.1.1).
decarboxylated.
35
A
Another posssibility to prod duce bio‐baseed methyl chloride of CO from biom mass, such a pathway wou uld have a mu uch
has been demonstratted using methyl h halide low
wer mass yield of CO than
n catalytic con
nversion of bio‐
transferases.419 These enzyymes occur in number of orrgan‐ maass into syngaas.
isms, including marine algae, fungi, and halop phytic
plants, and aare responsible for the natuural productio on of
methyl chloriide from Cl‐.4420 The methyyl group origin nates
16
6. SUMMARY
Y AND OUTLLOOK
from SAM. In n a metagenom mic screen, mmethyl halide t trans‐
ferase from BBatis maritima a, a halophytiic plant, displlayed Alrready 21 of thhe commodityy products treeated in this rre‐
the highest aactivity; ten‐ffold higher fo
or iodide than for vieew are comm mercially prod duced and att least 9 otheers
chloride, and d only methyyl iodide actiivity was cheecked havve been testeed at pilot scaale (Figure 199). In particullar,
after expressiion in S. cerevvisiae, which was further m meta‐ carrboxylic acidss, alcohols, an nd amino acid ds are produceed.
bolically engiineered. Produ uction of 0.19
9 g/(L h) of m methyl Abbout 5 of the c commercial prroduction pro ocesses and alll 9
iodide from g glucose was acchieved at a lo ow yield.419 pillot tests date back to the past 10 yearss. It is expectted
thaat production n of chemicalls from biomaass will becom me
mo ore importantt in the near future, and th hat biochemiccal
meethods will plaay a prominen nt role in thiss field. Compaari‐
15. INORGA
ANIC COMPO
OUNDS
sonn of the 2nd and
a 3rd colum
mn of Table 3 shows that bio‐
15.1. Hyd
drogen based biochemical productiion is alreadyy relevant wiith
In industry, m
most H2 is prroduced from m natural gas by a resspect to produ uction tonnag ge. To get an overall pictu ure,
combination of steam refforming and water shift reac‐ all major comm modity productts should have been includ ded
tions, accomp panied by neet release of C
CO2 to the attmos‐ in the table. Stiill, that could give a distorrted picture. R Re‐
phere. Curreent productio on is about 4 48 million t//a, of plaacement of peetrochemical synthesis by fermentation of
which half is used for amm monia synthessis.421 Reforming of rennewable reso ources would decrease to otal commod dity
bio‐based meethane could become an iimportant alterna‐ chemicals produ uction in such h statistics. Fo or example, fe fer‐
tive. meentative prod duction of 10 00,000 t/a off 1,4‐butaned diol
A
A biochemicaal alternative using biomasss is anaerobiic hy‐ woould eliminatee the requirem ment of severral petrochem mis‐
drogen fermeentation. In th he so‐called d
dark fermentaation, tryy‐based precursors of 1,4‐bu utanediol: ~110 0,000 t/a maleeic
different anaeerobic bacteriia use differennt reactions tto re‐ anhydride, ~633,000 t/a bu utane/butenee mixture aand
lease two e‐ eequivalents to o form H2 usin ng two H+ eq quiva‐ ~111,000 t/a H2.
lents. Strict aanaerobes obtaain the electrons from pyru uvate Am
mongst manyy issues that pplay a role before a produ uct
oxidation. Th hese are then
n transferred to ferrodoxin n and n be commerrcialized,426 a few will be aaddressed in tthe
can
further on to
o a hydrogenaase that catalyyzes H2 formaation. next sections.
Facultative H H2‐producers p primarily use formate oxidation
(Figure 2), w
which is catalyyzed by a form
mate‐hydrogeen ly‐
ase.422 The acchieved H2 yieeld on glucosee is 0.022 g/g,, cor‐
responding to o 2 mol/mol, which is abou ut half of the max‐
imum of thee pathway ussed. Reasonab ble productivvities,
about 0.6 g/(L h), are acchieved, howeever, and pro oduct
recovery is eaasy because of f the gas formaation.422
To increase tthe low yield
d, bioelectroch hemically asssisted
microbial fueel cell reactorrs are being used.
u So far tthese
consume relaatively much electricity, an nd require fu
urther
development.422
Cell‐free syntthetic enzymaatic pathways are also studiied to
produce H2. UUsing the enzzymes of the oxidative pen
ntose
phosphate cyycle and hydro ogenase purified from the b bacte‐
rium Pyrococccus furiosus, 11.6 mol H2 was
w generated d per
mol glucose‐6‐phosphate.4423 Polyphosp phate can be used Figgure 19. Comm merical and pilot
p productioon of commod dity
as alternativee to ATP to obbtain glucose‐6 6‐phosphate from cheemicals from biomass
b using enzymes or cells
c accordingg to
glucose.424 datta in this review
w.
15.2. Carbon monoxid
de
The final stepp in alkane biosynthesis in nvolves loss o
of CO
from a fatty aaldehyde (Fig
gure 3). This d
decarbonylatiion is
catalyzed by microsomes ffrom the greeen alga Botyro ococ‐
cus braunii.4255 Although th his could be ussed for produ uction
36
Table 3. Industrial production and biochemical yield data for formation of some major commodity chemicals from glucose.
16.1. Feasible yields Side‐products considered are CO2 and H2O. For each
product with the formula CaHbNcOd, the stoichiometric
In a bio‐based economy, there will be competition be‐
coefficients w, x, y, and z can be calculated in a reaction
tween product routes based on their ecologic, social, and
equation from glucose that does not involve other com‐
economic value. For a fixed raw material and product, the
pounds than indicated in (Equation 1:
highest yield of product on feedstock is likely to favor all
these values. For commodity products that are produced
by fermentation, the carbohydrate feedstock is the main Glucose + w NH3 x CaHbNcOd + y CO2 + z H2O
cost contributor.427 So when discussing biochemical
routes from carbohydrate to commodity products, it is (Equation 1)
useful to know which overall reaction yields would be
The calculation of the four coefficients can be done using
achievable according to stoichiometry calculations in case
the four elemental balances of C, H, N, and O. For the
that no side products are formed.
product and CO2, the resulting values (x and y) are shown
For simplicity, glucose is taken as C‐source and, in case in Table 3 for a number of products, and the values of x
the product contains nitrogen, ammonia as N‐source. are recalculated into mass yields of product on glucose.
37
The first entry, for example, indicates that 1 mol glucose product yield. Such additional ATP generating routes lead
might lead to 3 mol methane and 3 mol carbon dioxide. to undesired side‐products or they require aerobic condi‐
No ammonia is consumed or water is produced, so w = 0 tions, with part of the carbohydrate being fully oxidized
and z = 0, but for brevity w and z values are not included into CO2 and H2O.
in Table 3. Due to the low molar mass of methane as Once an optimized metabolic route to the product has
compared to carbon dioxide, a maximum yield of only been formulated that obeys biochemical and thermody‐
0.27 g per g of glucose is found, and such a yield can in‐ namic constraints, implementation of the pathway and
deed be achieved. For some products, a much higher yield achieving the assumed maximum yield sometimes in‐
can be theoretically achieved, in particular when negative volves very large efforts on metabolic engineering436,437
values of z are found, implying carbon dioxide incorpora‐ and protein engineering.438 This is outside the scope of
tion rather than production. For succinic acid this has this review. The last columns of Table 3 indicate that for
indeed been achieved (see section 9.3.4). For other com‐ some products there is still a lot of room for improvement
pounds, the yield calculated using Equation 1 is not ap‐ of the yield.
proached. The first reason is that no pathway has been
formulated yet that might lead to such a yield.
Table 4. High product concentrations achieved ac‐
For a pathway to a desired product to be feasible, the bio‐
cording to data in this review.
chemistry of each enzymatic conversion in the pathway
has to be feasible. This review illustrates the large syn‐
Product Concentration Fermentative
thetic potential of natural enzymes, and also the potential
(g/L) (mol/L) or enzymatic
to engineer enzymes in order to achieve nonnatural reac‐ conversion
tions using biocatalysis. Reaction databases have been
constructed of known enzymatic reactions,428,429,430 and gluconate 504 2.57 F
these databases can be used to generate biochemically FAME 400 1.3 E
feasible candidate pathways from biomass to a desired glycerol 302 3.28 F
product. This set of known reactions can be supplement‐
sorbitol 300 1.65 F
ed by hypothetical enzymatic reactions, which are, for
example, homologous to known enzymatic reactions with xylitol 244 1.60 F
respect to functional groups in substrate and erythritol 241 1.97 F
product.431,432 Obviously, candidate pathways need to in‐
citric acid 240 1.25 F
clude cofactor regeneration, and may require that NAD‐
dependent enzymes are replaced by NADP‐dependent mannitol 240 1.32 F
enzymes, for example. Too many candidate high‐yield L‐lactate 231 2.57 F
pathways may result, and methods are being developed to valine 227 1.94 F
determine the likeliness that a known enzyme will cata‐
lyze a hypothetical reaction upon protein engineering. GABA 224 2.17 E
These methods include molecular modeling to assess the D‐lactate 207 2.30 F
binding affinity and catalytic efficacy of the hypothetical acetic acid 203 3.38 F
reaction, and calculating the optimized performance of
fructose 200 1.11 E
candidate enzymes if they would be rationally re‐
engineered.432 A limitation of such an approach is the lack isomaltulose 200 0.58 E
of structural and mechanistic knowledge of some enzyme ethanol 170 3.70 F
types. 2,3‐butanediol 152 1.69 F
If the desired biochemistry (enzymes, cofactors, transport
succinate 146 1.24 F
proteins, etc.) is in place and active, the desired product
yield will still be unachievable if the thermodynamics of 1,3‐propanediol 141 1.85 F
the pathway are unfavorable. Methods are being devel‐ glutamate 141 0.96 F
oped to check this before any experimental work is start‐ pyruvate 135 1.53 F
ed.433 For many enzyme‐catalyzed reactions, equilibrium
threonine 118 0.99 F
positions have been measured at physiological or near‐
physiological conditions.434 Group contribution methods 3‐ 118 1.13 F
allow the estimation of equilibrium constants of reactions hydroxybutyrate
that have not been thermodynamically analyzed.435 Using 1,4‐butanediol 115 1.28 F
corrections of equilibrium constants to a specific physio‐
itaconate 113 0.87 F
logical pH, temperature, and ionic strength, equilibrium
positions of individual reactions and of whole pathways malate 113 0.84 F
can be determined. Occasionally it is found that part of 2‐KGA 113 0.41 F
the available carbohydrate has to be converted by other lysine 112 0.76 F
routes in order to generate sufficient ATP to drive the
fumarate 107 0.92 F
product pathway and drive reactions required for cell
maintenance. This will be at the expense of the achievable propionate 106 1.43 F
38
zyme concentrations are used. For cells, immobilization
16.2. Feasible product concentrations but also cell retention by membranes and cell recycling
are used to maximize cell concentration and hence
Table 4 convincingly shows that high product concentra‐
productivity. If O2 is consumed, the productivity can be
tions are no exception for enzymatic and fermentative
increased by supplying it as pure O2 rather than via air. If
conversions. In some cases not much optimization was
the product becomes inhibiting, ISPR (in‐situ product
performed but in other cases a lot of effort was required
removal) can be applied. All these process options be‐
to find or develop cells that would tolerate these concen‐
come important if enzyme and strain development have
trations. A notorious, so far unsuccessful case is 1‐butanol
led to sufficiently fast product pathways. For valine, the
fermentation, where final product concentrations remain
last entry of Table 5, rate‐limiting metabolic steps were
below 20 g/L despite all efforts to increase this. In con‐
addressed using metabolic engineering.
trast to the fermentative products in Table 4, 1‐butanol is
only slightly soluble in water and will therefore have rela‐
tively much tendency to interact with hydrophobic por‐ 16.4. Outlook
tions of cell membranes and proteins, thereby destabiliz‐ Transformation of biomass into commodity chemicals
ing these structures. By analogy, one cannot expect that it using enzymes or cells will be successful if the production
will be easy to achieve high product concentrations for process is more attractive than for alternative options to
the other butanols, for phenol, and for some undissociat‐ produce these chemicals. Competition is on production
ed carboxylic acids, for example. Full understanding of costs, but a constraint is that the results of life cycle anal‐
stabilizing cells against such toxic products is still lacking, ysis should not favor the traditional nonrenewable pro‐
though. Recovering toxic or inhibiting products in‐situ cesses.
can partly circumvent the associated problems.439 In‐situ
There are several critical factors:
product recovery may also improve conversions that are
constrained by the occurrence of product degradation or ‐ Sufficient second generation biomass should be
by the enzymatic reaction equilibrium. available for a reasonable price; the price will not
only be dictated by the biomass production but
also by competitive uses of this biomass such as
Table 5. Highest productivities cited in this review. combustion for energy generation.
Product Productivity Methods used ‐ All biomass components should be convertible
into product, or otherwise into valuable co‐
[g/(L h)]
product.
fructose 800 immobilized enzyme, packed ‐ There should be a large margin between cost of
bed
biomass and revenues from product, considering
lactic acid 150 cell recycling the anticipated yield of product on biomass.
ethanol 82 ISPR, cell recycling ‐ Too high bioreactor investments, due to high O2
isomaltulose 40 immobilized cells, packed bed requirements or too low productivities, should
be avoided.
FAME 35 immobilized enzyme, packed
bed ‐ Product recovery should not be too complicated.
Prerequisites are high product concentrations
sorbitol 38 ‐
and low contaminant concentrations, in particu‐
gluconate 19 cell retention, pure O2 lar when the product is nonvolatile and highly
succinate 15 cell retention, ISPR soluble in water.
1‐butanol ~10 immobilized cells ‐ Biochemical processes compete with chemical
valine 6.3 metabolic engineering
processes that aim at similar routes from bio‐
mass to product. The biochemical process should
be more selective or should avoid production
16.3. Feasible productivities and isolation of intermediate chemicals.
It is estimated that productivities below 2 g/(L h) are un‐ ‐ Although the potential of many biochemical
commercializable for commodity chemicals because this routes is high, approaching this potential for a
leads to too high capital costs for bioreactors.426 Higher particular product often requires a larger effort
values were indeed found, except for citric acid and ita‐ than anticipated. Vision, dedication, and capabil‐
conic acid. These traditional products require aerobic ity to build a team that can develop the whole
fermentations, but the achievable productivities may easi‐ value chain around the product are key require‐
ly be limited by oxygen transfer rates in the used fungal ments.
pellets. Scientific discoveries and method development have been
It is interesting to review the most productive conver‐ very important to increase the rate of development of
sions mentioned (Table 5). Some involve simple conver‐ biochemical routes; and they will continue to do so. This
sions catalyzed by highly active enzymes. When using the should be accompanied by increased understanding of
enzyme immobilized in a packed bed reactor, high en‐
39
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(8)) Guterl, J. K.; Garbe, D.; Carsten,, J.; Steffler, F.;
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Sommeer, B.; Reisse, S.; Philipp, A.; Haack, M.; M
* Phone: +31‐155‐2782330. E‐m
mail: [email protected]
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