Abstract
Succinic acid, as one of the important fermentation products, is commonly used as an acidulant, flavoring agent, and antimicrobial agent. However, the accumulation of organic acids in the later fermentation phase will restrict the production efficiency of strain. To address this issue, the directed evolution on the key transcription morphology factor bolA gene was performed to enhance acid resistance of Escherichia coli by ep-PCR library and CRISPR/Cas9. The best mutant strain 5-21-5 was screened from 4000 mutant colonies ep-PCR library, which showed higher cell survives in succinic acid stress. Its acid resistance ability was systematically illustrated from multiple aspects by transmission electron microscope (TEM), confocal laser scanning microscopy (CLSM), and three-dimensional excitation emission matrix (3D-EEM) analysis, respectively. Higher tyrosine and tryptophan protein level was beneficial to acid resistance, while cell tend to short rod shape. Subsequently, with the assistance of the CRISPR/Cas9 gene editing technology knocking the mutated sequences into the genome, the succinic acid yield of strain M-K10 reached 9.8 g/L, representing a 15.29% increase compared to the Escherichia coli MG1655 without gene editing.
Published Version
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