Abstract:

High-level production of malic acid by fermentation of corn cob hydrolysate with an isolated Rhizopus delemar
strain was expected. Then metabolic analysis and research of the underlying mechanism were carried out. Phylogenetic
analysis based on ITS region sequences was carried out for the identification of X-ray-induced mutant strains, and the key
enzyme activities and metabolic flux were also analyzed. The results showed 3 “TAA” codons in the alcohol dehydrogenase
gene of the anti-allyl mutant strain, which interrupt the pathway of ethanol metabolism. The breeding for the antifluoroacetate
mutant strain succeeded to decrease the flux of fumaric and succinic acid. Some NADP (H)-binding loci were
mutated in the G6PDH gene of the mutant, probably leading to increased interaction of Embden-Meyerhof-Parnas pathway
with Hexose-Monophophate pathway and improved pentose metabolism. After two-step breeding and regulation for this
isolated strain, the production of malic acid was enhanced markedly; the formation of ethanol, fumaric acid and succinic acid
were decreased simultaneously. Compared with the parent strain, the proportion of malic acid in mutant strain-2 increased to
91% from 71% and the yield of malic acid was increased markedly, which is of significance for deep industrial development.

Key words: malate, corn cob hydrolysate, metabolic flux analysis, glucose-6-phosphate dehydrogenase