Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B

Abstract

BackgroundPolylactic 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.ResultsIn 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.ConclusionsThese 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.

Metabolic engineering as a tool for enhanced lactic acid production

“…plantarum [55] At pH 3.8: 2.6-fold increase in cell growth; Titer -3.1-fold increase, Productivity -5.77AE0.05 g/l/h Lb. rhamnosus [31] Carbon source Xylose to produce L(+)-lactic acid Purity -99.5%, Titer -62.0 g/l, Yield -97.0%, Productivity -1.631 g/l/h (Xylose fermentation) E. coli [49] Sucrose to produce D(S)-lactic acid Titer -110 g/l, Yield -73.0%…”

“…casei [48] Carbon source Xylose to produce L(+)-lactic acid Purity -99.5%, Titer -62.0 g/l Yield -97.0%, Productivity -1.631 g/l/h (Xylose fermentation) E. coli [49] Glycerol to produce D(S)-lactic acid Purity -99.97%, Titer -100.3 g/l Yield -78.0%, Productivity -2.78 g/l/h (Glycerol fermentation) E. coli [17] Glycerol to produce L(+) lactic acid Purity -99.9%, Titer -50 g/l Yield -93.0%, Productivity -1.3 g/l/h (Glycerol fermentation) E. coli [16] Xylose to produce L(+)-lactic acid Purity -99.6%, Titer -50.1 g/l, Yield -94.6% (Xylose fermentation) L. lactis [39] Corn starch to produce D(S)-lactic acid Purity -99.6%, Titer -73.2 g/l Yield -0.85 g per g of consumed sugar Productivity -3.86 g/l/h (Starch fermentation)…”

Microbial production of lactic acid: the latest development

“…Multiple rounds of sub-cultivation evolved strain WL204 with improved anaerobic cell growth. WL204 strain produced 62 g/l L-LA (99.5% optical purity) from 70 g/l xylose at 1.63 g/l/h with a yield of 97% based on metabolized xylose (Zhao et al, 2013).…”

Enhancement of D-lactic acid production from a mixed glucose and xylose substrate by the Escherichia coli strain JH15 devoid of the glucose effect

“…Nevertheless, most studies reported the D-lactic acid production from glucose and/or sucrose by an engineered E. coli strain, with a titer, productivity and yield of 80–120 g L −1 , 2–6 g L −1 h −1 and 80–95 %, respectively. Few of these strains, however, have the ability to ferment xylose into D-lactic acid with a desired titer, yield and rate [3, 23, 24, 30, 31]. Furthermore, none, if any, of these strains have demonstrated the ability to co-metabolize both glucose and xylose for enhanced D-lactic acid fermentation.…”

“…In this study, we report reengineering E. coli WL204 (Δ frdBC Δ ldhA Δ ackA Δ pflB Δ pdhR ::pflBp6-acEF-lpd Δ mgsA Δ adhE , Δ ldhA :: ldhL ) [30] for D-lactic acid production by 1) replacing the L-lactate dehydrogenase gene ( ldhL ) with a D-lactate dehydrogenase gene ( ldhA ); 2) eliminating catabolite repression via deletion of the ptsG gene that encodes for IIBC glc , a major enzyme of the glucose PTS system; 3) adaptive evolution in screw-cap tubes for improved cell growth with glucose as the sole substrate. The resulting strain, E. coli JH15, is able to co-utilize both glucose and xylose for enhanced D-lactic acid production.…”