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Bioresour Technol. 2017 Dec;245(Pt A):1093-1102. doi: 10.1016/j.biortech.2017.09.063. Epub 2017 Sep 9.

Combinatorial promoter engineering of glucokinase and phosphoglucoisomerase for improved N-acetylglucosamine production in Bacillus subtilis.

Author information

1
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
2
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta 30332, USA.
3
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
4
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: gcdu@jiangnan.edu.cn.

Abstract

In previous work, a recombinant Bacillus subtilis strain was successfully constructed for microbial production of N-acetylglucosamine (GlcNAc). In this study, GlcNAc titer was further improved by combinatorial promoter engineering of key genes glck encoding glucokinase and pgi encoding phosphoglucoisomerase. First, three promoters including constitutive promoter P43, xylose inducible promoter PxylA, and isopropyl-β-d-thiogalactoside inducible Pgrac were used to replace the native promoters of glcK and pgi, yielding 12 recombinant strains. It was found that when glcK and pgi were both under the control of promoter PxylA, the highest GlcNAc titer in 3-L fed-batch bioreactor reached 35.12g/L, which was 52.6% higher than that of the initial host. Next, the transcriptional levels of the related genes in glycolysis, GlcNAc synthesis pathway, peptidoglycan synthesis pathway, and pentose phosphate pathway were investigated by quantitative real-time PCR analysis. Fine-tuning upper GlcNAc synthesis pathway by combinatorial promoter substitution significantly enhanced GlcNAc production in engineered B. subtilis.

KEYWORDS:

Bacillus subtilis; Metabolic engineering; N-acetylglucosamine; Promoter engineering

PMID:
28946392
DOI:
10.1016/j.biortech.2017.09.063
[Indexed for MEDLINE]

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