Microbial fermentation was widely
explored to produce malic acid.
Previously, Aspergillus niger has been successfully
engineered, and a high titer of malic acid was achieved with strain
S575, but it also produced a high level of byproduct citric acid.
Here, the capability of A. niger in malic acid biosynthesis
was further improved by eliminating the accumulation of citric acid
and enhancing glycolytic flux. Characterization of variant mutants
suggested that disruption of cexA, a gene encoding
citric acid transporter located on cell membrane, abolished citric
acid accumulation. However, cexA-deficient strain
S895 showed significantly decreased malic acid production. Further
analysis of S895 indicated that the transcription level of genes involved
in glucose transportation and glycolytic pathway was significantly
reduced, and the corresponding enzyme activity was also lower than
those of S575. Individual overexpression of genes encoding glucose
transporter MstC and key enzymes (hexokinase HxkA, 6-phosphofructo-2-kinase
PfkA, and pyruvate kinase PkiA) involved in irreversible reactions
of glycolic pathway increased malic acid production. Accordingly,
genes of mstC, hxkA, pfkA, and pkiA were overexpressed altogether in S895,
and the resultant strain S1149 was constructed. The titer of malic
acid in fed-batch fermentation with S1149 reached 201.13 g/L. Compared
with S575, the byproduct of citric acid was completely abolished in
S1149, and the ratio of malic acid/glucose was increased from 1.27
to 1.64 mol/mol, the highest yield reported so far, and the fermentation
period was shortened from 9 to 8 days. Thus, a strain with great industrial
application potential was developed by engineering nine genes in A. niger, and a pilot fermentation technology was exploited.
Malic acid, a four-carbon dicarboxylic acid, is widely used in the food, chemical and medical industries. As an intermediate of the TCA cycle, malic acid is one of the most promising building block chemicals that can be produced from renewable sources. To date, chemical synthesis or enzymatic conversion of petrochemical feedstocks are still the dominant mode for malic acid production. However, with increasing concerns surrounding environmental issues in recent years, microbial fermentation for the production of L-malic acid was extensively explored as an eco-friendly production process. The rapid development of genetic engineering has resulted in some promising strains suitable for large-scale bio-based production of malic acid. This review offers a comprehensive overview of the most recent developments, including a spectrum of wild-type, mutant, laboratory-evolved and metabolically engineered microorganisms for malic acid production. The technological progress in the fermentative production of malic acid is presented. Metabolic engineering strategies for malic acid production in various microorganisms are particularly reviewed. Biosynthetic pathways, transport of malic acid, elimination of byproducts and enhancement of metabolic fluxes are discussed and compared as strategies for improving malic acid production, thus providing insights into the current state of malic acid production, as well as further research directions for more efficient and economical microbial malic acid production.
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