Engineering rTCA pathway and C4-dicarboxylate transporter for l-malic acid production
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l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L−1, 0.30 g g−1, and 0.32 g L−1 h−1 in the resulting strain W4209 with CaCO3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L−1, 0.31 g g−1, and 0.32 g L−1 h−1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.
Keywordsl-Malic acid Saccharomyces cerevisiae Reductive TCA pathway C4-Dicarboxylate transporter Pathway optimization
This work was financially supported by the National Natural Science Foundation of China (21676118, 21422602), the Provincial Natural Science Foundation of Jiangsu Province (BK20160163), the Fundamental Research Funds for the Central Universities (JUSRP51611A), the Special Foundation for State Key Research and Development Program of China (2016YFD0400801), and the National Science Foundation for Post-doctoral Scientists of China (2016M600362).
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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