Abstract
β-Alanine is a naturally occurring β-amino acid that has been widely applied in the life and health field. Although microbial fermentation is a promising method for industrial production of β-alanine, an efficient microbial cell factory is still lacking. In this study, a new metabolically engineered Escherichia coli strain for β-alanine production was developed through a series of introduction, deletion, and overexpression of genes involved in its biosynthesis pathway. First, the L-aspartate a-decarboxylase gene, BtADC, from Bacillus tequilensis, with higher catalytic activity to produce β-alanine from aspartate, was constitutively expressed in E. coli, leading to an increased production of β-alanine up to 2.76 g/L. Second, three native aspartate kinase genes, akI, akII, and akIII, were knocked out to promote the production of β-alanine to a higher concentration of 4.43 g/L by preventing from bypass loss of aspartate. To increase the amount of aspartate, the native AspC gene was replaced with PaeAspDH, a L-aspartate dehydrogenase gene from Pseudomonas aeruginosa, accompanied with the overexpression of the native AspA gene, to further improve the production level of β-alanine to 9.27 g/L. Last, increased biosynthesis of oxaloacetic acid (OAA) was achieved by a combination of overexpression of the native PPC, introduction of CgPC, a pyruvate decarboxylase from Corynebacterium glutamicum, and deletion of ldhA, pflB, pta, and adhE in E. coli, to further enhance the production of β-alanine. Finally, the engineered E. coli strain produced 43.12 g/L β-alanine in fed-batch fermentation. Our study will lay a solid foundation for the promising application of β-alanine in the life and health field.
Key Points
• Overexpression of BtADC resulted in substantial accumulation of β-alanine.
• The native AspC was replaced with PaeAspDH to catalyze the transamination of OAA.
• Deletion of gluDH prevented from losing carbon flux in TCA recycle.
• A 43.12-g/L β-alanine production in fed-batch fermentation was achieved.
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Author contribution statement
JZ and ZF conceived and designed research. XZ, LG, LH, ML, and SZ conducted experiments. AY, YZ, and LZ analyzed data. JZ, ZF, and XZ wrote the manuscript. HZ polished the manuscript. All authors read and approved the manuscript.
Funding
This work was funded by the National Mega Project of GMO Crops of China (2016ZX08004-002-006) and the Major Scientific and Technological Innovation Project of Shandong Province (2019GSF107094). This work was also funded by the National Natural Science Foundation of China (31901987) and the Project of Shandong Province Higher Educational Science and Technology Program (J14LE07).
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Zou, X., Guo, L., Huang, L. et al. Pathway construction and metabolic engineering for fermentative production of β-alanine in Escherichia coli. Appl Microbiol Biotechnol 104, 2545–2559 (2020). https://doi.org/10.1007/s00253-020-10359-8
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DOI: https://doi.org/10.1007/s00253-020-10359-8