|Overexpression of genes encoding glycolytic enzymes in Corynebacterium
glutamicum enhances glucose metabolism and alanine production under oxygen
Appl. Environ. Microbiol. 78: 4447-4457. 2012.
S. Yamamoto, W. Gunji, H. Suzuki, H. Toda, M. Suda, T. Jojima, M. Inui and H. Yukawa.
|We previously reported that Corynebacterium glutamicum strain DeltaldhADeltappc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159?165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved
rates of glucose consumption and alanine formation compared to GLY1/pCRD500.
Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably,
both alanine productivity and yield increased after each overexpression
step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine
at a yield of 91.8%. This was 6.4-fold higher productivity than that of
the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream
of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression
resolved a bottleneck in glycolysis. Changing ratios of the extracellular
metabolites by overexpression of glycolytic genes resulted in reduction
of the intracellular NADH/NAD+ ratio, which also plays an important role
on the improvement of glucose consumption. Enhanced alanine dehydrogenase
activity using a high-copy-number plasmid further accelerated the overall
alanine productivity. Increase in glycolytic enzyme activities is a promising
approach to make drastic progress in growth-arrested bioprocesses.