N-acetylglucosamine (GlcNAc) is widely used in the fields of food, nutrition and drugs. In previous studies, our team constructed a chassis cell of Bacillus subtilis with high synthesis efficiency of GlcNAc through metabolic regulation. However, the excessive accumulation of GlcNAc precursor caused phosphosugar stress, which limited the yield of GlcNAc. In this study, the effect of phosphate stress on the transcription level of glcR-ywpJ operon was verified by qRT-PCR; the binding ability between GlcR and PglcR was determined by EMSA. Then, a PglcR-ep mutant library was constructed by error prone PCR, and a high-throughput screening system based on fluorescence detection was designed to identify the key regions of PglcR binding to GlcR. Finally, on the basis of FMIK strain, the key binding regions between PglcR and GlcR were knocked out, and the engineering strain FMIK-m4 was obtained. This strain effectively reduced the concentration of GlcNAc precursor, relieved the secondary growth phenomenon caused by phosphate sugar stress, and increased the yield of GlcNAc in the shake flask to 18.21 g/L.
FANG Jun
,
HUANG Ziyang
,
LIU Yanfeng
,
LI Jianghua
,
DU Guocheng
,
LYU Xueqin
,
LIU Long
. Improving N-acetylglucosamine synthesis ability of Bacillus subtilis by relieving phosphosugar stress[J]. Food and Fermentation Industries, 2023
, 49(6)
: 27
-34
.
DOI: 10.13995/j.cnki.11-1802/ts.032274
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