Biotechnology Advances 33 (2015) 648–665

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Biotechnology Advances

journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Lovastatin production: From molecular basis to industrial

process optimization
Kelly C.L. Mulder a, Flávia Mulinari a, Octávio L. Franco a, Maria S.F. Soares a,b,
Beatriz S. Magalhães a, Nádia S. Parachin a,b,⁎
a
Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasilia, Brasília, DF 70790-160, Brazil
b
Grupo de Engenharia Metabólica Aplicada a Bioprocessos, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF CEP 70790-900, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Lovastatin, composed of secondary metabolites produced by filamentous fungi, is the most frequently used drug
Received 25 September 2014 for hypercholesterolemia treatment due to the fact that lovastatin is a competitive inhibitor of HMG-CoA reduc-
Received in revised form 4 April 2015 tase. Moreover, recent studies have shown several important applications for lovastatin including antimicrobial
Accepted 5 April 2015
agents and treatments for cancers and bone diseases. Studies regarding the lovastatin biosynthetic pathway have
Available online 11 April 2015
also demonstrated that lovastatin is synthesized from two-chain reactions using acetate and malonyl-CoA as a
Keywords:
substrate. It is also known that there are two key enzymes involved in the biosynthetic pathway called polyketide
Lovastatin synthases (PKS). Those are characterized as multifunctional enzymes and are encoded by specific genes orga-
Aspergillus terreus nized in clusters on the fungal genome. Since it is a secondary metabolite, cultivation process optimization for
Hypercholesterolemia lovastatin biosynthesis has included nitrogen limitation and non-fermentable carbon sources such as lactose
HMG-CoA inhibitors and glycerol. Additionally, the influences of temperature, pH, agitation/aeration, and particle and inoculum size
Secondary metabolites on lovastatin production have been also described. Although many reviews have been published covering differ-
ent aspects of lovastatin production, this review brings, for the first time, complete information about the genetic
basis for lovastatin production, detection and quantification, strain screening and cultivation process optimiza-
tion. Moreover, this review covers all the information available from patent databases covering each protected
aspect during lovastatin bio-production.
© 2015 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
2. Lovastatin interactions in HGM-CoA reductase active sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
3. The lovastatin biosynthetic pathway: metabolite, genome and transcriptome analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
3.1. Metabolite analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
3.2. Genome analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
3.3. Transcriptome analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
4. Strategies for lovastatin purification and detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654
5. Screening for over-producing strains: naturally and chemically-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656
6. Fermentation mode and cultivation for optimized lovastatin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656
6.1. Solid state fermentation (SSF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657
6.2. Submerged fermentation (SmF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657
6.3. Medium composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
6.4. Cultivation parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
6.4.1. The effects of pH adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
6.4.2. The effect of aeration/agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
6.4.3. The effect of particle size in SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
6.4.4. The effect of pellet morphology in SmF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

⁎ Corresponding author at: Grupo de Engenharia Metabólica Aplicada a Bioprocessos, Instituto de Ciências Biológicas, Universidade de Brasília, CEP 70.790-900 Brasília-DF, Brazil.
Tel.: +55 61 3448 7126.
E-mail address: [email protected] (N.S. Parachin).

http://dx.doi.org/10.1016/j.biotechadv.2015.04.001
0734-9750/© 2015 Elsevier Inc. All rights reserved.
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 649

6.4.5. The effect of inoculum age and size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

6.4.6. The effect of biomass formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661
6.4.7. The effect of temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662
6.5. Biosynthesis of by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662
7. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663

1. Introduction corresponding acid form has an even strong inhibition action and it
has been called lovastatin. Thus in this manuscript mevinolinic acid is
The World Health Organization (WHO) reported that cardiovascular called lovastatin. Lovastatin is the active ingredient of Mevacor and is
disease is the leading cause of death worldwide. In 2008, about 17.3 the precursor for simvastatin, the active principle in Zocor. Since the
million people died from cardiovascular disease, accounting for 30% of discovery of natural statins, filamentous fungi extracts have been pat-
total world deaths. This number is expected to increase 34% by 2030 ented to be used as food additives, mainly in oriental diets as cholesterol
(www.who.int, 2013). One of the factors leading to cardiovascular dis- reducers (Hajjaj et al., 2003; Hong et al., 2003).
ease is hypercholesterolemia, which represents high blood cholesterol Statins can be produced via microbial or chemical synthesis. Among
levels (N200 mg/dL). In the U.S. one in every six Americans has high the ones produced via microbial synthesis, lovastatin is the most stud-
blood cholesterol levels (www.cdc.gov, 2012), and a study performed ied. Fig. 1 illustrates the main research focus in developing a bioprocess
in Brazil showed that about 40% of its population has high blood choles- for microbial lovastatin production. To date, many reviews have covered
terol levels (Martinez et al., 2003). different aspects of lovastatin including its discovery (Alberts et al.,
Statins are the most widely used drugs for hypercholesterolemia 1980; Manzoni and Rollini, 2002; Tobert, 2003), metabolic pathways in-
treatment. These compounds inhibit the enzyme hydroxymethylglutaryl volved in its production (Manzoni and Rollini, 2002), genomic organiza-
coenzyme A (HMG-CoA) reductase, the first enzyme in the cholesterol tion and regulation of lovastatin biosynthetic clusters (Barrios-González
biosynthesis pathway that catalyzes the reduction of HMG-CoA to and Miranda, 2010; Brakhage, 2013; Keller et al., 2005; Manzoni and
mevalonate with concomitant oxidation of 2NADPH molecules. Statin Rollini, 2002), process optimization for development of cultivation
treatment reduces cholesterol synthesis, preventing the buildup of medium, and establishment of fermentation modes (Bizukojc and
plaque inside the arteries (Barrios-González and Miranda, 2010). Nowa- Ledakowicz, 2009; Radha and Lakshmanan, 2013). Nevertheless, none
days, it is one of the best sold drugs in the U.S. with sales totaling of them compile all the information available on lovastatin biosynthesis.
US$11.6 billion by 2011 (www.drugs.com). In addition to cholesterol Therefore, this review brings a complete overview of the mecha-
reduction, statins have been reported to show other effects including nisms in which lovastatin inhibits active sites of HMG-CoA reduc-
nitric-oxide-mediated blood vessel growth (Shuto et al., 2011), femoral tase, the genetic basis for lovastatin production, detection and
osteolyses (Lubbeke et al., 2012), modification of low-density lipoprotein quantification protocols, the different strain screening assays in
quantity (Bojadzievski et al., 2012), and also anti-inflammatory activity addition to a complete vision on what has been done during optimi-
(Khanicheh et al., 2013). Recently lovastatin was also considered as a zation of cultivation conditions. Moreover, from a commercial stand-
candidate to inhibit methanogenic archea present in ruminants point, this review covers all the information available from patent
(Jahromi et al., 2013a,b). Archeas present in ruminant intestine are databases covering all protected aspects regarding lovastatin bio-
responsible for about 20% of methane production, one of the main production (Table 1).
gases responsible for the greenhouse effect. Those microorganisms
synthesize isoprenoid chains to be incorporated into its membrane cell 2. Lovastatin interactions in HGM-CoA reductase active sites
walls. Thus HMG-CoA reductase plays an essential role in isoprenoid
biosynthesis. Thus its inhibition by lovastatin leads to reduction of HMG-CoA reductase is the third enzyme in the cholesterol biosyn-
methanogenic archea. Therefore Aspergillus terreus strains were used to thetic pathway and the first rate-limiting step within this pathway
hydrolyze rice straw improving the quality of ruminant feed (Jahromi since the previous two reactions are reversible. It catalyzes the four-
et al., 2013a,b). Moreover ruminants feed with this hydrolizate was electron reductive HMG-CoA into mevalonate with the concomitant
shown to significantly reduce methane production. In a similar study, oxidation of 2NADPH molecules and the release of CoA-SH. Overall,
lovastatin was shown to inhibit growth rate of Methanobrevibacter statins compete with HMG-CoA by binding to the active sites of HMG-
smithii, one of the methanogen archeas present in ruminant intestine CoA reductase while keeping the NADPH binding site untouched. As
(Jahromi et al., 2013a). Thus besides its medical application, lovastatin can be seen in Fig. 2A, lovastatin hydrophobic-ring structure contacts
may play an important role in feed preparation maximizing biomass residues from the helical structures at the enzyme's large domain. Fur-
utilization. thermore, it has been described that HMG-CoA reductase is arranged
The first reported statin, mevastatin, also known as compactin, was in a strongly associated tetramer with bipartite active sites (Istvan and
first discovered in 1976 and was isolated from a Penicillum citrinum Deisenhofer, 2001). When the HMG-binding pocket is expanded in
strain using a screening assay of 6000 fungal extracts for cholesterol view (Fig. 2B), it can be observed that it is characterized by a cis-loop
biosynthesis inhibitors (Endo et al., 1976a,b). This molecule has a simi- formed by residues 682–694. Since lovastatin is a competitive inhibitor,
lar structure to the substrate HMG-CoA and thus acts as a competitive it seems that its HMG-like moieties bind to HMG-binding active sites.
inhibitor of HMG-CoA reductase. Mevastatin has been assayed in cell Nevertheless, in this complexation mode, their bulky hydrophobic
cultures (Brown et al., 1978) and in vivo (Endo et al., 1979); however, groups collide with amino acid residues that compose the fine pocket,
when it entered into clinical trials, high dosages led to side effects accommodating the pantothenic acid moiety of CoA (Istvan and
such as lymphoma formation in dogs. In addition, the parallel discovery Deisenhofer, 2001). Finally, it can be seen that no portion of the elongat-
of other statins impaired commercialization of mevastatin (Endo, ed NADP(H) binding site is occupied by lovastatin, clearly showing
2010). In the beginning of the 1970s, Merck started a program for isolat- competitive inhibition.
ing new antihypercholesterolemic compounds that resulted in the char- Multiple polar interactions are also formed between the HMG-
acterization of mevinolin (Stossel, 2008). This molecule was found in moieties and amino acid residues situated at cis loops (Asp690, Lys691,
the supernadant of A. terreus culture and it was shown to have a higher Lys692) (Fig. 2B). Lys691 also coordinates the hydrogen-bonding
inhibitory action than mevastatin (Alberts et al., 1980). Indeed its network formation with Glu559, Asp767 and the statin O5-hydroxyl.
650 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

Fig. 1. Main steps during lovastatin production optimization.

The HMG moiety termini carboxylate forms ionic interactions with bonds and also the ion pairs formed result in shape and complementary
Lys735 stabilizing the complex. Finally, and not least important, the charge between enzymes and statins (Istvan and Deisenhofer, 2001).
hydrophobic residue side chains of Leu562, Val683, Leu853, Ala856 and The study of statin interactions with HMG-CoA active sites
Leu857 are involved in van der Waals contacts with lovastatin. Hydrogen allowed for its utilization as biotechnological tools. Studies on the

Table 1
Patents related to lovastatin production and purification as well as the description of each protected process.

Patent number Title Protected process Reference

US2011/0223640 Improved statin production Introduction of LovE into an no Aspergillus terreus sp. aiming van ven Berg and Hans, 2011
increased production of compactin, lovastatin, pravastatin or
simvastatin
US2005/6943017 Method of producing antihypercholesterolemic agents Increased activity of LovA, LovC, LovD and LovF in Hutchinson et al. (2005)
lovastatin-producing and non-lovastatin producing organisms
US2003/0133920 Koji molds for preparing cholesterol lowering products Microorganisms that produces any cholesterol lowering agent Hajjaj et al. (2003)
without producing toxins to be used as food fermentation
additive
US2003/0194394 Methods for producing low cholesterol animal products Production of low cholesterol animal products using Hong et al. (2003)
using hypocholesterolemic feed supplements and hypocholesterolemic feed supplement
products therefrom
US2009/0197311 Method for the production of simvastatin Production of simvastin by introduction of malonate synthase van ven Berg et al. (2009)
activity in A. terreus ATCC 20542
US2005/6949356 Methods for improving secondary metabolite Increased poliketide production by conditional expression of CreA or
production in fungi homolog
US2012/0190038 LovD mutants exhibiting improved properties toward Mutants of acyltransferase (LovD) aiming at increased Tang et al. (2012)
simvastatin synthesis simvastatin and/or huvastatin production by microbial
fermentation
US3983140 Physiologically active substances Physiologically active substances ML-236 where R is a hydrogen Endo et al., 1976a
atom, hydroxyl group or 2-methylbutyryloxy group having
cholesterol and lipid lowering effects.
US4231938 Hypocholesterolemic fermentation products and Compound named MSD803 which has a lactone structure as well as Monaghan et al. (1981)
process of preparation its free hydroxyl form
US4323648 Preparation of monacolin K Antihypercholesterolemic compound of molecular formula C24H36O5 Tanzawa et al. (1982)
from Monascus sp.
WO2001039768 Process for recovering statin compounds from a Large scale lovastatin extraction from cultivation broth with butyl Keri et al. (2001)
fermentation broth acetate
WO199429292 Process for the Isolation of lovastatin Large scale lovastatin extraction from cultivation broth with butyl Hajko et al. (1994)
acetate
WO2006035295 Process for the purification of lovastatin Large scale lovastatin extraction from cultivation broth with toluene Kumar et al. (2006)
WO200017150 New salts of HMG-CoA reductase inhibitors Large scale lovastatin extraction from cultivation broth with ethyl Pflaum (2000)
acetate
WO200006341 Process for the isolation of lovastatin from fermentation Large scale lovastatin extraction from cultivation broth with a Jakubcova et al. (2000)
broth combination of butyl acetate and n-octane, or ethyl acetate and
cyclohexane
WO1997020834 Method of production of lovastatin Large scale lovastatin extraction from cultivation broth with Dimov et al. (1997)
chloroform
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 651

Fig. 2. HMG-CoA arranged in a strongly associated tetramer with bipartite active sites (A). Expanded in view HMG-binding pocket characterized by a loop formed by residues 682–694
named cis loop (B).

structure–activity relationships are also essential for developing et al., 2014). Moreover, structure-functional relationships also open
more potent drugs and avoiding collateral effects. Computer-aided new frontiers for statins and nanotechnology. For example, stents
molecular design screening tools have been used to redesign and have evolved through several generations with the most recent focus
synthesize novel cholesterol inhibitors as previously revised (Sun on completely bioresorbable to add drugs targeting restenosis, the
652 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

surface polymer distributing those drugs, and scaffolds on which those (Chan et al., 1983). In a subsequent study, sodium [1-13C]-, [2-13C]-,
pharmacies are displayed on the surface (Lemos et al., 2013). In [1,2-13C2]-, [1-13C,18O2]-, [1-13C, 2H3] and [2-13C,2H3] acetate, as well
this context a standard bioresorbable terpolymer was successfully as 1802 and [methyl-13C] methionine, were incorporated into mevinolin
modified by covalent incorporation of lovastatin as both a stent and a in A. terreus ATCC 20542 cultures. It allowed for the conclusion that
drug targeting impaired stent thrombosis and re-endothelialization mevinolin is formed from two chains of the polyketide acetate units,
(Kaesemeyer et al., 2013). Such a design allows lovastatin to be deliv- where each chain has a methionine derived from a methyl group
ered directly to injured vessel lumen. Simvastatin-hydroxyapatite (Moore et al., 1985) (Fig. 3). Using the same principle, it has been also
(sim-HA) coatings on a titanium surface was also generated in order demonstrated that biosynthesis of lovastatin has two distinguished
to improve osteoprogenitor cell responses (Shifang et al., 2014). branches, each of which uses acetyl-CoA and malonyl-CoA as substrates
(Fig. 3) (Hendrickson et al., 1999). Since it has been shown that
3. The lovastatin biosynthetic pathway: metabolite, genome and malonyl-CoA is a substrate for statin biosynthesis, genetic strategies
transcriptome analyses for increased intracellular malonyl-CoA, such as the over-expression of
malonate synthase, has been patented for lovastatin and its derivative
Lovastatin production has been reported in several fungal species, compound synthesis in A. terreus ATCC 20542 (van ven Berg et al.,
such as Monascus spp. (Kimura et al., 1990; Komagata et al., 1989; 2009).
Negishi et al., 1986), Penicillium citrinium (Endo et al., 1976a,b),
Paecilomyces viridis (Kimura et al., 1990), Penicillium purpurogenum,
Pleurotus sp. and Trichoderma viride (Javiel and Marimuthu, 2010). 3.2. Genome analyses
Nevertheless, most genomic and transcriptomic studies related to
lovastatin biosynthesis were performed in A. terreus ATCC 20542 After the identification of the main metabolites involved in the lova-
(Askenazi et al., 2003; Hendrickson et al., 1999; Kennedy et al., 1999). statin biosynthetic pathway, studies focused on the genes and enzymes
involved in the synthesis of those metabolites (Hendrickson et al., 1999;
3.1. Metabolite analyses Kennedy et al., 1999). Initial genetic research on lovastatin biosynthesis
identified an essential gene (lovB) that coded for a protein originally
Initial research on lovastatin biosynthesis was aimed at identifying called triol polyketide synthase (Vinci et al., 1998) and later named
intermediate metabolites involved in its pathway. The first study used lovastatin nonaketide synthase (LNKS) (Kennedy et al., 1999). This
the Monascus ruber strain M4681 fed with 14C-labeled followed enzyme consists of a multidomain protein exerting more than one cat-
by NMR analysis. This allowed the identification of monacolin J and alytic function. Identification of LNKS catalytic sites have been done
monacolin L as intermediate metabolites (Fig. 3) (Endo, 1985). An through homology studies between the amino acid sequences of Fatty
NMR study using the same strain utilized labeled 18O2 showed that acid Synthase and LNKS (Vinci et al., 1998). Initially, LNKS was predicted
monacolin L is the precursor of monacolin J where a monooxygenase to contain six catalytic domains: MAT (malonyl-CoA:ACP acyltransfer-
NADPH dependent was involved in the reaction (Fig. 3) (Komagata ase); DH (dehydratase); ER (enoylreductase); KR (ketoreductase);
et al., 1989). ACP (acyl carrier protein); MT (methyltransferase) and CON (condensa-
In parallel, nuclear magnetic resonance (NMR) studies with tion domain). Later, it was shown to contain seven catalytic sites with
A. terreus strain ATCC 20542 using labeled radioisotopes revealed that the additional site called KS (ketosynthase) (Ma and Tang, 2007).
biosynthesis of lovastatin required acetate and methionine as substrates When LNKS gene sequence (lovB) had been isolated, it was utilized
(Chan et al., 1983; Moore et al., 1985). Using sodium [1-13C] acetate, as probe for the isolation of cosmids containing this gene and its
sodium [2-13C] acetate and [methyl-13C] methionine as substrates in surrounding genomic DNA from the A. terreus (ATCC 20542) genomic
the cultivation media, it was shown, for the first time, that a major library. Comparisons of genome sequences allowed for the identifica-
portion of mevinolin consists of a polyketide chain containing nine tion of 18 genes arranged into a 64 kb clusters (Fig. 4) (Kennedy et al.,
intact acetate units and a methionine-derived methyl group from C-6 1999).

Fig. 3. Biosynthetic pathway for lovastatin in A. terreus. LovB is the lovastatin nonaketide synthase (LNKS) that synthesizes dihydromonacolin L together with the dissociated
enoylreductase LovC. Dihydromonacolin L is oxidized to monacolin L and monacolin J by one or more cytochrome P450 enzymes. LovF is the lovastatin diketide synthase (LDKS) that syn-
thesizes 2-methylbutyryl-S-LovF. The side chain is transferred by LovD to monacolin J to yield lovastatin acid form. KS, ketosynthase; MAT, malonyl-CoA:acyl carrier protein transacylase;
ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; MT, methyltransferase; KR, ketoreductase.
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 653

Fig. 4. Lovastatin biosynthetic gene cluster. The blue genes are encoding enzymes that operate in the metabolic pathway of lovastatin; green genes are encoding carriers; orange genes
encode regulatory molecules; gray genes are encoding proteins involved in drug resistance and red genes have unknown function.

In an attempt to obtain strains of A. terreus, which produce increased the direct conversion of Monacolin J into simvastatin in only one step
amounts of lovastatin, random mutagenesis was performed in ATCC (Tang et al., 2012).
20542 (Kennedy et al., 1999). It resulted in the isolation of mutant
strains with alterations in the lovastatin biosynthetic pathway 3.3. Transcriptome analyses
(Hendrickson et al., 1999). Among these mutants, the so-called BX102
did not produce any detectable intermediates in the lovastatin biosyn- Genomic analysis has suggested that ORFs of lovE and lovH are
thetic pathway, however it did produce the oktaketide emodin, indicat- transcription factors since they contain sequences encoding for “zinc
ing that other secondary metabolite pathways were still active. finger” motifs known to bind DNA (Table 2). The deletion and overex-
However, when BX102 was fed with monacolin J, lovastatin synthesis pression of each have individually shown a reduction and increase,
was restored, which indicated that the gene coding for the enzyme respectively, of lovastatin biosynthesis (Kennedy et al., 1999). This idea
responsible for synthesizing and binding of methylbutyryl group is consistent with the fact that lovE and lovH mutants do not produce
was being functionally expressed. On the other hand, the mutant was any of the intermediates of lovastatin biosynthesis (Kennedy et al.,
deficient of genes responsible for the nonaketide part of lovastatin 1999). In another study where the transcriptional levels of the genes
(Fig. 3). As defects in the biosynthetic pathway were reported as lovE (transcription factor) and lovF (LDKS) were compared when using
being either at the diketide portion or the nonaketide portion the both SSF (solid state fermentation) and SmF (submerged fermentation),
biosynthesis of lovastatin, it was concluded that two different enzymes it was shown that transcription of lovE and lovF was 20- and 6-fold
were involved in the pathway (Hendrickson et al., 1999). From this higher, respectively, in SSF than in SmF. Moreover, transcription of lovF
study, two types of polyketide synthases could be described: lovastatin was only detected 5 to 7 days after the beginning of the SmF process.
nonaketide synthase (LNKS) and lovastatin diketide synthase (LDKS), These findings, together with the higher production of lovastatin in SSF
although only the gene encoding for LNKS could be identified in this indicate, at least partially, that a higher transcription factor of its biosyn-
study (Hendrickson et al., 1999). thetic genes, most importantly lovE, enhances lovastatin production
As mentioned before, the 18 genes related to the lovastatin biosyn- (Barrios-González et al., 2008). Indeed, genetically modified strains
thetic pathway are organized into a 64 kb clusters (Kennedy et al., showing LovE overexpression were patented for lovastatin and its analog
1999). This contains the genes encoding for LNKS and LDKS enzymes production (van ven Berg and Hans, 2011).
that are essential for lovastatin biosynthesis Table 2. It has been
shown that the enzyme encoded by the gene lovB concomitant with Table 2
the enoyl reductase enzyme encoded by lovC are essential for the Present ORFs in lovastatin biosynthetic cluster and its described function.
synthesis of dihydromonacolin L (Fig. 3) (Kennedy et al., 1999; Ma Gene Function References
et al., 2009). It has been shown that in the absence of a functional
ORF1 Encodes resistance genes Kennedy et al. (1999)
LovC, the LNKS enzyme encoded by LovB is still able to assemble part
ORF2 Encodes cytochrome 450 monooxygenases Kennedy et al. (1999)
of the monacolin L skeleton, but it results in the production of unstable lovA Encodes cytochrome 450 monooxygenases Kennedy et al. (1999)
conjugated pyrones (Burr et al., 2007). lovastatin diketide synthase lovB Encodes lovastatin nonaketide synthase Vinci et al. (1998)
(LDKS) encoded by lovF has seven catalytic domains: KS, MAT, DH, (LNKS) (cyclization of the main polyketide
MT, ER (enoyl reductase), KR and ACP. This enzyme is involved in the chain, to form the hexahydro naphthalene
ring system)
synthesis of 2 methylbutyryl-CoA as shown in Fig. 3. lovG Encodes a multifunctional esterase Xu et al. (2013)
The last step of the lovastatin biosynthetic pathway is catalyzed by a lovC Encodes a enoylreductase (to catalyze the Kennedy et al. (1999);
transferase encoded by LovD. This transferase connects Monacolin J reactions in the first part of the biosynthetic Burr et al. (2007)
and 2 methylbutyryl-CoA resulting in the acid form of lovastatin pathway, leading to dihydromonacolin L)
lovD Encodes transesterase (catalyzes the Xie et al. (2006)
(Fig. 3) (Hendrickson et al., 1999; Kennedy et al., 1999). A study using
attachment of the 2-methylbutyric acid to
protein–protein interaction has shown that LovD and LovF have an monacolin J, derived from monacolin L).
essential role in transferring 2 methylbutyryl-CoA from LDKS to the ORF8 Unknown function Kennedy et al. (1999)
transferase encoded by LovD, thus ensuring efficient lovastatin synthe- lovE Encodes regulatory genes Kennedy et al. (1999);
sis (Fig. 3) (Xie et al., 2006). All these studies together have shown that Barrios-González et al.
(2008)
the genes LovA, LovC, LovD and LovF are essential for lovastatin biosyn- ORF10 Encodes resistance genes Kennedy et al. (1999)
thesis. Therefore, its overexpression in both lovastatin-producing lovF Encodes lovastatin diketide synthase (LDKS) Hendrickson et al. (1999)
and non-producing microorganisms has been protected for the (transfer of the methylbutyryl side chain to
improvement of statin biosynthesis (Hutchinson et al., 2005). In monacolin J).
ORF12 Unknown function Kennedy et al. (1999)
addition, it has also been possible to develop new processes for the pro-
lovH Encodes regulatory genes Kennedy et al. (1999)
duction of not only lovastatin, but semisynthetic statins, such as simva- ORF14 Encodes transporter genes Kennedy et al. (1999)
statin, demonstrating that facilities currently producing lovastatin can ORF15 Unknown function Kennedy et al. (1999)
also be utilized for the production of simvastatin other related ORF16 Encodes transporter gene Kennedy et al. (1999)
compounds with minimal modifications. For instance, a strain overex- ORF17 Encodes cytochrome 450 monooxygenases Kennedy et al. (1999)
ORF18 Unknown function Kennedy et al. (1999)
pressing acetyltransferase encoded for LovD has been developed for
654 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

Similar results have also been obtained using different genes from methanol have also been employed when extracting lovastatin from
lovastatin clusters (Sorrentino et al., 2010). In a study, lovastatin Pu Erh tea (Yang and Hwang, 2006). Lovastatin extraction from
production was correlated with the increased expression levels of lovB M. purpureus grown on different solid phase substrates has been opti-
and lovF genes encoding for LNKS and LDKS, respectively (Sorrentino mized with 75% ethanol and was 20 times more efficient than extraction
et al., 2010). More recently, an A. terreus strain defective in producing with ethyl acetate (Ajdari et al., 2011a,b). Solid-state fermentation of
LovG could detect lovastatin in the supernatant, however, at a much A. terreus was also performed with an inert polyurethane foam. In this
lower concentration compared to the wild strain type (b5%) (Xu et al., case, extraction proceeded with 50% acetonitrile, but no extraction
2013). Moreover, the production of metabolites such dihydromonacolin optimization was performed (Baños et al., 2009). When using agro-
L, monacolin L or monacolin J has not been observed, indicating that biomass as substrate, the extraction A. terreus SSF was achieved with
LovG is involved in the metabolite synthesis upstream from those inter- MeOH (Jahromi et al., 2012). Lovastatin has been extracted from
mediates. Thus, it has been hypothesized that LovG was involved in the wheat bran substrate through supercritical CO2 (SC-CO2) (Pansuriya
hydrolysis of dihydromonacolin L since homology analyses have shown and Singhal, 2009). The study compared its efficiency with acetonitrile
that LovG belonged to the esterase-lipase family of serine. Genetic and extraction and found that SC-CO2 extraction was similar to that
enzymatic studies have therefore shown that lovG encodes a multifunc- employed with other common organic solvents, but a fourfold increase
tional esterase necessary for the formation of dihydromonacolin L from in purity was achieved. In another study, techniques such as accelerated
lovB (Xu et al., 2013). solvent extraction was proposed to extract lovastatin from red yeast rice
cultures at higher pressure and temperature conditions as a more
4. Strategies for lovastatin purification and detection efficient alternative with limited solvent consumption (Liu et al.,
2010). More recently, lovastatin has also been extracted with acetoni-
Protocols for lovastatin extraction have been optimized over the trile from mycelia and fruiting bodies of several edible mushroom
years according to the biological matrices under study. In general, the species (Chen et al., 2012). Among tested species, Antrodia salmonea
critical step involves an initial extraction phase for which several and Cordyceps sinensis contained the highest amount of lovastatin
protocols exist. When considering liquid cultivation broths, in particular in mycelia: over 1000 mg/kg of dry weight. Other studies have also
from A. terreus, M. ruber and Pleurotus ostreatus, the most common highlighted the fact that extracting lovastatin from fungal mycelium
workflow to date involves a liquid–liquid extraction step with ethyl instead of just culture filtrate can enhance recovery yields by 8-fold
acetate as the solvent of choice (Alarcon et al., 2003; Greenspan and in A. terreus (Chegwin-Angarita et al., 2013; Manzoni et al., 1998,
Yudkovitz, 1985; Jia et al., 2009; Kumar et al., 2000a; Lee et al., 2006; 1999).
Li et al., 2011; Manzoni et al., 1998, 1999; Samiee et al., 2003). Some Extraction methods have also been shown to yield different
studies have also diluted liquid cultivation broths in acetonitrile before forms of lovastatin. Generally, lovastatin is mostly present in the cultiva-
proceeding with detection steps (Askenazi et al., 2003; Lopéz et al., tion broth in its hydroxy acid form. The commercial availability of
2003a,b; Porcel et al., 2006). Although the liquid–liquid extraction is the lactone form of lovastatin makes it the standard choice for subse-
sought to concentrate lovastatin present in the cultivation broth and quent quantification protocols. Therefore, lowering the pH is generally
hence facilitate its detection from a simplified sample, protocols that sought to convert most of the acid form to the lactone-quantifiable
detect lovastatin directly from the liquid broth are also employed. In lovastatin (Alarcon et al., 2003; Friedrich et al., 1995; Manzoni et al.,
some cases screening processes need to be accelerated (Ferrón et al., 1998; Morovjan et al., 1997). However, most reports acknowledge
2005; Hajjaj et al., 2001; Kysilka and Kren, 1993) or simultaneous detec- that incomplete hydrolysis often occurs under these conditions, and
tion with other metabolites is needed (Bizukojc and Ledakowicz, that equilibrium between lactone and acid forms is still present. For
2007a). Unfortunately, the literature lacks a direct comparison between this reason, some studies have focused on converting the lactone form
the exact amount of lovastatin recovered when assayed directly from of lovastatin to the acid form under alkaline conditions. In such cases,
the broth and when extracted with an organic solvent. most lovastatin is present in the more stable hydroxy acid form
Nevertheless, several studies have focused on optimizing lovastatin (Ajdari et al., 2011a; Chegwin-Angarita et al., 2013; Friedrich et al.,
liquid–liquid extraction efficiency by testing pH, temperature and 1995; Jia et al., 2009; Kittell et al., 2005; Lee et al., 2006; Manzoni
organic solvent conditions. Methanol and ethanol extractions have et al., 1998, 1999; Nigovic et al., 2013; Pansuriya and Singhal, 2009;
been shown to yield the highest amount of lovastatin, up to 20 times Yang and Hwang, 2006). In addition, acetonitrile is the solvent of choice
higher when compared to ethyl acetate extraction from Monascus in this case since methanol can react with lovastatin forming the
purpureus cultures (Ajdari et al., 2011a). Furthermore, the highest lovastatin methyl ester derivative (Friedrich et al., 1995; Yang and
amount of monacolin derivatives was obtained using methanol and Hwang, 2006).
ethanol as extraction solvents from both cultivation broth and biomass Protocols for lovastatin detection and quantification invariably use
(Ajdari et al., 2011a). Optimum conditions in this case included heating high performance liquid chromatography (HPLC) coupled with UV
to 60 °C and extracting for 2 h under shaking conditions (Ajdari et al., detection at 238 nm. This method is widely used in fermentation
2011a). This result is also in accordance with previous studies for laboratories, whereby C18 analytical reverse-phase columns are condi-
which methanol and ethanol also extracted lovastatin more efficiently tioned in different mobile phases, and lovastatin acid and lactone forms
from red yeast rice at 60 °C for 30 min (Lee et al., 2006). Recently, are readily separated and quantified. For a detailed description see
extraction conditions of both acid and lactone lovastatin producing Table 3. However, a few reports have focused on capillary electrophore-
A. terreus were optimized at 23 °C and pH 3.0 using ethyl acetate as an sis for quantifying lovastatin in cultivation broths (Kittell et al., 2005;
extraction solvent (Lisec et al., 2012). However, when looking to Nigovic et al., 2013). The advantages over HPLC quantification lie in
improve the extraction of lactone lovastatin, the higher hydrophobicity the fact that columns are unnecessary, less solvent is used, and chro-
of butyl acetate or isopropyl acetate significantly improved yields by at matographic runs are shorter, which allow for a high throughput
least twofold. These authors, however, did not include methanol or screening of A. terreus mutant strains (Kittell et al., 2005). More recently,
ethanol in their studies (Lisec et al., 2012). however, protocols that employ electrospray mass spectrometry (ESI-
When considering solid-state fermentation processes, organic MS) coupled to liquid chromatography (LC) for additional detection
solvent extraction is also employed but no common protocol exists. and quantification have become routine. Although more prominent in
Ethyl acetate was used to extract lovastatin from M. ruber grown on the clinical field, LC-MS detection and quantification of lovastatin
long grain rice (Lee et al., 2006). In another study, 80% methanol has have been used successfully for M. purpureus strains (Lee et al., 2006;
been employed to extract lovastatin from red yeast rice supplements Nigovic et al., 2013; Song et al., 2012) and for A. terreus (Askenazi
(Nigovic et al., 2013). Solid-phase extraction cartridges conditioned in et al., 2003).
Table 3
Lovastatin extraction and quantification protocols from different biological matrices.

Matrix Extraction method Sample lactonization or Quantification References

hydrolysis
LC LC-MS

Monascus purpureus EtOH:H2O solutions for 2 h at 60 °C Sample hydrolysis with 0.1 N NaOH and Symmetry C18 (150 × 3.9 mm). Gradient elution Zorbax SB C18 (100 × 2.1 mm). Gradient Ajdari et al. (2011a)
fermented products under stirring kept for 2 h at 30 °C. with ACN/0.1% TFA elution with ACN/0.2% AcCOOH
M. purpureus fermented 75% MeOH, sonication for 30 min None – Intersil ODS 3 (150 × 2.1 mm). Gradient elution Song et al. (2012)
products with MeOH/0.05% FA/4 mM of NH4 formate
M. purpureus fermented 80% MeOH at room temperature for 1 h Hydrolysis of the lovastatin stock with 0.1 N – Symmetry C18 (150 × 4.6 mm). Gradient Nigovic et al. (2013)
products in an ultrasound bath NaOH in 50% ACN at 45 °C for 1 h elution with ACN/0.1% FA
M. purpureus Accelerated solvent extraction with None High-speed counter-current chromatography with Symmetry Shield C18 (250 × 4.6 mm) with Liu et al. (2010)
AcOEt at 120 °C, for 7 min 8:2:5:5 (v/v/v/v) of n-hexane/ethyl isocratic elution with 70% ACN/0.1% H3PO4
acetate/MeOH/H2O
Monascus sp. strains AcOEt at 70°°C for 1.5 h Lactonization is mentioned but conditions Ultrasphere ODS (150 × 4.6 mm). Isocratic elution Discovery C18 (250 × 4.6 mm) with isocratic Lee et al. (2006)
not stated with ACN 65% and H3PO4 0.5% elution 55% ACN
Monascus sp. Strains AcOEt at 70 °C for 1.5 h Sample lactonization with TFA 1%. Ultrasphere ODS (150 × 4.6 mm). Isocratic elution None Su et al. (2003)
with 72% ACN

K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

Monascus rubber and MeOH at 30 °C for 1 h under stirring None Spherisorb ODS (250 × 4.0 mm). Isocratic elution None Friedrich et al. (1995)
Aspergillus terreus with 60% ACN/0.1% H3PO4
A. terreus No extraction None Separon SGX (150 × 3 mm). Isocratic elution with None Kysilka and Kren (1993)
77.5% MeOH/18 mM H3PO4
A. terreus No extraction None Nucleosil 100-5 C18 (250 × 4 mm). Gradient Detection by MS is mentioned but conditions Hajjaj et al. (2001)
elution with ACN/0.05% H3PO4 not stated
A. terreus No extraction None Ultrasphere ODS (250 × 4.6 mm). Isocratic elution None Ferrón et al. (2005)
with 60% ACN/0.1% H3PO4
A. terreus No extraction None Symmetry Shield C18 (250 × 4.6 mm). Gradient None Bizukojc and Ledakowicz
elution with ACN/0.1% H3PO4 (2007a)
A. terreus 30% ACN dilution None – Xterra MS-C18 (250 × 2.1 mm). Isocratic Askenazi et al. (2003)
elution with 60% ACN/0.1% FA
A. terreus 50% ACN dilution Hydrolysis of the lovastatin stock with 0.1 N Ultrasphere ODS (250 mm × 4.6 mm) Isocratic Lopéz et al. (2003a,b); Porcel
NaOH in 50% EtOH at 50 °C for 20 min elution with 60% ACN/0.1% H3PO4 et al. (2006)
A. terreus 50% ACN at 50 °C for 30 min under None Novapack C18 column (150 × 3.9 mm). Isocratic None Baños et al. (2009)
stirring elution with 60% ACN/0.1% H3PO4
A. terreus ACN for 60 min Sample lactonization with concentrated Ultrasphere ODS (50 × 4.6 mm). Isocratic elution None Morovjan et al. (1997)
H3PO4 for 60 min with 50% ACN/0.1% H3PO4.
A. terreus AcOEt for 2 h under stirring None Hypersil BDF C18 (250 × 4.6 mm). Isocratic elution None Kumar et al. (2000a)
with 55% ACN/12% MeOH/33% phosphate buffer pH
4.0
A. terreus AcOEt at 23 °C for 1 h, pH 3.0 Lactonization by refluxing the sample at Eurosphere C18 (250 × 4.6 mm). Gradient elution None Lisec et al. (2012)
reduced pH and elevated temperature with ACN/0.1% H3PO4
A. terreus AcOEt for 2 h under stirring Hydrolysis of the lovastatin stock with 0.1 Eurosphere-100 C18 (250 × 4.0 mm). Gradient None Samiee et al. (2003)
M NaOH at 50 °C for 1 h elution with 60% ACN
A. terreus MeOH for 2 h under stirring Sample hydrolysis with 0.1 N NaOH in 50% Acclaim 120 C18 (150 × 4.0 mm). Isocratic elution None Sorrentino et al. (2010)
ACN at 45 °C for 1 h 55% ACN/0.1% H3PO4
A. terreus Super critical CO2 extraction with MeOH Sample hydrolysis with 0.025 N NaOH in Hamilton C18 (250 × 4.6 mm). Isocratic elution None Pansuriya and Singhal (2009)
at 40 °C, 300 bar, 60 min MeOH at 45 °C for 30 min under stirring with 60% ACN/0.1% H3PO4
Pleurotus ostreatus Supernatant extracted with AcOEt; Sample and mycelial mass lactonization LiChrospher 100 C18. Gradient elution with None Alarcon et al. (2003)
mycelium with CH2Cl2 and AcOEt at 40 with HCl MeOH/H2O
°C for 1 h
P. ostreatus, Culture filtrate and mycelial mass Hydrolysis of the lovastatin stock with 0.1 Hypersil GOLD C18 (150 × 4.6 mm). Gradient None Chegwin-Angarita et al.
P. pulmonarius, extracted with AcOEt M NaOH in 50% EtOH at 50 °C for 20 min elution with ACN/0.1% FA (2013)
P. djamor cold under reflux
Pu-Erh tea DSC-C18 solid-phase extraction Supernatant was mixed with 0.1 N NaOH in Luna C18 (250 × 4.6 mm). Isocratic elution with None Yang and Hwang (2006)
cartridges in MeOH/H2O 50% ACN at 45 °C for 1 h 70% ACN/0.5% AcCOOH
Mycelia and fruiting ACN at 25 °C for 24 h None LiChrospher 100 C18 (250 × 4.6 mm). Gradient None Chen et al. (2012)
bodies of mushrooms elution with ACN/H2O

655
656 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

Moreover, when lovastatin has to be purified from cultivation broth ATCC 20542 for improvement of lovastatin production using cycles of
in large-scale, most patents describe a pre-alkaline treatment to UV. The resulting strain named L4414 was obtained after 4 cycles of
enhance lovastatin acid extraction followed by an acidification step UV exposure and resulted in a threefold increase in lovastatin titers as
prior to organic solvent extraction. Lovastatin lactone extraction is well as increased resistance to lovastatin concentrations up to threefold
then achieved with various solvents, such as butyl acetate (Hajko when compared to the wild type (Jia et al., 2010).
et al., 1994; Keri et al., 2001), toluene (Kumar et al., 2006), a combina- Strain engineering and cultivation optimization processes have been
tion of butyl acetate and n-octane, or ethyl acetate and cyclohexane used together with A. terreus ATCC 20542 to improve lovastatin produc-
(Jakubcova et al., 2000), ethyl acetate (Pflaum, 2000) or chloroform tivity (Kumar et al., 2000a). Initially, ATCC 20542 was incubated with
(Dimov et al., 1997). Most protocols proceed with the distillation Ethyl Methanesulfonate (EMS) followed by UV exposure. In a second
or evaporation of the solvent and describe the use of crystallization round, the resulting strain was incubated with N-methyl-N-nitro-N-
procedures for lovastatin lactone recovery. nitroso-guanidine (NTG) followed by UV treatment. The resulting strain
was named DRCC122 and was used in repeated fed-batch fermentations
5. Screening for over-producing strains: naturally using maltodextrin as a feeding carbon source. Although results from
and chemically-induced strain improvement are not shown independently, it has claimed an
improvement of 83% in lovastatin production with maximum produc-
Lovastatin producing A. terreus was first reported more than 30 years tivity of 7.64 mg/L ∙ h (Kumar et al., 2000a). Attempts were made to
ago on a strain isolated from soil in Madrid, Spain, and subsequently increase lovastatin titers when A. terreus NRRL265 was submitted to
deposited in the ATCC collection with reference number 20542 random mutagenesis using UV irradiation followed by incubation with
(Alberts et al., 1980). Studies that report screening for hyper nitrous acid (Mukhtar et al., 2014). After exposure to both mutagens,
producing strains are limited and are summarized in Table 3. Though 38 conidia were selected and tested for lovastatin production. Among
encompassing other species, the highest lovastatin production was the isolates, one produced about 3.5 times more lovastatin than
reported in a strain of A. terreus (Jaivel and Marimuthu, 2010a,b) NRRL265. The isolate producing higher lovastatin titers was further
followed by other strains of Aspergillus sp. (Mangunwardoyo et al., utilized for media optimization. Altogether, strain modification and
2012; Samiee et al., 2003). However, on average, these levels are process design resulted in 8 times greater lovastatin production when
lower than those obtained in different studies with strain ATCC 20542 compared to the wild strain (Mukhtar et al., 2014).
(98 mg–1 g/L) (Bizukojc and Ledakowicz, 2007a,b; Jia et al., 2009; Most of the studies cited above used chromatography methods in
Lopéz et al., 2003a,b; Lopéz et al., 2004a,b; Pecyna and Bizukojc, order to detect lovastatin super producers. Nevertheless, a novel meth-
2011). Yet, it is important to emphasize that none of the screening odology using the antimicrobial property of lovastatin has been used to
studies used ATCC 20542 as a control strain. Therefore, considering screen for spores submitted to UV treatment. In the developed setup,
different growth conditions of the fungus and different lovastatin the spore solution was trapped into agar plugs and placed in petri dish
extraction and quantification protocols, a direct comparison of the plates containing Neurospora crassa. Hyperproducers were screened in
results is not possible. a larger inhibition zone. The method was able to isolate one particular
Although only a limited number of studies report novel lovastatin strain that produced almost 1.5 times more lovastatin than the wild
producing strains, it is more frequent to isolate one particular type (Kumar et al., 2000a). Another study utilized a similar methodolo-
producing strain and perform random mutagenesis in order to gy but used Candida albicans instead (Ferrón et al., 2005). In this study,
increase lovastatin titers. The most common methodology to generate spores of A. terreus strain ATCC 20542 were exposed to EMS at different
lovastatin-producing strains is to expose them to UV followed by exten- concentrations and incubation times. After two mutation rounds,
sive screening. The first study that used this experimental setup was extracts prepared from different strains were used in bioassays against
published more than 20 years ago (Vinci et al., 1991). It used A. terreus C. albicans. Although the number of strains that were screened using
ATCC 20542 and aimed to reduce secondary metabolite production this methodology has not been reported, the resulting strain produced
other than lovastatin. More specifically, it focused on the reduction of about four times more lovastatin than the parental strain (Ferrón
sulochrin production. During the mutagenesis cycle, the A. terreus strain et al., 2005).
was incubated with N-Methyl-N′-nitro-N-nitrosoguanidine (NTG) as
chemical mutagen. Initial screening was performed using high perfor- 6. Fermentation mode and cultivation for optimized lovastatin
mance thin layer chromatography (HPTLC) for detection of both production
lovastatin and sulochrin, using a total of 1623 mutants. From this
screening, 39 strains were used in a secondary screen where strains The industrial process for lovastatin production was initially set up
were grown in shake flasks for 16 days and the supernatant was in 1980 using glycerol as a carbon source in a fed-batch cultivation
analyzed in HPLC for lovastatin and sulochrin detection and quantifica- mode (Mevacor, Merck). Using the A. terreus strain ATCC 20542, maxi-
tion. The final strain resulted in 20% increased lovastatin production, mum lovastatin production was 180 mg/L (Buckland et al., 1989).
concomitant to an 83% sulochrin reduction (Vinci et al., 1991). Adjustments of process parameters such as culture homogeneity,
In another study, a strain isolated in Korean soil that produced lova- carbon source, pH, aeration, and agitator design yielded a fivefold in-
statin was treated with UV and selected for cerulenin and L-methionine crease in lovastatin production (Manzoni and Rollini, 2002). Aeration
resistance (Hong et al., 1999). These molecules are known to inhibit related to high viscosity of the cultivation broth and slow use of the
polyketide enzymes. Selection was initially done separately for both carbon source, particularly glycerol, were parameters shown to limit
molecules, then a mutant resistant to both cerulenin and L-methionine the scaling-up processes from 800 L to 19,000 L (Buckland et al.,
analog was prepared by the protoplast fusion of both strains. The 1989). The current lovastatin production level of the strain A. terreus
resulting strain increased lovastatin production 15-fold, while reducing ATCC 20542 found in the literature is around 7000–8000 mg/L,
other secondary metabolites by about 90% (Hong et al., 1999). produced through mutagenesis approaches by the Metkinen Group
Resistance to lovastatin and fatty acid synthase (FAS) inhibitors such (Metkinen Oy, Finland).
as iodoacetamide and N-ethylmaleimide was also been utilized to Twenty years ago, Biocon (Biocon India, Bangalore, India) began a
improve lovastatin production in an A. terreus strain isolated after distinct and successful industrial-scale production method of lovastatin
having been screened for 134 fungal isolates. After three cycles of muta- by A. terreus using wheat-bran SSF (Suryanarayan, 2003). It had obtain-
genesis, a hyper-producing strain called EM19 had about 7.5 times ed United States FDA approval for lovastatin production in January
greater lovastatin production when compared to an isolated strain 2001, which went off patent in June 2001. The technology is based on
(Kaur et al., 2009). In a recent study, exogenous lovastatin was used in the use of the Plafractor, a large-scale SSF bioreactor (Barrios-González
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 657

and Miranda, 2010). Under precise conditions, this bioreactor gathers use of a high-density polyurethane foam (PUF) led to about twofold
both solid substrate and submerged fermentation, and allows the higher lovastatin production than that obtained by sugarcane bagasse
reduction of downstream processing problems during product extrac- (19.95 mg/g versus 9.94 mg/g, respectively) (Baños et al., 2009). More-
tion (Manzoni and Rollini, 2002). over, wheat bran was found to be the best solid substrate for increased
The lovastatin synthesis pathway utilizes the carbon source more lovastatin production (0.9823 mg/g), followed by sorghum grains, rice
slowly than the biomass growth pathway. Thus, the use of a slowly bran (0.7904 mg/g) and paddy straw (0.7257 mg/g) (Jaivel and
metabolized C-source such as lactose and glycerol has been shown to Marimuthu, 2010a,b). Wheat bran has also yielded the highest (up to
be very effective for obtaining elevated amounts of lovastatin (Lopéz approximately 30-fold) lovastatin amount among cotton oil cake,
et al., 2003a,b), especially when lactose is fed at the early stationary gram husk, corn hull, nut oil cake, rice husk, orange peel and pulp, and
phase since it is used for both lovastatin biosynthesis and biomass sugar cane bagasse using an A. terreus strain (Pansuriya and Singhal,
formation (Bizukojc and Ledakowicz, 2007a,b; Lopéz et al., 2003a,b; 2010). In a different study, wheat bran led to up to 13-fold higher
Novak et al., 1997). However, the most suitable quantity and type of lovastatin amounts by an Aspergillus flavipes strain among barley,
carbon source for lovastatin super-production varies in the literature. gram bran, soybean meal, wheat bran (bagasse, barley and gram bran)
They include the use of over 100 g/L of glucose (Novak et al., 1997), and orange and pineapple epicarp (Valera et al., 2005). High lovastatin
glycerol at 70 g/L (Manzoni et al., 1998), sucrose at 50 g/L, lactose at yields on wheat bran has been associated to higher mycelia growth,
70 g/L (Lai et al., 2005), or lactose at 100 g/L (Lopéz et al., 2005). Regard- since lovastatin is an intracellular product, and higher mycelia growth
ing the nitrogen source, production of lovastatin is generally associated is directly associated to a higher lovastatin yield (Wei et al., 2007).
with N-limited growth conditions, when carbon is not limiting but
growth has been arrested by nitrogen limitation, and excess carbon 6.2. Submerged fermentation (SmF)
can be channeled into secondary metabolism production (Bizukojc
and Ledakowicz, 2007a,b; Lopéz et al., 2003a,b). Among different SmF fermentation modes, the most utilized are
The two most common fermentation modes reported for lovastatin batch and fed-batch. Regarding fed-batch fermentation, the use of
production are solid state fermentation (SSF) and submerged fermenta- carbon sources such as lactose or glycerol, which are slowly assimilated,
tion (SmF). has been shown to be more effective for lovastatin production than
readily consumable C-sources such as glucose (Hajjaj et al., 2001). How-
6.1. Solid state fermentation (SSF) ever, it is worthwhile to mention that in some strains a higher formation
of (+)-geodin, also a product of the polyketide pathway, has been
SSF has gained researchers' attention due to its many advantages observed with the use of lactose (Bizukojc and Ledakowicz, 2007a,b,
over the conventional SmF, like higher yields of secondary metabolites 2008).
and enzymes that are only produced by SSF (Jaivel and Marimuthu, Therefore, alternatives have been reported for more cost-effective
2010a,b; Miranda et al., 2013). The molecular and physiological details batch and fed-batch processes. A repeated fed-batch process using
related to the behavior of microorganisms in SSF, which are different 50% maltodextrin and 37.5% corn steep liquor as C- and N-sources
from the those in SmF, are not well understood and are often referred resulted in a significant increase in lovastatin yield of 73% over the
to as “physiology of solid medium” (Barrios-González et al., 2008). batch process (Kumar et al., 2000a). This sort of result has been
One feature related to the molecular behavior of SSF is the transcript previously observed where lovastatin concentration in the fed-batch
accumulation level of genes lovE and lovF. The higher production of phase was increased by 37% over batch fermentation when a second
lovastatin by SSF compared to SmF (20 mg/g vs. 650 mg/L, respectively) feeding was applied. However, although repeated fed-batch fermenta-
has also been shown to correlate with higher transcription levels of lovE tion raised the lovastatin production, the overall productivity was
(4.6-fold) and lovF (twofold) under specific growth conditions (Barrios- claimed not to be an improvement over the batch process (Novak
González et al., 2008). et al., 1997). In another study where a initial batch/fed-batch phase
The sort of solid substrates used in SSF has been demonstrated to be followed by a semi-continuous operation was performed lovastatin
a limiting factor for the production of lovastatin. Substrate is mainly production was enhanced by 50% when an initial batch phase lasting a
composed of cellulose, hemicellulose, and lignin, all of which have to minimum of 4 days was utilized, followed by a semi-continuous culture
be degraded by microorganisms through enzymatic hydrolysis phase that was employed to sustain biomass near zero growth (Porcel
(Table 4). For instance, it has been shown that when rice straw (RS) is et al., 2007). A year later, the same research group proposed a new
used as substrate, it resulted in a fourfold increase in lovastatin produc- fermentation strategy consisting of a 96 h batch/fed-batch phase follow-
tion than when oil palm frond (OPF) was used as substrate (Jahromi ed by a semi-continuous operation at a dilution rate of 0.42 day for a fur-
et al., 2012). This significant difference is likely due to the composition ther 140 h. The rate of lovastatin production increased with time in the
of the biomass of both substrates, with OPF having twice the amount batch phase as biomass decreased slightly (less than 2 g/L) in the semi-
of lignin present in RS and lower hemicellulose content. Therefore, the continuous phase. This strategy resulted in threefold higher lovastatin
lower recorded production of lovastatin by OPF is likely due to the production than batch fermentations (Porcel et al., 2008).
absence of enzymes that degrade the lignin component of the biomass The time of initial carbon feeding is considered of highest impor-
(Jahromi et al., 2012). Another comparative study reported that the tance in fed-batch experiments and has been debated in several studies.

Table 4
Screened strains for lovastatin production.

Screened genus Isolated Number of studied Maximum lovastatin Reference

strains production

Aspergillus, Monascus, Penicillium, Pleurotus and Trichoderma. Coimbatore, South India 10 116.8 mg/L Jaivel and Marimuthu (2010a)
Aspergillus University of Indonesia 40 85.8 mg/L Mangunwardoyo et al. (2012)
Culture Collection
Acremonium, Aspergillus, Penicillium and Trichoderma. PTCC, Tehran, Iran 110 55.0 mg/L Samiee et al. (2003)
Aspergillus, Biospora, Cylindrocarpon, Penicillium, Trichoderma Egypt 23 48.4 μg/L Osman et al. (2011a)
and Mycelia.
Soil isolates India soil 134 190 mg/L Kaur et al. (2009)
Aspergillus and Rhizopus Karnataka and Tamil Nadu of India 130 996.6 mg/L Upendra et al. (2013)
658 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

Usually, the feeding of the carbon source must initiate prior to carbon crude glycerol (45% glycerol) and pure glycerol, it has been observed
depletion during the batch phase. This strategy has been previously that the last yielded a significantly higher lovastatin production than
reported and has resulted in increased lovastatin production by differ- the former in a range from 10 to 50 g/L of substrate, reaching optimal
ent A. terreus strains (Kumar et al., 2000a; Novak et al., 1997). In agree- production at 30 g/L (Rahim et al., 2015).
ment with previous studies, it has been shown that when lactose is It has been reported that the main limiting substrate for lovastatin
depleted from cultivation media, the biosynthesis of lovastatin ceases production is the N-source rather than the C-source (Lopéz et al.,
(Bizukojc and Ledakowicz, 2007a,b). However, one particular study 2003a,b, 2004a,b; Porcel et al., 2007; Valera et al., 2005). As lovastatin
has shown that lovastatin biosynthesis starts only when lactose does not contain any nitrogen in its structure (C24H36O5), its biosynthe-
consumption has stopped, suggesting that lovastatin synthesis is sis is connected with nitrogen use only to the extent of nitrogen
enhanced when the substrate is depleted from the cultivation media influence in the biomass formation (Bizukojc and Ledakowicz, 2007a,
(Hajjaj et al., 2001). b). Indeed, depending on the nitrogen concentration, lovastatin produc-
A lactose feeding strategy starting at the early stationary phase was tion may be inhibited. Therefore, the N-source must not be high in the
proposed to be very effective for lovastatin production since this carbon system but at the same time not too low, which might also impair
source is utilized for both biomass and lovastatin production (Bizukojc lovastatin production (Bizukojc and Ledakowicz, 2007a,b; Jahromi
and Ledakowicz, 2007a,b). In a following study, an improved strategy et al., 2012). In the literature, adequate levels of N sources vary among
for lovastatin production was developed by combining lactose- and the type of the source; a range from 2 to 10 g/L of yeast extract can be
glycerol-fed batches, in which lactose was the initial carbon source found, as well as various concentrations of inorganic sources such as
and glycerol was used in the feed step (Pecyna and Bizukojc, 2011). In glutamic acid and sodium nitrate, and of organic sources such as
this strategy, the highest lovastatin concentration (122.4 mg/L) was soybean, corn meal, etc. (Table 5). Production of lovastatin is generally
obtained when the feed started while lactose was still present in the associated with the stationary phase of nitrogen-limited growth when
medium (Pecyna and Bizukojc, 2011). excess carbon can be channeled into secondary metabolism. In view of
these results, lovastatin yields can be improved when carbon is not lim-
6.3. Medium composition ited and growth has been arrested by nitrogen limitation. The best N-
source for lovastatin production is controversial among several studies.
Optimization of the cultivation media is important for both lovastat- Complex nitrogen sources, such as yeast extract, corn steep liquor and
in yields and production rates, as is shown by the following studies. soybean meal are considered more suitable than single amino acids or
Carbon and nitrogen sources as well as the C/N ratio play a significant salts containing ammonium ions (Bizukojc and Ledakowicz, 2007a,b).
role as sources of precursors and cofactors since these nutrients are Inorganic N-source like sodium nitrate or urea (at 4, 4, and 4. 5 g/L,
directly involved in the synthesis of biomass building blocks and metab- respectively) led to an inhibition of lovastatin production (b 1 mg/L),
olites (Hajjaj et al., 2001; Kumar et al., 2000a; Li et al., 2011). Moreover, likely due to its consumption driven to biomass formation (Hajjaj
it has been reported that the nature and concentration of the carbon et al., 2001). Moreover, the use of amino acids such as glutamate and
source regulates lovastatin biosynthesis (Jia et al., 2009; Lopéz et al., histidine, and to a lesser extent glycine (all at 12.5 g/L) better supported
2003a,b). For instance, Hajjaj et al. (2001) tested two carbon sources lovastatin biosynthesis than inorganic N-sources (at 47, 46, and
(glucose and lactose) in complex medium on lovastatin production. 17 mg/L, respectively) (Hajjaj et al., 2001). Furthermore, the use of
Initially the highest lovastatin production (54 mg/L) was obtained in response surface methodology has been employed to perform experi-
an experiment with 20 or 45 g/L of glucose. This range of initial glucose mental design for media composition and it has been shown that this
concentration had little effect on lovastatin yield, and, increasing the approach might reach a four-fold increase of lovastatin production
initial glucose concentration to 70 g/L led to a significant decrease in (Lai et al., 2003; Lopéz et al., 2004a,b).
lovastatin production. It was also shown that lactose was only Other media components have been evaluated in order to improve
consumed after glucose starvation, about at the same time lovastatin lovastatin production, such as the presence and concentration of differ-
synthesis was initiated. This time, the initiation of lovastatin production ent divalent metal cations (Jia et al., 2009), exogenous lovastatin, antibi-
is likely due to relief from carbon metabolite repression. Finally when otics (Jia et al., 2010), linoleic acid (Sorrentino et al., 2010), and vitamins
lactose was used as sole carbon source lovastatin biosynthesis only (Bizukojc et al., 2007). The role of divalent metal cations such as Fe2+,
initiated after lactose consumption stopped also indicating that the Ca2+, Zn2+, Cu2+, Mg2+ and Mn2+ has been analyzed and demonstrat-
synthesis of this secondary metabolite initiates under growth limited ed to be important for cell growth and lovastatin production (Jia et al.,
conditions (Hajjaj et al., 2001). In a different study, sucrose and glycerol 2009). It has also been reported that Cu2+ (at 1, 2 and 5 mM) inhibited
were each shown to significantly enhance lovastatin production (over 4 cell growth and hardly affected lovastatin biosynthesis, while all other
and 5 mg/g, respectively) compared to glucose and lactose (b 4 mg/g) cations tested were stimulated by the increase in biomass from 1.5-
(Xu et al., 2005). up to 2.5-fold compared with the other elements, and led to the highest
Production of lovastatin has been shown to be highly influenced by production rate of lovastatin (472 mg/L). The interaction of all the metal
carbon source (lactose, glycerol, and fructose), nitrogen source (yeast cations (at 2 mM) had a certain negative effect on lovastatin biosynthe-
extract, corn steep liquor, and soybean meal) and the C:N mass ratio sis, compared with their sole action (Jia et al., 2009).
in the medium (Lopéz et al., 2003a,b). The pathway in which carbon is It has been shown that supplementation of exogenous lovastatin at
directed to lovastatin synthesis is slower than the one that uses carbon the early stage of cultivation did not delay the onset of lovastatin
for biomass since lovastatin is a product of secondary metabolism. Thus, production, rather, it markedly inhibited 20% of its final biosynthesis
the use of lactose, a slowly-metabolized C-source, in combination with (Jia et al., 2010). On the other hand, other polyketide antibiotics such
either soybean meal or yeast extract under N-limited conditions as tylosin, erythromycin, tetracycline, and rifamycin all improved the
gave the highest titers and specific productivity of lovastatin net yields of final lovastatin by approximately 20–25%, probably due
(0.1 mg g− 1 h−1) (Lopéz et al., 2003a,b). Other studies have shown to a similar mechanism of action. This stimulation has been associated
that the highest lovastatin production was obtained when glycerol to precursor formation in the early stage of lovastatin biosynthesis (Jia
was used as the C-source, reaching up to a concentration of 916 mg/L et al., 2010).
with 3% glycerol supplementation (A. terreus Z15-7) (Li et al., 2011), Linoleic acid is able to regulate the transcription of lovastatin by its
and 244 mg/L by adding a “shot” of glycerol of 50 g/L (A. terreus MIM potential role as a quorum-sensing molecule, and it has increased
A2) (Manzoni et al., 1998). It must be considered that the quality of lovastatin production up to 1.8-fold when added into the media at low
glycerol is essential for substrate uptake efficiency. Comparing the cell density. Conversely, the synthesis of lovastatin was reduced when
utilization of crude glycerol and production efficiency. By comparing a high cell density of linoleic acid was added (Sorrentino et al., 2010).
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 659

Table 5
Parameters utilized for lovastatin production optimization in solid state fermentations.

Strain Lovastatin Incubation time Initial Temp. Inoculum size Substrate Reference
concentration (days) pH (°C) (spores/mL)

Aspergillus terreus TUB F-514 17.1 mg/mL 8 6.5 30 108 High-density polyurethane foam Baños et al. (2009)
(PUF) — inert support
A. terreus ATCC 74135 260.85 mg/kg DM 8 6.0 25 107 Rice straw Jahromi et al. (2012)
A. terreus JPM 3 982.3 μg/g−1 15 5.0 RT NR Wheat bran Jaivel and Marimuthu (2010a,b)
A. terreus ATCC 20542 2.9 mg/g dry 11 5.5 28 107 Rice Wei et al. (2007)
substrate
Aspergillus flavipes BICC 5174 16.65 mg g−1 6 5.0 30 108 Wheat bran Valera et al. (2005)
A. terreus TUB F-514 118 mg/dm 7 6.5 NR 2 × 106 High-density polyurethane foam Miranda et al. (2013)
M. purpureus MTCC 369 and 2.83 mg/g 14 6.0 29 4.95 mL Rice Panda et al. (2010)
M. ruber MTCC 1880
A. terreus UV 1718. 3723.4 ± 49 μg/g−1 10 6.0 28 5 × 107 Wheat bran Pansuriya and Singhal (2010)

In order to evaluate the impact of the supplementation of cul- A. terreus (Bizukojc and Ledakowicz, 2008; Bizukojc et al., 2012;
tivation media with B-vitamins on the biosynthesis of lovastatin, five Pawlak et al., 2012).
vitamins (thiamine, riboflavin, pyridoxine hydrochloride, calcium The initial pH strongly influences the substrate use by A. terreus. It
D-pantothenate and nicotinamide) were used. It was demonstrated seems that the rapid use of carbon substrates at higher initial pH values
that three of these, calcium D -pantothenate (1 mg/L), pyridoxine is evidence of the faster metabolism. Therefore, neutral and alkaline pH
(5 mg/L), and nicotinamide (1 mg/L) exerted a significant positive values give better lovastatin titers than acidic ones (Bizukojc et al.,
effect on the biosynthesis of lovastatin (Bizukojc et al., 2007). This 2012). The initial pH value of the medium also influences the biosynthe-
study has reported the costs of B-vitamin supplementation, and sis of other polyketide metabolites of A. terreus, such as the significant
these were very low due to the fact that the vitamins were added decrease of (+)-geodin (Bizukojc and Ledakowicz, 2008). Moreover,
at low amounts as trace elements. The prices of B-vitamins range the control of pH at the levels of 7.6 and 7.8 has been successfully
from about 100 €/kg for nicotinamide to about 3000 €/kg for Ca- applied to repress the formation of the by-product (+)-geodin in both
pantothenate (Sigma, Germany), resulting in a total cost of about batch and fed-batch experiments (Bizukojc and Ledakowicz, 2008).
0.50 € per 100 L of the cultivation medium (800 mg of the vitamin Nevertheless, it is important to mention that this effect is weaker than
mixture in 100 L medium). The cost of 1 mg of the analytical grade compared with pH effects on lovastatin synthesis, where it increases
lovastatin is about 4.50 € (Sigma, Germany). The authors claim that 10-fold when pH is controlled at 7.5 (Bizukojc et al., 2012).
if pharmaceutical grade lovastatin is ten times cheaper, its increased
yield due to B-vitamins supplementation, even by several milligrams 6.4.2. The effect of aeration/agitation
per liter, seems to be cost-effective (Bizukojc et al., 2007). Molecular oxygen is required in the biosynthesis of lovastatin mole-
cules via the polyketide pathway, and it has been suggested that an
increase in the oxygen tension affects some equilibrium step in the bio-
6.4. Cultivation parameters synthetic route, enhancing the final lovastatin concentration (Lopéz
et al., 2004a,b). Therefore, due to the important role of oxygen availabil-
6.4.1. The effects of pH adjustment ity, dissolved oxygen (DO) has to be controlled to guarantee high
Although it has been claimed in most scientific reports that the lovastatin productivity. It has been reported that a high titer of lovastat-
optimum initial pH value for lovastatin production medium is 6.5 in is obtained when oxygen-enriched cultivation are carried out instead
(Tables 4 and 5), the effects of pH control have been debated. As of a cultivations sparged with air (Lai et al., 2005; Lopéz et al., 2004a,b).
described below, a number of studies have shown that pH control is of Moreover, increasing DO above 20% of air saturation helps to advance
little significance in the final lovastatin titer. When comparing SSF and production by almost one day, and the DO level during the first 48 h
SmF pH, the kinetics are similar, indicating that the difference in lova- (in 5 L fermentations) has been shown to be critical for lovastatin
statin production is only due to the culture system (Baños et al., synthesis (Lai et al., 2001). It has also been shown that if DO is reduced
2009). It has also been reported that there is no significant difference to 10%, maximal lovastatin production only achieves 11% of that at 20%
in lovastatin production when the initial pH ranges from 5 to 8 of DO (Lai et al., 2005). On the other hand, if DO is maintained at ≥70% of
(Jahromi et al., 2012). In addition, Lai et al. (2005) showed that pH is saturation, the high shearing field produced by the high agitation rate
gradually stabilized toward 6.1 by the culture itself in the 5-L fermenter, disrupts the mycelia, consequently reducing the lovastatin yield in the
where no adjustments are needed. On the other hand, there are studies process (Novak et al., 1997).
reporting negative/positive effects of pH adjustments. Experiments car- Considering differences in stimuli sensed by cells growing in SSF and
ried out in shake-flask cultures have shown that lovastatin production SmF, an evident one is the direct contact with air (and O2) in the former
decreased by 16–48% when an additional pH adjustment of 5.5 or 7.5 (Miranda et al., 2013). However, high concentrations of mycelium and
was applied, and product formation seemed not to be influenced if the spores growing on the surface of solid materials might reduce the
culture was run either naturally or at 6.5 (Lai et al., 2005). At low initial flow of air in the culture, consequently decreasing the available oxygen
pH values, even at pH ranges of 3.5 to 5.5, lovastatin is still, although for growth of A. terreus (Jahromi et al., 2012). In SmF cultures, A. terreus
inefficiently, produced, and, of course, this formation is far less efficient can grow in a variety of morphological forms, varying between a net-
than batches with neutral or alkaline initial pH values (Bizukojc et al., work of freely dispersed mycelia to tightly packed and discrete pellets,
2012; Osman et al., 2011a,b). In fact, the effect of increasing pH has determined by the culture conditions and the inoculation method.
been reported as positively correlated with lovastatin production. Besides, the rapid increase in viscosity greatly impairs oxygen transfer,
Over a range of pH 7–8.5, a maximum peak of lovastatin concentration and this is believed to explain the low titers of lovastatin produced
was reached at pH 8.5 (66.69 mg/L), and at pH 9 productivity was under dispersed growth conditions. In order to better understand this
reduced (45.89 mg/L) (Osman et al., 2011a,b). These results are in limitation, lovastatin production has been studied under a range of agi-
accordance with those previously reported, where pH control of the cul- tation speeds (Lopéz et al., 2005). It was found that oxygen transfer did
tivation medium at 7.0–7.5 was applied to produce lovastatin by not limit lovastatin synthesis at agitation speeds ≥300 rpm. However, in
660 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

oxygen-enriched cultures, agitation at 800 rpm reduced lovastatin titers production was observed due to unfavorable morphological changes in
to the level of air-sparged titers, due to the shear-induced alterations in the fungal cells (Lai et al., 2002).
pellet morphology (Lopéz et al., 2005). Another study has shown that On the contrary, it has also been shown that excessive aeration is not
while using 325 rpm of agitation, lovastatin production achieved beneficial for lovastatin production. In a study performed with SSF using
305 mg/L, which was both 29% and 33% higher than that using high-density polyurethane foam (PUF), cultures with limited aeration
225 rpm or 425 rpm, respectively. Furthermore, when the impeller or without aeration have presented the highest lovastatin yields. It is
speed was reduced lower than 225 rpm, O2 limitation (less than 3% of likely that O2 limitation triggered slower but steadier respiration and
DO) occurred, and lovastatin production was reduced to one-fifth (Lai growth rates that were more conducive to secondary metabolite
et al., 2005). production (Baños et al., 2009). Furthermore, it has also been reported
It is important to emphasize that previous studies not only imply that higher aeration rates favor the formation of (+)-geodin (over
that an efficient supply of DO is inevitably required for lovastatin 60 mg/L) more than lovastatin, so that the decrease of aeration rate is
production, it also indicates that there is an optimum between mor- preferred for lovastatin biosynthesis (Bizukojc and Ledakowicz, 2008).
phology and agitation for process optimization. Regarding the effect of In two-step submerged fermentation (Table 6), lovastatin productivity
the shear on the cell surface at a different physiological age, it has has been shown to be negatively affected by aeration, in which non-
been reported that it is inadequate to apply constant agitation through- aerated mediums yielded higher rates of lovastatin production than
out the process. Thus, a sufficient DO supply without affecting pellet those produced in aerated production medium (Osman et al., 2011a,b).
formation is essential (Lai et al., 2005). Indeed, addition of n-dodecane
as oxygen vector in shake-flask culture has been shown to increase 6.4.3. The effect of particle size in SSF
lovastatin production thorough morphological changes in the fungal Water availability is a typical condition in SSF fermentations and
cells. However, by adding oxygen carrier to experiments carried in 5 L could be one of the inducing factors of the lovastatin biosynthetic
fermenter cultivation presenting high DO level (N 60%), lower lovastatin genes. Both positive and negative effects of moisture content have

Table 6
Parameters utilized for optimization of lovastatin production in submerged fermentations.

Strain Lovastatin Incubation Initial Agitation Temp. (°C) Inoculum size C source N source Reference
(A. terreus) concentration time (days) pH (rpm) (spores/mL)

ATCC 20542 952.7 ± 24.3 mg/L 8 6.5 220 28 108 67.56 g/L of soluble 10 g/L of yeast Jia et al. (2010)
starch extract powder
3
M. purpureus 315 mg/L 14 6.0 110 30 5.7 × 10 29.59 g/L dextrose Sayyad et al. (2007)
MTCC 369
6
TUB F-514 570 mg/L 7 6.5 250 30 10 4% lactose or 3.4% 0.3% soybean Baños et al. (2009)
glucose + 0.6% meal
lactose
TUB F-514 650 mg/L 7 6.5 250 30 106 Glucose 0.6% Barrios-González
et al. (2008)
ATCC 20542 70 mg/L 7 6.5 150 30 107 Lactose 20 g/L 4 or 2 g/L yeast Bizukojc and
extract Ledakowicz (2008)
ATCC 20542 55 mg/L 10 NR 150 30 NR Lactose 20 g/L 4 g/L yeast Bizukojc et al. (2007)
extract
ATCC 20542 120 mg/L 10 NR 110 30 107 Lactose 20 g/L 4 g/L yeast Bizukojc and
extract Ledakowicz (2007a,b)
ATCC 20542 112 mg/L 7 7.5 110 30 109 Lactose 20 g/L 4 g/L yeast Bizukojc et al. (2012)
extract
ATCC 74135 220 mg/L 13 6.5 400 28 NR Lactose + glucose 9.8 g/L glutamic Hajjaj et al. (2001)
20 g/L acid
9
DRCC 122 2200 mg/L 10 6.5–7.2 150–165 28 3.5 × 10 Maltodextrin Corn steep liquor Kumar et al. (2000a)
ATCC 20542 1.050 mg/L 7 6.5 200 28 5 × 106 Lai et al. (2003)
ATCC 20542 572 mg/L 10 5.5–7.5 325 28 (shift to 23) NR Lactose 70 g/L Yeast extract Lai et al. (2005)
(8 g/L)
ATCC 20542 873 mg/L 10 6.5 200 28 107 Lactose 70 g/L NR Lai et al. (2007)
Z15-7 916.7 mg/L 15 6.0 150 30 NR Glycerol 3% 1% corn meal and Li et al. (2011)
0.2%
sodium nitrate
ATCC 20542 30 mg/g 10 6.5 150 28 2 × 108 Lactose 20 g/L Soybean meal Lopéz et al. (2003a,b)
3.84 g/L
ATCC 20542 230 mg/dm3 7 6.5 150 28 9 × 107 Lactose 48 g/L Soybean meal Lopéz et al. (2004a,b)
0.46 g/L
MIM A2 256 mg/L 21 6.4 60 25 NR Glycerol 70 g/L Soybean flour Manzoni et al. (1998)
strokes/min Glucose 30 g/L 30 g/L
6
TUB F-514 30 mg/dm 7 6.5 200 30 2 × 10 0.6% glucose + 0.3% soybean Miranda et al. (2013)
3.4% lactose meal,
ATCC 20541 160 U/L 6 6.8 400–700 30 5 × 108 Glucose 100 g/L Corn steep liquor Novak et al. (1997)
20 g
wet wt/L,
188.3 mg/L 7 7.0 180 30 NR Glucose 50 20 yeast extract, Osman et al.
20 oat meal (2011a,b)
ATCC 20542 122.4 mg/L 9 NR 110 min−1 30 109 Lactose 10 g Yeast extract Pecyna and Bizukojc
4 g L−1 (2011)
7
ATCC 20542 186.5 ± 20.1 mg/L 7 6.5 150 28 8.6 × 10 Lactose 114.26 g/L Soybean meal Porcel et al. (2005)
5.41 g/L
−1
ATCC 20542 ≈2.5 mg/L/h 10 6.0 300 28 NR Lactose 114.26 g/L 5.41 g/L soybean Porcel et al. (2007)
meal
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 661

been described in previous studies. Low water availability has been observed that addition of n-dodecane as oxygen carrier in 5 L cultivation
claimed as one of the factors responsible for high levels of gene did not enhance lovastatin production as in shake-flask experiment due
transcripts involved in lovastatin production (Barrios-González et al., to different morphological changes in the fungal cells. Together with
2008). On the other hand, it has also been found that initial moisture shear of cells caused by the impellers, pH–DO interactions in the cultiva-
content (from 75 to 85%) not only increases mycelium growth, but tion caused changes from smooth to fluffy loose pellets, described as
also extends production time from five to seven days, and moreover, star-like ones (Lai et al., 2002). Rheological studies using A. terreus
twofold higher lovastatin production is achieved in cultures with higher have shown that rheological characteristics are directly related to cell
initial moisture contents (Baños et al., 2009). morphology regarding pellet size and, to a lesser extent, fluffiness, and
Throughout several studies, the importance of the interaction effect lovastatin production (Porcel et al., 2005; Gupta et al., 2007). It has
between particle size and moisture content in SSF fermentation has been shown that pellet presenting intermediate hairiness and rough-
been emphasized (Jahromi et al., 2012; Pansuriya and Singhal, 2010; ness is required for better production of lovastatin (Gupta et al., 2007).
Valera et al., 2005; Wei et al., 2007). Two opposing effects of particle
size on the SSF process at any given moisture content have been
6.4.5. The effect of inoculum age and size
proposed. The first is that small particle size increases the surface area
Spore concentration and age have been found to be the main source
of solid materials and therefore provides for better attachment, allowing
of variation in lovastatin production using SmF. It has been claimed that
fungal growth (Wei et al., 2007). The second effect is that smaller parti-
the increase of initial spore concentration gradually increased lovastatin
cle size reduces space between particles, consequently decreasing
titers, as well as developed smaller pellets (Bizukojc and Ledakowicz,
oxygen transfer and also increasing localized heat generation in the
2010). It has been also reported that increasing spore age from 9 to
bed of the solid substrate, eventually reducing the growth potential of
16 days has raised the lovastatin titer by 52%. As expected, this behavior
the aerobic microorganisms and lovastatin production (Jahromi et al.,
was associated with the fact that lovastatin is produced mainly during
2012; Pansuriya and Singhal, 2010; Valera et al., 2005; Wei et al., 2007).
the stationary phase of the growth (Porcel et al., 2006)
The particle size for lovastatin production by SSF depends on the
In some SSF experiments, inoculum size has been reported to have
solid substrate, as well as on the species of Aspergillus sp. used. It has
both positive and negative effects. Different inoculum sizes within the
been found for A. terreus (ATCC 20542 and ATCC 74135), optimum
range of 5 × 107 to 10 × 107 spores/mL did not affect lovastatin produc-
particle size of rice straw is between 1.4 and 2 mm (Jahromi et al.,
tion in three strains of A. terreus: ATCC 74135, ATCC 20542, and UV 1718
2012) or 840 μm (ATCC 20542) (Wei et al., 2007), and for wheat bran
(Jahromi et al., 2012); while either higher or lower inoculum size de-
it is 0.35 mm (UV 1718) (Pansuriya and Singhal, 2010). For A. flavipes,
creased lovastatin production, from 1845 μg/g to under 1800 μg/g or
solid wheat bran particles should be no bigger than 0.4 mm (Valera
1300 μg/g, respectively (Pansuriya and Singhal, 2010).
et al., 2005). Therefore, a certain combination of particle size and mois-
ture content should be considered in order to obtain the best result in
terms of lovastatin yield. It has been proposed that 50% moisture 6.4.6. The effect of biomass formation
content is optimum for lovastatin production (Jahromi et al., 2012). By construction a kinetic model for lovastatin production Bizukojc
On the other hand, a different study has shown that an increased in and Ledakowicz observed that lovastatin biosynthesis is only partially
the moisture level from 60 to 70% increased lovastatin production. growth-associated. First, the maxima of biomass volumetric formation
This may be due to the appropriate amount of oxygen and water within rate (rx) and lovastatin volumetric formation rate (rmev) did not coin-
the substrate to support fungal growth. However, when the moisture cide. Second, in the early hours of the run, the sensitivity of μmax
level was increased from 80 to 90%, lovastatin production decreased. (maximum specific biomass growth rate) achieved its highest value,
This has been associated with poor oxygen availability caused by the and it was followed by increasing kmev (constant rate for lovastatin
excessive replacement of air by water (Pansuriya and Singhal, 2010). formation), therefore reflecting the partial association of lovastatin bio-
synthesis with biomass growth (Bizukojc and Ledakowicz, 2007a,b).
6.4.4. The effect of pellet morphology in SmF Later, the same research group confirmed this observation, and more-
It has been reported that lovastatin synthesis is likely to occur in the over, it reported that the obtained yields of lovastatin over biomass
filamentous zone of the pellet and not in the central core when SmF fer- occurred to be significantly better in bioreactor experiments than the
mentations are carried out. Interestingly, it has been reported that pellet ones reported for shake flask cultures (Bizukojc and Ledakowicz, 2008).
size is inversely proportional to the initial number of spores; indeed, the It is also important to consider both the N- and C-sources of the
smaller the pellets, the faster the biosynthesis as well as better titers of medium when analyzing the effects between biomass growth and lova-
lovastatin (Bizukojc and Ledakowicz, 2010). Moreover, the agitation statin production. When compared among fructose, glycerol and lactose
speeds correlated with the size of pellets are significant parameters as C-sources, fructose was shown to be the source which yielded the
that can affect fungus morphology, which, in consequence, is very im- highest concentration of biomass (N5 g/L compared to b 5 g/L using
portant for the formation of lovastatin (Kumar et al., 2000a; Lopéz the other C-sources) in N-limited growth using either yeast extract or
et al., 2005; Gupta et al., 2007). For instance, it has been shown that ag- corn steep liquor, as well as the highest titers of lovastatin (N 120 mg/L
itation speed of ≥600 rpm reduced pellet size and damaged pellet mor- compared to b 100 mg/L).
phology; moreover, much smaller pellets were produced under higher It has been reported that for SmF experiments carried out in shake
agitation (≥ 800 rpm), which resulted in poor lovastatin productivity flasks, the cells directed more building blocks for biomass production
(approximately 40 mg/L). On the contrary, at lower agitation speeds rather than lovastatin production when compared with SmF carried
(300 rpm) stable maximum pellet diameter exceeded 2500 μm, and out in a 5 L fermenter leading to a an enhanced by 38%, lovastatin
this size produced high lovastatin titers (approximately 40 mg/L). This production together with a decrease in biomass production and by in-
pattern was also reported by Porcel et al. (2005) where it has been creasing sugar utilization (Lai et al., 2005). However, two other ap-
found that stirred tanks agitated at up to 300 rpm keep the hydrody- proaches performed in order to reduce biomass production have
namic shear comparable and stable presence of pellets of up to shown either negative or no effects on lovastatin production. First, it
2300 μm. Still corroborating with previous studies, it was stated that was shown that the biomass growth profile between a wide range of ag-
the compactness and fluffiness of pellets are affected by the agitation itation speeds, from 300 to 800 rpm, was slightly affected (varied from
intensity, and that small and dense pellets are formed under 600 rpm 11 to 13 g/L, respectively) (Lopéz et al., 2005). Fortunately, the associa-
agitation (Porcel et al., 2005). This suggests that a high oxygen concen- tion of product formation with biomass growth has been reported to be
tration in the pellet is necessary but not sufficient for attaining a high weaker for the biosynthesis of by-products than for lovastatin (Bizukojc
titer of lovastatin (Lopéz et al., 2005). Indeed, Lai et al. (2002) had and Ledakowicz, 2007a).
662 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665

In an SSF experiment, it has been reported that the lovastatin yield (Pecyna and Bizukojc, 2011). This study has used either lactose or
increased quickly from day 1 to day 5 (from 0 to 2 mg/g), which glycerol as the C-source and only yeast extract as the N-source. It has
might explain that although lovastatin is a secondary metabolite, its been shown that the maximum (+)-geodin concentration, about
accumulation in mycelia seems to be growth related, which is different 255.5 mg/L, twofold higher than the lovastatin concentration, was ob-
from phenomena observed in SmF. Since lovastatin is an intracellular tained when the initial C-sources lactose and glycerol were utilized in
product, it has been proposed that product accumulation almost simul- feed (Pecyna and Bizukojc, 2011). Moreover, the earlier glycerol was
taneously increases as does cell growth (Wei et al., 2007). added, the earlier (+)-geodin was formed, and its final concentration
was higher since its specific production rate was sustained for a longer
6.4.7. The effect of temperature period of time by the glycerol feed (Pecyna and Bizukojc, 2011). It is im-
In order to evaluate the effects of the temperature, lovastatin portant to note that initial amount of carbon source has a stronger influ-
production was investigated in a range from 23 °C to 28 °C in SmF (Lai ence on (+)-geodin production compared to lovastatin, as it has been
et al., 2005). When the temperature was changed from 28 °C to 23 °C, observed by a 338% rise on (+)-geodin and 48% inhibition on lovastatin
maximal lovastatin production increased by 25% compared to the production using crude glycerol (45% glycerol) as carbon source (Rahim
control grown at 28 °C. Thus, temperature was indeed shown to be an et al., 2015).
important environmental factor that improves lovastatin titers, likely It is also known that lactose feeding also increases final (+)-geodin
by inducing genes or activating enzymes involved in lovastatin produc- yield by up to 90% (Bizukojc and Ledakowicz, 2007a). The difference in
tion (Lai et al., 2005). It is important to emphasize that the temperature- the production of both (+)-geodin and lovastatin was about 40% of final
shift might be strain and/or fermentation mode dependent and might lovastatin amount produced before lactose feeding started, while 90% of
be strictly correlated to the physiological age of the fungus. For instance, the final yield of (+)-geodin was produced during lactose feeding
it has been recently reported that 25 °C is the optimum temperature for (Bizukojc and Ledakowicz, 2007a). Therefore, if lactose is the C-source
lovastatin production by two different strains of A. terreus (ATCC 74135 of choice, batch fermentation mode is more efficient than the fed-
and ATCC 20542) in SSF (Jahromi et al., 2012). Moreover, it was shown batch mode for high production of lovastatin and inhibition of (+)-
that by increasing the incubation temperature over 25 °C, the effect on geodin formation. Regarding the N-source effect, it has been observed
lovastatin production was negative, with ATCC 74135 more sensitive that the highest yield for (+)-geodin was obtained in nitrogen limiting
to higher temperatures (Jahromi et al., 2012). For the M. purpureus conditions of 2 g/L of yeast extract. This result is explained by the bio-
strain MTCC 369 and the M. ruber strain MTCC 1880, 29.46 °C has mass content at this N-source concentration, which is about 36% lower
been shown to be the optimum cultivation temperature that resulted than when higher concentrations of yeast extract are utilized
in the highest yields in lovastatin production, about 0.83 mg/g in co- (Bizukojc and Ledakowicz, 2007a).
culture of M. purpureus and M. ruber SSF (Panda et al., 2010).
In studies carried out via SmF, it was shown that 30 °C was the opti- 7. Conclusions and perspectives
mum and maximum temperature for lovastatin production, resulting in
916.7 mg/L of lovastatin when using the A. terreus strain Z15-7 (Li et al., Lovastatin, a secondary metabolite commonly produced by filamen-
2011). Lovastatin production by A. terreus ATCC 20542 was highly tous fungi with various applications within health and agro industries.
affected by incubation temperatures, where lovastatin concentration in- Initial studies on this molecule have demonstrated its ability to bind
creased concomitantly with the increase of incubation temperature to the active site of HMG-CoA reductase, the first limiting step in the
from 15 °C (17.6 mg/L) until reaching the maximum lovastatin concen- cholesterol biosynthetic pathway. Although lovastatin production is re-
tration at 30 °C (50.9 mg/L) (Osman et al., 2011a,b). ported in trace amounts in many fungi genera such as Monascus, Peni-
cillium, Paecilomyces and Trichoderma, industrial production is done
6.5. Biosynthesis of by-products using an A. terreus strain. A. terreus is also the most common species uti-
lized in molecular, physiological and process optimization studies. It has
It is known that A. terreus is a rich secondary metabolite producer been previously reported that the genes that encode protein within the
and is able to synthesize a wide variety of by-products such as 6- lovastatin biosynthetic pathway are located in a 64 kb cluster. In total,
methylsalicylic acid, pigment precursors, citrinin, sulochrin, asterric there are 18 genes, of which only half have described functions.
acid, and (+)-geodin (Couch and Gaucher, 2004; Hutchinson et al., Lovastatin is synthesized from two reaction branches that use acetyl-
2000; Schimmel and Parsons, 1999). (+)-Geodin is the most produced CoA malonyl-CoA and methionine as substrates. Each branch of the
by-product and is therefore more often reported in the literature metabolic pathway has polyketide synthases involved. These enzymes
(Askenazi et al., 2003; Bizukojc and Ledakowicz, 2007a,b, 2008). It is no- contain seven catalytic domains and are conserved among other micro-
ticeable that (+)-geodin is produced later than lovastatin, and it in- organisms that produce secondary metabolites. To date, there is no
creases at the end of the cultivation process, indicating that its knowledge on the variability of the lovastatin genomic cluster. More-
production may be related to nitrogen deprivation phase (Rahim et al., over, there is only one genome sequence available for A. terreus with
2015). In the mid-1930s, it was first reported that A. terreus strains limited knowledge on which genes are truly necessary for lovastatin
were capable of producing (+)-geodin (Raistrick and Smith, 1936). Sig- biosynthesis.
nificant amounts (up to 2.66 M concentrations) of this compound were Regarding the improvement of lovastatin titers, both strain modifi-
found in several Aspergillus sp. by investigation of the transcriptional cation and cultivation conditions have been optimized. At the strain
and metabolite profiles of lovastatin-producing fungi. Genetic engineer- level, the most frequent methodology utilized is directed evolution
ing has been applied to disrupt (+)-geodin biosynthesis, and those using mutagen agents such as UV. After that, a screening strategy is
engineered did not produce detectable levels of (+)-geodin (Askenazi necessary in order to isolate a hyper-producing strain. Although lova-
et al., 2003). Simultaneous lovastatin and (+)-geodin biosynthesis statin is commonly detected and quantified using reverse chromatogra-
were evaluated regarding medium composition in a fed-batch process. phy methods, antimicrobial detection assays have also been developed
It has been found that the highest (+)-geodin concentration, approxi- based on the properties of lovastatin that inhibit yeast growth. At the
mately 18 mg/L, followed by the same amount of lovastatin, were process design level, lovastatin production has been attempted in both
obtained when the lowest amount of nitrogen source, 2 g/L of yeast ex- SSF and SmB where lovastatin production is about 5 times higher
tract, was present in the medium (Bizukojc and Ledakowicz, 2007a, using SmB. Within this fermentation setup, fed-batch using glycerol as
2008). the carbon source and nitrogen limitation have shown to be the most
Moreover, not only the quantity but also the type of carbon used as promising strategies for lovastatin production. Moreover, the use of
substrate is shown to be of high importance to (+)-geodin formation more complex substrates such as maltodextrin and corn steep liquor
 K.C.L. Mulder et al. / Biotechnology Advances 33 (2015) 648–665 663

has shown production higher than 1 g/L. Besides carbon and nitrogen Chegwin-Angarita C, Jeannette Nieto-Ramirez I, Diaz GJ, Rojas LJ, Sepulveda L, Atehortua
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