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Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid

Tsuge, Yota, Kato, Naoto, Yamamoto, Shogo, Suda, Masako, Jojima, Toru, Inui, Masayuki
Applied microbiology and biotechnology 2019 v.103 no.8 pp. 3381-3391
Corynebacterium glutamicum, NAD (coenzyme), NADH dehydrogenase, Zymomonas mobilis, alcohol oxidoreductases, antibiotic resistance genes, carbon, cost effectiveness, fructose-bisphosphate aldolase, gene overexpression, glucokinase, glucose, glyceraldehyde-3-phosphate dehydrogenase, glycolysis, heat tolerance, metabolic engineering, phosphofructokinases, phosphopyruvate hydratase, plasmids, polylactic acid, salts, triose-phosphate isomerase
Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner–Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.