Methylglyoxal Bypass Identified as Source of Chiral Contamination in l(+) and d(−)-lactate Fermentations by Recombinant Escherichia coli

Abstract

Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both L: (+)- and D: (-)-lactate. Strain TG113 contains only native genes and produced optically pure D: (-)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only L: (+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield.

Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

“…1B). The bacterial population was maintained at the exponential or early stationary phase in a fermentation vessel by serially transferring cultures into new media as previously described (20,21). In all three evolutionary trajectories, a rapid adaptation occurred that simultaneously increased xylose catabolism, lactate titer, and cell growth (Fig.…”

“…To further demonstrate the application of our discovery in converting sugar mixtures into renewable chemicals, we substituted wildtype xylR with xylR (R121C and P363S) of a previously engineered D-lactate E. coli producer TG114, which efficiently converts glucose into D-lactate (21). Fermenting TG114 in a glucose-xylose mixture, 50 g·L −1 of each sugar, showed that only 34% of the xylose was used due to CCR (Fig.…”

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

“…This is more critical as we attempt to replace petroleum-based plastics with renewable PLA-based plastics, which would require production of the base chemical in large quantities and lignocellulosic biomass as the feedstock for fermentation. Biocatalysts currently being developed to reduce the cost of production of optically pure isomers of lactic acid include Kluyveromyces (Bianchi et al, 2001;Porro et al, 1999), Saccharomyces (van Maris et al, 2004;Saitoh et al, 2005), Pichia (Ilmen et al, 2007), Rhizopus (Skory, 2000) and Lactobacillus (Liu et al, 2007); however, Escherichia coli stands out as an excellent microbial biocatalyst for this purpose (Dien et al, 2001;Grabar et al, 2006). E. coli is a well studied bacterium that uses glycolysis via the Embden-Meyerhoff-Parnas (EMP) pathway to convert hexose sugars into a mixture of acids (lactic, acetic, formic and succinic) and ethanol (Clark, 1989).…”

“…Zhou et al (2003b) further engineered E. coli by deleting the native d-ldh gene and replacing it with the gene encoding L-(+)-LDH from Pediococcus to produce optically pure L-(+)-lactic acid. Further metabolic engineering and directed evolution led to E. coli strains, TG114 and TG108, that produced optically pure (99.9%) D-()) and L-(+)-lactate, respectively, from 12% w/v glucose at greater than 95% of the theoretical yield (Grabar et al, 2006). The very high lactic acid production titre and yield achieved by engineered E. coli strains TG114 and TG108 in simple salts medium is equal to or higher than the best values reported for lactic acid bacteria in complex medium (Table 2).…”

“…Apparently, these E. coli derivatives have overcome some of the growthand fermentation-inhibitory properties of high concentrations of lactic acid (Pieterse et al, 2005). The engineered lactic acid-producing E. coli demonstrate the power of directed evolution of the fermenting organism coupled with metabolic pathway engineering for production of desired product (Grabar et al, 2006).…”