[Application of a metabolic-pathway-analysis based breeding strategy enhances the production of pyruvate by Torulopsis glabrata].
To further enhance the pyruvate productivity by multi-vitamin auxotrophic yeast Torulopsis glabrata, a breeding strategy aiming at decreasing the activity of pyruvate decarboxylase but increasing the activity of acetyl-CoA synthetase was developed based on analysis of pyruvate-related metabolic pathways. Nitrosoguanidine mutagenized cells of T. glabrata WSH-IP303 were screened for mutants that require acetate for complete growth on glucose minimum medium. A mutant, T. glabrata CCTCC M202019, produced pyruvate 21% higher than that of the parent strain and was genetically stable in flask cultures, was selected as a working strain. To elucidate the metabolic changes that led to the increase of pyruvate production, the activities of enzymes that involved in pyruvate-related metabolic pathways of the mutant and the parent strain were determined. Enzymatic analysis revealed that, compared with the parent strain WSH-IP303, the activity of pyruvate decarboxylase of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase of the latter increased by 103.5% or 57.4%, respectively, in the presence or absence of acetate. When 6 g/L sodium acetate was added to the medium, pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g/L at 62 h (yield on glucose, 0.651 g/g) in fermentations performed in a 7-L jar fermentor, indicating the shortage of cytosolic acetyl-CoA resulted from the disruption of pyruvate decarboxylase was properly compensated by the increase of the activity of acetyl-CoA synthetase.
Liu
Li-ming
LM
Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology Southern Yangtze University, Wuxi 214036, China. mingll@sytu.edu.cn
Li
Yin
Y
Chen
Jian
J
D000085:Acetates / Q000494:pharmacology; D041221:Candida glabrata / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003470:Culture Media / Q000494:pharmacology; D005285:Fermentation; D016296:Mutagenesis; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*
D000085:Acetates; D003470:Culture Media; D019289:Pyruvic Acid; D011767:Pyruvate Decarboxylase; D003066:Coenzyme A Ligases; C101958:acetate-CoA ligase (ADP-forming)