Metabolic engineering of Escherichia coli to produce D-Lactate from sugar mixtures contained in lignocellulosic hydrolysates

Escherichia coli MG1655 was metabolic engineered to produce D-Lactate from hexoses, pentoses and sugar mixtures contained in lignocellulosic hydrolysates. Two genetic backgrounds were evaluated, strain JU15 (MG1655, ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, gatC S184L, ΔmidarpA, Δreg 27.3 kb) and the derivative strain AV01 (JU15, ΔpoxB, ΔmgsA, ΔackA-pta). The strain JU15 was designed and evolved to produce D-Lactate from xylose: through the elimination of the xylose ABC transporter and metabolic pathways competing by pyruvate, and the use of an alternative xylose symporter obtained from a mutated version of the galactitol transporter (GatC S184L). The strain JU15 was modified to avoid the formation of acetate and L-Lactate (by the methylglyoxal pathway). JU15 and AV01 produced D-Lactate with conversions yields above 90% and 95% of the maximum theoretical, respectively, using mineral media supplemented with glucose or xylose (40 g/L); however, the volumetric production rate was 40% lower when xylose was used as the only carbon source. JU15 was used to ferment sugars mixtures from sugar cane diluted acid hydrolysates, producing above 70 g/L of D-Lactate in 72 h. AV01 was used to ferment sugar mixtures from corn stover diluted acid hydrolysates obtaining similar rates as JU15, but avoiding the formation of acetate. Both strains efficiently metabolize pentose-hexose mixtures to D-Lactate in hydrolysates containing above 5 g/L of acetate.