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Journal of Biotechnology 134 (2008) 154–159

Genome shuffling enhanced l-lactic acid production by improving

glucose tolerance of Lactobacillus rhamnosus
Lei Yu a,b , Xiaolin Pei a , Ting Lei a , Yuhua Wang a,b , Yan Feng a,∗
a Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University,
2519 Jiefang Road, ChangChun 130023, PR China
b College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, PR China

Received 17 July 2007; received in revised form 16 December 2007; accepted 11 January 2008

Abstract
Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we applied the
genome shuffling to improve the glucose tolerance of Lactobacillus rhamnosus ATCC 11443 while simultaneously enhancing the l-lactic acid
production. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for the recursive
protoplast fusion. The positive colonies from library created by fusing the inactivated protoplasts were more likely to be screened on plates
containing different concentrations of high glucose and 2% CaCO3 . Characterization of all mutants and wild-type strain in the shake flask indicated
the compatibility of two optimal phenotypes of glucose tolerance and lactic acid enhancement. The lactic acid production, cell growth and glucose
consumption of the best performing strain from the second round genome shuffled populations were 71.4%, 44.9% and 62.2% higher than those of
the wild type at the initial glucose concentration of 150 g/l in the 16 l bioreactor. Furthermore, the higher lactic acid concentrations were obtained
when the initial glucose concentrations increased to 160 and 200 g/l in batch fermentation.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Genome shuffling; Lactobacillus rhamnosus; l-Lactic acid; Glucose tolerance

1. Introduction The various environmental stresses such as extremes in tem-

perature, pH, osmotic pressure (NaCl), oxygen and high pressure
Lactic acid has been widely used in food, industrial, pharma- have already been largely considered for the improvement of the
ceutical and cosmetic applications. As a general purpose food lactic acid bacteria to meet industrial requirements. However,
additive, l-lactic acid is generally recognized as safe (GRAS) there is little information available in literature about tolerance
(Datta et al., 1995). In fact, interest toward l-lactic acid is to high substrate concentration (glucose) for lactobacilli. It is
rapidly increasing because l-lactic acid is used in a very large desirable to produce lactic acid with concentrations of substrate
amount of chemical intermediate for biodegradable polylactic as high as possible because it will lead to the increase of lac-
acid, a typically high crystalline polymer with attractive mar- tic acid concentration and the reduction of downstream process
ket prospect. Polylactic acid shows great potential in a wide costs (Senthuran et al., 1997). However, it can be expected that
range of material uses such as food packaging, garbage bags, the lactic acid production would suffer from substrate inhibition
and agricultural plastic sheeting. This is due to its mechanical at high glucose concentrations. Therefore, it is necessary to gen-
properties (Ohara, 2003; Thomsen, 2005). In poly-l-lactic acid, erate glucose-tolerant strains with an enhanced ability of lactic
a biocompatible thermoplastic polyester with high strength, it acid fermentation.
can also be used for dental and drug delivery systems, sur- Classical strain improvement methods have succeeded in
gical sutures and implantable surgical devices (Di Lorenzo, obtaining many industrial strains, but it is time-consuming
2005). and laborious for many repeated rounds of random muta-
tion and selection methods. Recently, an efficient technology
named genome shuffling has made a major advance in the
∗ Corresponding author. Tel.: +86 431 8849 9722; fax: +86 431 8898 7975. construction of mutants with distinctly significant improved
E-mail address: [email protected] (Y. Feng). phenotype. The tylosin production from Streptomyces fradiae

0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2008.01.008
 L. Yu et al. / Journal of Biotechnology 134 (2008) 154–159 155

has been rapidly improved using two rounds of genome shuf- When the glucose concentration increased, the rest nutrients
fling which previously required 20 rounds of mutagenesis and increased by the same proportion.
screening (Zhang et al., 2002). Genome shuffling allows many
parental strains with certain phenotypic improvements recom- 2.3. Strain mutagenesis and mutant screening
bine through recursive protoplast fusion. A library of shuffled
bacteria with genetic exchange is achieved by the repetition of The cells of Lr-WT were grown in a resting tube containing
the above process. Since the limited knowledge about genome a 10 ml MRS medium at 37 ◦ C for 24 h. For NTG mutagenesis,
sequence information impairs rational application of recombi- a 0.2 ml culture broth was spread onto the solid MRS with two
nant DNA techniques to manipulate the strain, genome shuffling grains of NTG placed at the center of the plate. The lawn around
exhibits the advantage of recombination between genomes in the inhibition zone was scraped after 48 h incubation and culti-
uncharacterized organisms. This approach has also been used to vated in liquid MRS for 6 h. The enriched culture was serially
improve the acid tolerance in Lactobacillus (Patnaik et al., 2002), diluted in sterile saline and spread on YE agar plates containing
degradation of pentachlorophenol in Sphingobium chloropheno- 2% CaCO3 at 37 ◦ C with 5% CO2 in a CO2 incubator. UV irra-
licum (Dai and Copley, 2004) and production of hydroxycitric diation was performed by exposing Lr-WT spread on YE agar
acid in Streptomyces (Hida et al., 2007). Moreover, our research plates containing 2% CaCO3 directly to UV light at a distance
group has already applied genome shuffling to improve the acid of 20 cm for 30 s. The plates were also incubated at 37 ◦ C with
tolerance and volumetric productivity in Lactobacillus rhamno- 5% CO2 in a CO2 incubator. The fast grown colonies under both
sus (Wang et al., 2007). conditions which had the bigger transparent haloes compared to
In the present study, we aim to develop glucose-tolerant colony diameter, were picked off for the shake-flask analysis.
mutants of L. rhamnosus ATCC 11443 (Lr-WT) capable of The mutants with high l-lactic acid production were selected as
producing enhanced levels of l-lactic acid by using genome the starter for the genome shuffling.
shuffling. The mutant strains were obtained with subtle improve-
ments for L. rhamnosus wild-type strain ATCC 11443 by 2.4. Genome shuffling
ultraviolet (UV) irradiation and nitrosoguanidine (NTG) muta-
genesis, and then used for recursive protoplast fusion. All mutant Protoplast formation was carried out as described by
strains were screened on YE plates containing high glucose Cocconcelli et al. (1986), except that 1.2% glycine was added
and 2% CaCO3 . The shake-flask test was applied for the fur- into the MRS broth before the inoculation. When all lactobacil-
ther screening. The behavior of the Lr-WT and mutants was lus became the spherical cells in the visual field of phase contrast
investigated in a shake-flask and bioreactor. microscopy, the enzyme treatment was for 1 h with 10 mg/ml
lysozyme and 30 ␮g/ml mutanolysin in lactobacillus protoplast
2. Materials and methods buffer (LPB:20 mM HEPES, pH 7.0, 20 mM MgCl2 and 0.5 M
sucrose). Then the protoplasts were diluted in the LPB to stop the
2.1. Materials enzyme reaction and harvested by concentration at 4500 × g for
10 min. Protoplasts from different populations were mixed and
Standard l-lactic acid, mutanolysin and lysozyme were pur- divided equally into two parts. They were inactivated and shuf-
chased from Sigma, USA. Corn steep liquor (dry solids) was fled as described (Wang et al., 2007). The mixture was diluted,
obtained from Huanglong Food Industrial, Co., and molasses centrifuged and resuspended in liquid RM and shaken at 100 rpm
from Dacheng Group, Co., Jilin province, China. All other chem- for 6 h before plating on YE plates containing high glucose
icals used in this work were of analytical grade and procured concentration and 2% CaCO3 . The plates were incubated for
locally. 3 days with 5% CO2 incubator at 37 ◦ C. Based on the same
method of the above mutant screening and shake-flask analysis,
2.2. Organism and growth media the best shuffled mutants were taken for the subsequent round
of genome shuffling. The pooled fusion libraries were named F1
The L. rhamnosus ATCC 11443, a homofermenter of l- and F2.
(+)-lactic acid, was obtained from the American Type Culture
Collection. The strain was stored in a deMan, Rogosa and Sharpe 2.5. Shake flask and bioreactor cultivation
(MRS) broth with 20% (v/v) glycerol at −80 ◦ C and grown in
liquid MRS from a 1% inoculum with 16-h incubation at 37 ◦ C. Lr-WT and mutants were transferred to flask culture in orbital
At least two generations of precultures were required just before shakers at 37 ◦ C, 200 rpm and up to 36 h. Calcium carbonate
the experiments. (8%) was added into 250 ml Erlenmeyer flasks with 100 ml YE
Yeast extract fermentation medium (YE) contained 150 g/l medium to neutralize the lactic acid. Each strain was grown in
glucose and 20 g/l yeast extract. Regeneration medium (RM) three separate shake flasks, and the mean values were reported.
was MRS agar medium without Tween80 and supplemented Batch fermentations of different substrate (glucose) concentra-
with 0.5% bovine serum albumin (BSA), 25 mM MgCl2 , 25 mM tions were carried out in a 16.0 l bioreactor (Bioengineering,
CaC12 , 2.5% gelatin and 0.5 M sucrose. Fermentation medium Sweden) with a working volume of 10.0 l. A 6% (v/v) seed cul-
in bioreactor consisted of 150 g/l glucose, 46 ml/l molasses, ture was inoculated in the fermentation medium. The agitation
53 g/l corn steep liquor, 2 ml/l Tween80 and 0.4 g/l MnSO4 . speed and culture temperature was controlled at 200 rpm and
156 L. Yu et al. / Journal of Biotechnology 134 (2008) 154–159

37 ◦ C. The pH was maintained at 6.5 by automatic addition of

ammonia solution (25%).

2.6. Analysis for lactic acid, glucose and cell dry weight

l-lactic acid and glucose concentrations were measured by

a SBA-40C biosensor analyzer (Institute of Biology, Shandong
Province Academy of Sciences, PR China). The total biomass
concentration was determined by Cary 50 spectrophotometer
(Australian) at 600 nm (OD600) which was converted to biomass
dry weight after cell washing and lyophilization. Each sample
was analyzed three times.

3. Results

3.1. Strain mutagenesis and mutant screening

Fig. 1. Comparison of wild-type and mutant strains for lactic acid production in
Genome shuffling practically mimics the features of natural
YE medium in shake flask. Lr-WT: wild-type of L. rhamnous; UV: UV mutant
evolution through the recursive genetic recombination. Thus, it strains; NTG: NTG mutant stains; F1: strains from the first round of genome
requires a diverse population of mutants with an improvement shuffling; F2: strains from the second round of genome shuffling. Dotted line
of the desired phenotype compared with the wild-type as the represents the production level of Lr-WT.
starting point. NTG mutagenesis was introduced to generate the
first population of glucose-tolerant variants of Lr-WT. The cells in shake-flask. Consequently, these eight mutants were used as
of the wild-type were found to be very sensitive to NTG treat- the starting population for genome shuffling.
ment for the clear inhibition zone. The treated cells appeared to
have different morphologies compared to the Lr-WT. Colonies 3.2. Genome shuffling to generate the glucose-tolerant
were picked from plates with a spread of 40–50 colonies. The strains
diversity of the NTG treated cells was ensured when the cells
were scraped at different places from the lawn around inhibi- Genome shuffling is dependent upon the recursive fusion of
tion zone. About 43 colonies showing bigger transparent haloes protoplasts to allow recombination. This recursive strategy per-
were further tested for the lactic acid production in shake flasks. mits the obtaining the phenotype of interest quickly. The high
UV irradiation was used as the mutagenizing agent to develop frequency of protoplast formation and regeneration is the basis
the second population of mutants of Lr-WT. The cells spread on of the efficiency of genome shuffling. Increasing the concentra-
plates were exposed to UV irradiation at 254 nm for 30 s where tion of mutanolysin in the LPB to 30 ␮g/ml and lysozyme to
an approximately 90% killing was observed. The irradiated cells 10 mg/ml was necessary to improve the frequency of protoplast
characteristic of making bigger zone of lactic acid production formation as judged by osmotic fragility. The frequencies of
were selected. There are 21 colonies conforming to this stan- protoplast formation, regeneration and fusion are presented in
dard. Each of these two populations of mutants were believed Table 1. The frequency of protoplast regeneration was low and
to preparatorily possess the capability of resistance to high glu- the attempt to replace sucrose in the RM with 0.3 M raffinose was
cose, because the glucose concentration of YE agar plate was not feasible for improvement of protoplast regeneration (data not
7.5 time higher than that of the regular MRS medium. During shown).
the secondary screening in shake flask evaluations, five NTG The protoplasts of NTG and UV mutants were subjected
mutants and three UV mutants were obtained from NTG and to the first round of pool-wise recursive protoplast fusion.
UV populations. These mutants showed a small increase, from The resulting populations were screened for individuals with
6.1 to 11.2 g/l, in production of l-lactic acid in comparison with improved glucose tolerance using the selective plates. After the
the Lr-WT (Fig. 1). In addition, their glucose-tolerant stability first fusion, four colonies from the F1 with bigger haloes on YE
was maintained after at least 20 transfers in YE liquid medium plates containing 30% glucose and 2% CaCO3 were identified

Table 1
Frequencies of protoplast formation, regeneration and fusion in L. rhamnosus
Expt. CFU/ml of nonprotoplast CFU/ml of cells CFU/ml of cells CFU/ml of Protoplast formation Regeneration Fusion frequency
control (C) on RM (A)a on MRS (B)b fusants (D) frequency (A−B/A) frequency (A−B/C) (D/A)

1 1.2 × 109 2.3 × 108 3.2 × 106 7.8 × 102 9.9 × 10−1 1.9 × 10−1 3.4 × 10−6
2 7.1 × 108 9.7 × 107 4.6 × 106 2.6 × 102 9.5 × 10−1 1.3 × 10−1 2.7 × 10−6
a Colonies on RM plates from regenerated and nonprotoplasted cells after protoplast regeneration.
b Colonies on MRS plates after dilution of protoplasts with distilled water.
 L. Yu et al. / Journal of Biotechnology 134 (2008) 154–159 157

as producing more lactic acid (104.7 ± 3.3 g/l) than the mutated
parents (85.5 ± 1.7 g/l) in shake-flask (Fig. 1). These four strains
were then used for an additional round of shuffling.
Four colonies from the second shuffled library with big-
ger haloes on plates containing 40% glucose and 2% CaCO3
were identified as F2 after the shake-flask test. The F2 mutants
selected on the criterion of resistance to high glucose exhibited
67–77% improvement in lactic acid production (Fig. 1). There-
fore, the differences of lactic acid between the shuffled strains
and mutated strains are obvious. One of the best performing
shuffled strain from F2, F2-2, was selected for the scale-up fer-
mentation. A control experiment was carried out by plating the
selected populations of NTG and UV mutants and F1 without
exposure to PEG on the YE plates containing 30% and 40% glu-
cose. This was to determine whether acclimatization technique
could lead to adaptive growth on high glucose. In contrast to
the shuffled strains, no colonies were found on the correspond-
ing plates during the same cultivation period. Otherwise, the
protoplasts exposure to PEG, which promotes fusion, generated
recombinants on the plates.

3.3. Characterization of lactic acid production, cell growth

and glucose consumption of Lr-WT and F2-2 in bioreactor

The performance of F2-2 was compared with the Lr-WT

strain in the 16 l bioreactor. In an attempt to evaluate the effects
of scale-up fermentation on the genome shuffled strain for lactic
acid production with improved substrate concentration, dif-
ferent glucose concentration of mediums were used. At first,
150 g/l glucose was used. Consistent with shake flask results, the
genome shuffled strain produced about 71.4% improvement of
lactic acid over the parent strain at 150 g/l glucose concentration
after 40 h. Moreover, the F2-2 obtained the lactic acid produc-
tivity of 3.6 g/h l, which was 71.4% increase compared to the
Lr-WT. It also exhibited better growth and more rapid glucose
consumption under the same condition (Fig. 2). The growth char-
acterization was consistent with the lactic acid production. The
cell dry weight of F2-2 reached 11.3 g/l, whereas the Lr-WT was
7.8 g/l. The specific growth rate of F2-2 was 9.1% higher than
Lr-WT, when it was determined from the beginning to the end of
the batch fermentation. The cell mass yield on substrate of F2-2
was 0.075 g/g at 150 g/l glucose, a 1.5 fold improvement over
the Lr-WT. The rate of glucose depletion of genome-shuffled
strain was 62.2% higher than that of Lr-WT with 150 g/l initial
glucose. There was 60 g/l residual glucose left in the medium
when cultivated with Lr-WT, while only 4 g/l glucose left with
F2-2 at the end of the fermentation. The results showed the F2-
2 was not only more tolerant to high glucose in medium, but
also produced more lactic acid compared with Lr-WT. Fig. 2a
shows that the final lactic acid concentration of F2-2 increased
Fig. 2. Characterization of lactic acid production (a), cell growth (b) and glucose
2.2 times when the initial glucose concentration increased up
consumption (c) with different initial glucose by Lc-WT and F2-2 in 16 l bioreac-
to 200 g/l. Although there was no marked difference in lactic tor. Fermentation of Lc-WT with 150 g/l glucose (open squares); Fermentation
acid production between the experiments with initial glucose of F2-2 with 150 g/l glucose (closed squares); Fermentation of F2-2 with 160 g/l
concentration of 150 and 160 g/l, the lactic acid productivity of glucose (closed circles); Fermentation of F2-2 with 200 g/l glucose (closed tri-
the former process (3.60 g/h l) was higher than that of the latter angles); The samples of the experiments with initial glucose concentrations of
150 and 160 g/l were analyzed every 4 h; The samples of the experiment with
process (3.36 g/h l). The profiles of cell growth during lactic acid
initial glucose concentration of 200 g/l were analyzed every 6 h.
fermentations are shown in Fig. 2b.The genome shuffled strain
158 L. Yu et al. / Journal of Biotechnology 134 (2008) 154–159

had a shorter lag phase compared with the wild-type strain at the wild type strain was indiscernible during the same culti-
150 g/l glucose concentration. Dry cell weight decreased slightly vation time. This result suggests that the residual cells free
with the increase of initial glucose concentration up to 200 g/l. from protoplast formation and populations without shuffled
However, the specific growth rates of F2-2 at 150 g/l glucose can be eliminated on a high glucose medium. The recursive
concentration was 0.12 h−1 , twofold higher than that at 200 g/l protoplast-induced mutagenesis had been shown that it could
glucose concentration. As shown in Fig. 2c, although the glucose not achieve substantial improvements as genome shuffling in
consumption patterns of both 150 and 160 g/l glucose were sim- the protoplast populations (Patnaik et al., 2002). In our control
ilar, the latter was a little slower than the former. It also can be experiment, even the acclimatization technique failed to achieve
confirmed that F2-2 had lower cell mass yield on the substrate at the improved phenotypes as genome shuffling had done due to
160 g/l glucose concentration (0.067 g/g). When the initial glu- the sharp increase of glucose concentration in plates. One the
cose concentration increased to 200 g/l, the fermentation time other hand, recombination was the apparent outcome of the shuf-
of F2-2 increased to 90 h, about two times longer than those fled mutants with glucose tolerance because of the inactivated
with 150 and 160 g/l initial glucose. The better performance of parental protoplast fusion (Zhou et al., 1999). The mutants with
F2-2 at 150 g/l glucose may be as a result of the same glucose a high ratio of the diameter of transparent haloes to the diame-
concentration in shake-flask for secondary screening other than ter of colony on surface culture generally produced more lactic
gradual glucose increases in solid medium. Further experiments acid in the liquid medium. This result demonstrates that genome
are still needed to investigate whether the increase of glucose in shuffling can achieve the compatibility of the optimal genomic
shake-flask will lead to more improved mutants. These charac- profiles for two phenotypes, i.e. glucose tolerance and lactic acid
terizations indicated that F2 still had potential to tolerate more enhancement. Stephanopoulos (2002) once discussed this issue,
glucose if more rounds of genome shuffling and higher selective considering the practical application of the genome shuffling.
pressures were conducted. Characterization of mutant and wild type strains in the shake
flask showed the lactic acid production was closely correlated
4. Discussion with the glucose resistance (Fig. 1).
The advantage of the higher final lactic acid production and
In the present study, genome shuffling proved to be an effec- less concentration required for the product at higher initial glu-
tive strategy for generating the mutants tolerant to the high cose concentration was profitable for the industrial processes.
glucose concentration that was considered as one of the extreme Among the previously published results about lactic acid pro-
process conditions. For lactic acid fermentation, the selection duction through batch fermentation, few were able to satisfy the
of mutant strains with acid tolerance in Lactobacillus (Patnaik demand of giving the highest lactic acid concentration in such an
et al., 2002) and ammonia tolerance in Rhizopus (Miura et extreme process condition. Kadam et al. (2006) produced 135 g/l
al., 2004) have been well studied. As substrate inhibition is lactic acid from 150 g/l cane sugar with 90% yield by using L.
one of conventional traits for the batch fermentation of lactic delbrueckii. A maximum of 118.6 g/l lactic acid was obtained
acid, isolating a glucose tolerant mutant becomes of consid- with 160 g/l glucose, resulting in a 74% yield by L. casei
erable importance for industrial applications. Åkerberg et al. (Hujanen et al., 2001). The highest lactic acid concentration of
(1998) reported that substrate inhibition played great role in 184.6 g/l was obtained with an initial glucose concentration of
the glucose concentration ranging from 40 to 82 g/l for l-lactic 200 g/l by Enterococcus faecalis (Yun et al., 2003). Goncalves et
ATCC19435. Here, we successfully used genome shuffling to al. (1991) once studied the substrate inhibition kinetics in lactic
achieve increased substrate availability and l-lactic acid pro- acid production with glucose range of 50–340 g/l. The maximum
duction of the L. rhamnosus. In our study, the cell growth and of 140 g/l lactic acid was reached with 200 g/l initial glucose with
lactic acid concentration of Lr-WT decreased 33.6% and 41.7% 70% yield in 80 h. In the present work, genome shuffled F2-2 was
compared with the second round genome shuffled strain, F2-2, at further examined with high initial glucose concentration rang-
initial glucose concentration of 150 g/l. Thus the genome shuf- ing from 150 to 200 g/l in 16 l bioreactors. As shown in Fig. 2,
fled strain active in glucose-rich media opens the new avenue F2-2 produced 144, 148 and 184 g/l l-lactic acid when the initial
for the enhanced production of lactic acid. The technological glucose concentrations were 150, 160 and 200 g/l, respectively.
amelioration of industrial microorganisms leaves infinite room And the corresponding lactic acid yields were all more than 90%.
for genome shuffling, because the genome of organism con- Furthermore, it is noteworthy that F2-2 only produced optically
tains the potential to evolve novel functions that will allow it to pure l-lactic acid (99% enantiomeric purity) without other side
thrive in alternate environments (Hall, 1999). Unlike the rational products and avoided using the expensive yeast extract in fer-
methods for improvement of microbial strains, genome shuffling mentation medium mentioned in the above cited studies. Yeast
causes simultaneous changes broadly distributed throughout the extract leads to the highest lactic acid concentrations in a vari-
genome based on genome plasticity, without the need to know ety of nitrogen sources (Nancib et al., 2001; Rivas et al., 2004).
the genome sequence data or network information (Petri and However, the high cost of yeast extract causes lactic acid fer-
Schmidt-Dannert, 2004). mentation to be economically unattractive because yeast extract
The high glucose YE plate with 2% CaCO3 screening method was estimated to account for about 38% of the total production
turned out to be effective for obtaining two phenotypes simulta- cost (Hujanen et al., 2001). Considering the good performance
neously. The strains utilized after the second round of genome of F2-2, the substrate inhibition in lactic acid fermentation was
shuffling can grow on plates containing 400 g/l glucose, while overcome to a great extent in the bioreactor.
 L. Yu et al. / Journal of Biotechnology 134 (2008) 154–159 159

In conclusion, genome shuffling successfully improved the Hujanen, M., Linko, S., Linko, Y.Y., Leisola, M., 2001. Optimisation of media
tolerance of L. rhamnosus towards glucose. When exposed and cultivation conditions for l(+)(S)-lactic acid production by Lactobacillus
to high concentrations of glucose, the second round genome casei NRRL B-441. Appl. Microbiol. Biotechnol. 56, 126–130.
Kadam, S.R., Patil, S.S., Bastawde, K.B., Khire, J.M., Gokhale, D.V., 2006.
shuffled F2-2 exhibited dramatically enhanced l-lactic acid Strain improvement of Lactobacillus delbrueckii NCIM2365 for lactic acid
production. The research here demonstrated that genome shuf- production. Proc. Biochem. 41, 120–126.
fling could greatly accelerate the improvement of important Miura, S., Dwiarti, L., Arimura, T., Hoshino, M., Tiejun, L., Okabe, M., 2004.
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circumvent the extreme process condition. Rhizopus sp. MK-96–1196. J. Biosci. Bioeng. 97, 19–23.
Nancib, N., Nacib, A., Boudjelal, A., Benslimane, C., Blanchard, F., Boudrant,
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Acknowledgements the production of lactic acid from date juice by Lactobacillus casei subsp.
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