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

Lu H, Zhao X, Wang Y, Ding X, Wang J, Garza E, Manow R, Iverson A, Zhou S - BMC Biotechnol. (2016)

Bottom Line: To achieve this goal, however, it is imperative to produce optically pure lactic acid isomers using a cost-effective substrate such as cellulosic biomass.This result represents a 46 % improved sugar consumption rate, a 26 % increased D-lactic acid titer, and a 48 % enhanced productivity, compared to that achieved by JH13.These results demonstrated that JH15 has the potential for fermentative production of D-lactic acid using cellulosic biomass derived substrates, which contain a mixture of C6 and C5 sugars.

View Article: PubMed Central - PubMed

Affiliation: Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Wuhan, 430068, P. R. China. 183459815@qq.com.

ABSTRACT

Background: A thermal tolerant stereo-complex poly-lactic acid (SC-PLA) can be made by mixing Poly-D-lactic acid (PDLA) and poly-L-lactic acid (PLLA) at a defined ratio. This environmentally friendly biodegradable polymer could replace traditional recalcitrant petroleum-based plastics. To achieve this goal, however, it is imperative to produce optically pure lactic acid isomers using a cost-effective substrate such as cellulosic biomass. The roadblock of this process is that: 1) xylose derived from cellulosic biomass is un-fermentable by most lactic acid bacteria; 2) the glucose effect results in delayed and incomplete xylose fermentation. An alternative strain devoid of the glucose effect is needed to co-utilize both glucose and xylose for improved D-lactic acid production using a cellulosic biomass substrate.

Results: A previously engineered L-lactic acid Escherichia coli strain, WL204 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA ΔadhE, ΔldhA::ldhL), was reengineered for production of D-lactic acid, by replacing the recombinant L-lactate dehydrogenase gene (ldhL) with a D-lactate dehydrogenase gene (ldhA). The glucose effect (catabolite repression) of the resulting strain, JH13, was eliminated by deletion of the ptsG gene which encodes for IIBC(glc) (a PTS enzyme for glucose transport). The derived strain, JH14, was metabolically evolved through serial transfers in screw-cap tubes containing glucose. The evolved strain, JH15, regained improved anaerobic cell growth using glucose. In fermentations using a mixture of glucose (50 g L(-1)) and xylose (50 g L(-1)), JH15 co-utilized both glucose and xylose, achieving an average sugar consumption rate of 1.04 g L(-1)h(-1), a D-lactic acid titer of 83 g L(-1), and a productivity of 0.86 g L(-1) h(-1). This result represents a 46 % improved sugar consumption rate, a 26 % increased D-lactic acid titer, and a 48 % enhanced productivity, compared to that achieved by JH13.

Conclusions: These results demonstrated that JH15 has the potential for fermentative production of D-lactic acid using cellulosic biomass derived substrates, which contain a mixture of C6 and C5 sugars.

No MeSH data available.


Related in: MedlinePlus

Engineering an E. coli strain devoid of the glucose effect for D-lactic acid production from a mixture of glucose and xylose. Genes encoding important enzymes are indicated by italics. The relevant genes/enzymes are: ldhL, L-lactate dehydrogenase; ldhA, D-lactate dehydrogenase; adhE, alcohol dehydrogenase; ackA, acetate kinase; pflB, pyruvate formate lyase; frdABCD, fumarate reductase; glk, glucokinase; xylA, xylose isomerase; xylB, xylulokinase; xylFGH, xylose ABC transporter; xylE, xylose/proton symporter; ptsG, subunit of glucose PTS permease; EI-Hpr-IIA, phosphoenolpyruvate-protein phosphotransferase system; AC-P, adenylate cyclase; ~P, high-energy phosphate from a phosphorylated compound; Crp, cAMP receptor protein; Crp-cAMP, transcriptional dual regulator; GalP, galactose transporter; Mgl, galactose transporter. Symbols: the stop and delta (∆) signs indicated the relevant genes (adhE, frdBC, ackA, pflB, ptsG, ldhL) were deleted
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Fig1: Engineering an E. coli strain devoid of the glucose effect for D-lactic acid production from a mixture of glucose and xylose. Genes encoding important enzymes are indicated by italics. The relevant genes/enzymes are: ldhL, L-lactate dehydrogenase; ldhA, D-lactate dehydrogenase; adhE, alcohol dehydrogenase; ackA, acetate kinase; pflB, pyruvate formate lyase; frdABCD, fumarate reductase; glk, glucokinase; xylA, xylose isomerase; xylB, xylulokinase; xylFGH, xylose ABC transporter; xylE, xylose/proton symporter; ptsG, subunit of glucose PTS permease; EI-Hpr-IIA, phosphoenolpyruvate-protein phosphotransferase system; AC-P, adenylate cyclase; ~P, high-energy phosphate from a phosphorylated compound; Crp, cAMP receptor protein; Crp-cAMP, transcriptional dual regulator; GalP, galactose transporter; Mgl, galactose transporter. Symbols: the stop and delta (∆) signs indicated the relevant genes (adhE, frdBC, ackA, pflB, ptsG, ldhL) were deleted

Mentions: E. coli WL204, a xylose fermenting homo-L-lactate producing strain previously engineered from E. coli B [30], was used as the starting strain (Fig. 1). Although a one-step gene replacement (ldhL (encodes for L-lactate dehydrogenase) replaced by ldhA (encodes for D-lactate dehydrogenase)) in WL204 would allow the resulting strain produce D-lactic acid, the selection process will be complicated because the correct replacement (of ldhL by ldhA) will have to rely on the fermentation and/or sequence results. To make a simple selection, a two-step strategy was used to allow plate selection. L-lactate production was first eliminated through deletion of the recombinant L-lactate dehydrogenase gene (ldhL) using an ldhA’-FRT-kan-FRT-ldhA’ DNA fragment. The ldhL gene was replaced by the kanamycin resistance marker (kan) through double homologous recombination facilitated by the λ red recombination system [5, 22, 30]. The resulting strain, JH12 (∆ldhL:: FRT-kan-FRT), lost L-lactic acid production as well as anaerobic cell growth due to the loss of L-lactate dehydrogenase, blocking NADH oxidation (Fig. 1). Nevertheless, it grows well aerobically on either glucose or xylose plates.Fig. 1


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

Lu H, Zhao X, Wang Y, Ding X, Wang J, Garza E, Manow R, Iverson A, Zhou S - BMC Biotechnol. (2016)

Engineering an E. coli strain devoid of the glucose effect for D-lactic acid production from a mixture of glucose and xylose. Genes encoding important enzymes are indicated by italics. The relevant genes/enzymes are: ldhL, L-lactate dehydrogenase; ldhA, D-lactate dehydrogenase; adhE, alcohol dehydrogenase; ackA, acetate kinase; pflB, pyruvate formate lyase; frdABCD, fumarate reductase; glk, glucokinase; xylA, xylose isomerase; xylB, xylulokinase; xylFGH, xylose ABC transporter; xylE, xylose/proton symporter; ptsG, subunit of glucose PTS permease; EI-Hpr-IIA, phosphoenolpyruvate-protein phosphotransferase system; AC-P, adenylate cyclase; ~P, high-energy phosphate from a phosphorylated compound; Crp, cAMP receptor protein; Crp-cAMP, transcriptional dual regulator; GalP, galactose transporter; Mgl, galactose transporter. Symbols: the stop and delta (∆) signs indicated the relevant genes (adhE, frdBC, ackA, pflB, ptsG, ldhL) were deleted
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4759849&req=5

Fig1: Engineering an E. coli strain devoid of the glucose effect for D-lactic acid production from a mixture of glucose and xylose. Genes encoding important enzymes are indicated by italics. The relevant genes/enzymes are: ldhL, L-lactate dehydrogenase; ldhA, D-lactate dehydrogenase; adhE, alcohol dehydrogenase; ackA, acetate kinase; pflB, pyruvate formate lyase; frdABCD, fumarate reductase; glk, glucokinase; xylA, xylose isomerase; xylB, xylulokinase; xylFGH, xylose ABC transporter; xylE, xylose/proton symporter; ptsG, subunit of glucose PTS permease; EI-Hpr-IIA, phosphoenolpyruvate-protein phosphotransferase system; AC-P, adenylate cyclase; ~P, high-energy phosphate from a phosphorylated compound; Crp, cAMP receptor protein; Crp-cAMP, transcriptional dual regulator; GalP, galactose transporter; Mgl, galactose transporter. Symbols: the stop and delta (∆) signs indicated the relevant genes (adhE, frdBC, ackA, pflB, ptsG, ldhL) were deleted
Mentions: E. coli WL204, a xylose fermenting homo-L-lactate producing strain previously engineered from E. coli B [30], was used as the starting strain (Fig. 1). Although a one-step gene replacement (ldhL (encodes for L-lactate dehydrogenase) replaced by ldhA (encodes for D-lactate dehydrogenase)) in WL204 would allow the resulting strain produce D-lactic acid, the selection process will be complicated because the correct replacement (of ldhL by ldhA) will have to rely on the fermentation and/or sequence results. To make a simple selection, a two-step strategy was used to allow plate selection. L-lactate production was first eliminated through deletion of the recombinant L-lactate dehydrogenase gene (ldhL) using an ldhA’-FRT-kan-FRT-ldhA’ DNA fragment. The ldhL gene was replaced by the kanamycin resistance marker (kan) through double homologous recombination facilitated by the λ red recombination system [5, 22, 30]. The resulting strain, JH12 (∆ldhL:: FRT-kan-FRT), lost L-lactic acid production as well as anaerobic cell growth due to the loss of L-lactate dehydrogenase, blocking NADH oxidation (Fig. 1). Nevertheless, it grows well aerobically on either glucose or xylose plates.Fig. 1

Bottom Line: To achieve this goal, however, it is imperative to produce optically pure lactic acid isomers using a cost-effective substrate such as cellulosic biomass.This result represents a 46 % improved sugar consumption rate, a 26 % increased D-lactic acid titer, and a 48 % enhanced productivity, compared to that achieved by JH13.These results demonstrated that JH15 has the potential for fermentative production of D-lactic acid using cellulosic biomass derived substrates, which contain a mixture of C6 and C5 sugars.

View Article: PubMed Central - PubMed

Affiliation: Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Wuhan, 430068, P. R. China. 183459815@qq.com.

ABSTRACT

Background: A thermal tolerant stereo-complex poly-lactic acid (SC-PLA) can be made by mixing Poly-D-lactic acid (PDLA) and poly-L-lactic acid (PLLA) at a defined ratio. This environmentally friendly biodegradable polymer could replace traditional recalcitrant petroleum-based plastics. To achieve this goal, however, it is imperative to produce optically pure lactic acid isomers using a cost-effective substrate such as cellulosic biomass. The roadblock of this process is that: 1) xylose derived from cellulosic biomass is un-fermentable by most lactic acid bacteria; 2) the glucose effect results in delayed and incomplete xylose fermentation. An alternative strain devoid of the glucose effect is needed to co-utilize both glucose and xylose for improved D-lactic acid production using a cellulosic biomass substrate.

Results: A previously engineered L-lactic acid Escherichia coli strain, WL204 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA ΔadhE, ΔldhA::ldhL), was reengineered for production of D-lactic acid, by replacing the recombinant L-lactate dehydrogenase gene (ldhL) with a D-lactate dehydrogenase gene (ldhA). The glucose effect (catabolite repression) of the resulting strain, JH13, was eliminated by deletion of the ptsG gene which encodes for IIBC(glc) (a PTS enzyme for glucose transport). The derived strain, JH14, was metabolically evolved through serial transfers in screw-cap tubes containing glucose. The evolved strain, JH15, regained improved anaerobic cell growth using glucose. In fermentations using a mixture of glucose (50 g L(-1)) and xylose (50 g L(-1)), JH15 co-utilized both glucose and xylose, achieving an average sugar consumption rate of 1.04 g L(-1)h(-1), a D-lactic acid titer of 83 g L(-1), and a productivity of 0.86 g L(-1) h(-1). This result represents a 46 % improved sugar consumption rate, a 26 % increased D-lactic acid titer, and a 48 % enhanced productivity, compared to that achieved by JH13.

Conclusions: These results demonstrated that JH15 has the potential for fermentative production of D-lactic acid using cellulosic biomass derived substrates, which contain a mixture of C6 and C5 sugars.

No MeSH data available.


Related in: MedlinePlus