Metabolic engineering of Corynebacterium glutamicum for L-alanine production

Abstract

The production of L-alanine was enhanced in Corynebacterium glutamicum ATCC13869 through metabolic engineering of the biosynthesis pathways of L-alanine and fatty acids. Strains ΔfasB, ΔfasBR, ΔfasBΔpks13 and ΔfasBRΔpks13 were constructed and exhibited increased L-alanine yields up to 17.29 g/L. Different from ΔfasB mutant constructed from C. glutamicum ATCC13032 in which L-glutamate production accumulated, the muatnt ΔfasB constructed from C. glutamicum ATCC13869 in this study significantly produce L-alanine without L-glutamate accumulation. Transcriptional level analysis revealed that the knockout of fasB upregulated the expression levels of the genes related to L-alanine synthesis but downregulated those associated with fatty acid synthesis, confirming the redirection of metabolic flux from fatty acid synthesis to L-alanine synthesis in these strains. L-alanine productions were further enhanced in strains ΔfasB and ΔfasBR through the combinatorial expression of heterologous genes Bacillus subtilis alaD encoding alanine dehydrogenase and Escherichia coli alaE encoding alanine export protein, and the yields reached 55.21 g/L and 54.95 g/L, respectively. Finally, 69.9 g/L L-alanine was obtained in ΔfasB/pJYW-5-alaDE after 60 h of fermentation by supplementing glucose. Our data indicate that disrupting the fatty acid biosynthesis could redirect metabolic flux towards L-alanine biosynthesis. These results provide a new strategy for increasing the production of L-alanine in C. glutamicum.

Funding

National Key Research and Development Program of China,(2021YFC2100900)