A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (DeltafrdBC DeltaldhA DeltaackA DeltafocA-pflB DeltapdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.
Homofermentative production of reduced products requires additional reducing power output (NADH) from glucose catabolism. Anaerobic expression of the pyruvate dehydrogenase complex (PDH, encoded by aceEF-lpd, a normal aerobic operon) is able to provide the additional NADH required for production of reduced products in Escherichia coli fermentation. The multiple promoters (pflBp(1-7)) of pyruvate formate lyase (pflB) were evaluated for anaerobic expression of the aceEF-lpd operon. Four chromosomal constructs, pflBp(1-7)-aceEF-lpd, pflBp(1-6)-aceEF-lpd, pflBp(6,7)-aceEF-lpd, and pflBp6-aceEF-lpd efficiently expressed the PDH complex in anaerobically grown cells. Doubling the reducing power output was achieved when glucose was oxidized to acetyl-CoA through glycolysis and pyruvate oxidation by the anaerobically expressed PDH complex (glucose -->2 acetyl-CoA + 4 NADH). This additional reducing power output can be used for production of reduced products in anaerobic E. coli fermentation.
BackgroundA 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.ResultsA 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 IIBCglc (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−1h−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.ConclusionsThese 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.
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