Abstract
AbstractSuccinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Biophysics
Reference50 articles.
1. Ahn JH, Seo H, Park W, Seok J, Lee JA, Kim WJ, Kim JB, Kim KJ, Lee SY (2020) Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase. Nat Commun 11:1. https://doi.org/10.1038/s41467-020-15839-z
2. Banta AB, Ward RD, Tran JS, Bacon EE, Peters JM (2020) Programmable gene knockdown in diverse bacteria using mobile-CRISPRi. Curr Protol Microbiol 59(1):e130. https://doi.org/10.1002/cpmc.130
3. Chen XJ, Zhou YJ, Zhang D (2019) Engineering Corynebacterium crenatum for enhancing succinic acid production. J Food Biochem. https://doi.org/10.1111/jfbc.12645
4. Chen CM, Zhang Q, Qian JZ, Wu D, Chen PC, Zheng P (2022) Effect of the gad system on Actinobacillus succinogenes during acid stress. Syst Microbiol Biomanufact 2(1):9
5. Choi S, Song H, Lim SW, Kim TY, Ahn JH, Lee JW, Lee MH, Lee SY (2016) Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification. Biotechnol Bioeng. https://doi.org/10.1002/bit.25988
Cited by
3 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献