Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade l- and d-lactic acid

• Published in: Applied microbiology and biotechnology Vol. 103; no. 8; pp. 3381 - 3391
• Main Authors: Tsuge, Yota, Kato, Naoto, Yamamoto, Shogo, Suda, Masako, Jojima, Toru, Inui, Masayuki
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.04.2019; Springer; Springer Nature B.V
• Subjects: 2-dehydro-3-deoxyphosphogluconate aldolase; Additives; Aldolase; Amino acids; Antibiotic resistance; Antibiotics; Biodegradable materials; Biomedical and Life Sciences; Biopolymers; Biotechnological Products and Process Engineering; Biotechnology; Chemical properties; Corynebacterium glutamicum; Dehydration; Dehydrogenase; Dehydrogenases; Enzymes; Genes; Glucokinase; Glucose; Glucose 6 phosphate dehydrogenase; Glucosephosphate dehydrogenase; Glyceraldehyde-3-phosphate dehydrogenase; Glycolysis; Gram-positive bacteria; Isoenzymes; Lactic acid; Life Sciences; Metabolic engineering; Metabolism; Methods; Microbial drug resistance; Microbial Genetics and Genomics; Microbiology; Microorganisms; Monosaccharides; NADH; NADH dehydrogenase; Nicotinamide adenine dinucleotide; Organic acids; Organic chemistry; Phosphates; Phosphofructokinase; Phosphogluconate dehydratase; Phosphopyruvate hydratase; Physiological aspects; Plasmids; Polylactic acid; Polymer industry; Polymers; Salts; Triose-phosphate isomerase; Zymomonas mobilis; Metabolic engineering; Lactic acid; Entner–Doudoroff pathway; Glycolytic enzymes; Corynebacterium glutamicum
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-019-09737-8

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.