Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B
© Zhao et al.; licensee BioMed Central Ltd. 2013
Received: 17 January 2013
Accepted: 21 May 2013
Published: 7 June 2013
Polylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing “white pollution”. However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass.
In this study, an ethanologenic Escherichia coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA), was reengineered for homofermentative production of L-lactic acid from xylose (1.2 mole xylose = > 2 mole L-lactic acid), by deleting the alcohol dehydrogenase gene (adhE) and integrating the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici. The resulting strain, WL203, was metabolically evolved further through serial transfers in screw-cap tubes containing xylose, resulting in the strain WL204 with improved anaerobic cell growth. When tested in 70 g L-1 xylose fermentation (complex medium), WL204 produced 62 g L-1 L-lactic acid, with a maximum production rate of 1.631 g L-1 h-1 and a yield of 97% based on xylose metabolized. HPLC analysis using a chiral column showed that an L-lactic acid optical purity of 99.5% was achieved by WL204.
These results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates, which contain a significant amount of xylose.
Lactic acid, a widely used chemical, exists as a mixture of D and L isomers when synthesized through a chemical route . The requirement of an optically pure L isomer for applications in pharmaceutical and poly-lactic acid (PLA) bioplastic industries favors fermentative production of L-lactic acid using chiral-specific L-lactate dehydrogenase.
Glucose derived from starch biomass such as corn, is currently used for fermentative production of L-lactic acid by lactic acid bacteria like Lactobacillus. However, due to its competition with food resources, an alternative non-food substrate is needed for cost-effective production of L-lactic acid, in order to enable the environmentally friendly PLA to compete economically with petroleum based plastics .
Cellulosic biomass, the most abundant non-food resource, is a potential substrate for L-lactic acid fermentation. However, in addition to glucose, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria . The microorganisms that do ferment xylose to L-lactic acid, such as Lactococcus lactis IO-1  and Enterococcus mundtii, need improvements in yield, productivity, optical purity, and/or the requirement of complex nutrients.
Escherichia coli, a candidate with minimal nutrient requirements, is able to use all biomass derived hexose and pentose sugars. Derivative E. coli strains have been engineered for production of lactic acid [5–12]. However, few of these E. coli strains have demonstrated the ability to ferment xylose into L-lactic acid at high yields and/or optical purity. Furthermore, E. coli naturally produces D-lactic acid and lacks an endogenous L-lactate dehydrogenase gene. A plasmid bearing an exogenous L-lactate dehydrogenase gene from Streptococcus bovis[6, 13], Lactobacillus casei, or Clostridium thermocellum has been cloned into E. coli (pfl ldhA) to produce L-lactic acid. These plasmid bearing recombinants, however, may lack strain stability due to plasmid curing.
In this study, we report reengineering an ethanologenic E. coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) , for homofermentative production of L-lactic acid from xylose. The resulting strain, WL204, contains a chromosomal integrated ldhL gene, without any antibiotic marker or plasmids.
Results and discussion
Eliminate ethanol pathway
E. coli SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR::pflBp6-acEF-lpd ΔmgsA), a xylose fermenting homoethanol strain previously engineered from E. coli B, was selected for reengineering to produce L-lactic acid using xylose. The ethanol pathway of SZ470 was eliminated through deletion of the endogenous alcohol dehydrogenase gene (adhE) using the adhE’-FRT-kan-FRT-adhE’ DNA fragment and the λ red recombinase system [10, 16]. The antibiotic marker (kan) of the resulting kanamycin resistant colonies was then removed from the chromosome through FRT-recognizing site specific recombinase (flipase), producing the strain WL202 (∆adhE::FRT) which lacks the ethanol pathway and antibiotic marker.
Establish lactate pathway
To regain anaerobic growth and the ability to produce L-lactic acid by WL202, a L-(+)-lactate dehydrogenase enzyme was needed to convert pyruvate to L-lactate and oxidize NADH, and establish a homolactate pathway with a balanced NADH/NAD redox (1 glucose or 1.2 xylose + 2 NAD (glycolysis) = > 2 pyruvate + 2 NADH (L-lactate dehydrogenase) = > 2 L-lactate + 2 NAD) (Figure 1). To this end, the L-(+)-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici was amplified by PCR using E. coli SZ85 chromosomal DNA as the template (a strain containing integrated ldhL) . The amplified DNA fragment contained the ldhL coding region flanked by the promoter and terminator of the native E. coli ldhA gene. This hybrid DNA fragment (ldhA promoter-ldhL -ldhA terminator) was then transformed into WL202 (pKD46) through electroporation. The double homologous recombination with ldhL integrated at the ldhA locus was selected through anaerobic cell growth in screw-cap tubes. The temperature sensitive plasmid, pKD46, was then cured by incubation at 42°C. The resulting strain was designated WL203 (∆ldhA::ldhL).
WL203 produced L-lactic acid from xylose in screw cap tubes and in small scale fermentation with limited anaerobic growth (OD600nm of 0.5-1.0 after 24 h). A growth based metabolic evolution process was then carried out by growing and serial transferring (at 24 h intervals) WL203 in a LB-xylose medium for three months, resulting in strain WL204 with a one-fold improvement in anaerobic cell growth in screw cap tubes.
L-lactic acid production from xylose
Summary of xylose fermentations by E. coli WL204
Xylose (g L-1)
Volumetric productivity (g L-1h-1)
Specific productivity (g g-1h-1)
By-product (Acetic acid) (g L-1)
Mass (g L-1)
Titer (g L-1)
1.619 ± 0.112
0.271 ± 0.032
62.04 ± 0.92
1.631 ± 0.039
0.780 ± 0.017
1.05 ± 0.032
0.623 ± 0.097
1.01 ± 0.383
1.901 ± 0.110
0.291 ± 0.030
66.03 ± 1.51
1.934 ± 0.011
1.092 ± 0.010
1.02 ± 0.054
0.653 ± 0.052
0.992 ± 0.087
2.487 ± 0.150
0.205 ± 0.020
42.9 ± 0.80
0.768 ± 0.021
0.358 ± 0.012
0.309 ± 0.018
0.144 ± 0.010
0.596 ± 0.026
100 g L-1 xylose fermentation was also carried out to evaluate if osmotic pressure (from xylose) would be a challenge for cell growth and lactic acid production by WL204. As the results show in Figure 2B and Table 1, the patterns of cell growth, xylose consumption and lactic acid production were similar to those of the 70 g L-1 xylose fermentation. The cell growth rate (0.291 h-1), biomass (1.901 g L-1), and the maximum (1.934 g L-1h-1) and average (1.092 g L-1h-1) volumetric productivities of 100 g L-1 xylose fermentation were 7.4%, 17.4%, 18.6%, and 40% higher, respectively, than those of the 70 g L-1 xylose fermentation, demonstrating that osmotic pressure from 100 g L-1 xylose presented little challenge for WL204. However, the 66 g L-1 lactic acid titer achieved was almost the same as that achieved (62 g L-1) in the 70 g L-1 xylose fermentation, indicating that 62-66 g L-1 lactate inhibited WL204 from further growth and fermentation.
WL 204 was further evaluated for its ability for L(+)-lactic acid production in mineral salts medium using 70 g L-1 xylose NBS fermentation. As demonstrated in Figure 2C and Table 1, the engineered WL204 maintained its ability to grow and produce lactic acid with over 90% product yield in mineral salts medium. However, cell growth rate, product titer, and productivity achieved in mineral salts medium (NBS) were 32%, 44%, and 117% lower, respectively, than those obtained in complex LB medium under the same condition, suggestion that WL204 needs further adaptive evolution to improve its growth rate and lactic acid production in NBS medium.
Optical purity of L-lactic acid
Only the L-lactic acid isomer was detected in the fermentation product when analyzed by HPLC using a chiral column. However, a D-isomer peak was observed if it was intentionally added into the fermentation product at a ratio of 0.5% of the total lactic acid. These results indicated that the optical purity of L-lactic acid produced was at least 99.5%.
The current biocatalysts used for commercial L-lactic acid production are either unable to metabolize xylose or they metabolize xylose through phosphoketolase, which leads to a heterofermentative pathway of equal molar lactic acid and acetic acid . Attempting to engineer homolactic acid production from xylose has met limited success in lactic acid bacteria [18, 19] and in E. coli strains [6, 14, 17].
Comparison of E. coli strains engineered for L-lactic acid production using xylose
Initial xylose (g L-1)
Titer (g L-1)
Productivity (g L-1h-1)
Optical purity (%)
E. coli FBR9
E. coli FBR11
E. coli FBR19
E. coli SZ85
E. coli WL204
Strains, plasmids and growth conditions
E. coli strains, plasmids and primers used in this study
E. coli B, ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA
E. coli W3110, △focA-pflB △frdBC △adhE △ackA △ldhA::ldhL
SZ470, △adhE, lost anaerobic growth
WL202, △ldhA::ldhL, regained anaerobic growth
WL203, metabolically evolved in xylose with improved anaerobic growth
bla, red recombinase, temperature-dependent replication
bla, flp, temperature-dependent replication
Verify insertion ldhL-P1
Verify insertion ldhL-P2
Standard methods were used for DNA transformation, electroporation, PCR amplification, and analyses of DNA fragments. Chromosomal gene deletion and integration was carried out using previously described λ red homologous recombination procedures [10, 17]. The gene deletions and integrations were verified by using appropriate antibiotic markers and analysis of PCR and fermentation products.
Seed cultures were prepared by inoculating fresh colonies from LB-xylose plates into 500 ml flasks containing 200 ml LB medium with 2% (w/v) xylose, and incubated for 10 h (37°C, 150 rpm) until they achieved an OD600nm of ~1.9 (0.665 g L-1 cell dry weight). Seed cultures were inoculated (with a starting OD600nm of 0.1) into a 7-L fermenter (Sartorius Stedim Biotech GmbH 37070, Germany) containing 4-L LB medium with 70 g L-1 xylose. The fermentation was carried out for 96 h (37°C, 200 rpm, and pH 7). The pH was controlled by automatic addition of 6 N KOH. Samples (1.5 ml) were taken periodically for analysis of cell growth, sugar consumption and lactic acid production. Data presented were the averages of three replicated fermentations.
Cell growth was estimated from the optical density (1-L cells with an OD600nm of 1 is equivalent to 0.35 g dry cell weight). Fermentation samples were centrifuged at 8,000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm membrane, and used for HPLC analysis of sugar and organic acids concentrations (BioRad HPX 87H column, 35°C, 0.5 ml min-1 of 4 mM H2SO4 as the mobile phase). Optical purity was determined by HPLC using a chiral column (EC 250/4 NUCLEOSIL Chiral-1, Germany) (35°C, 0.5 ml min-1 of 0.2 mM CuSO4 as the mobile phase) and D (-) and L (+)-lactic acids (Sigma-Aldrich) as the standards.
This work was supported by a grant from the China National Natural Science Foundation (NSFC31070094), the Hubei Provincial Science Foundation (2011CDA008), the Chutian Scholar Program, Hubei University of Technology, P. R. China, and Northern Illinois University, USA.
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