Metabolic engineering of Bacillus amyloliquefaciens for efficient production of α-glucosidase inhibitor1-deoxynojirimycin

Xujie Li; Meng Zhang; Yu Lu; Ningyang Wu; Jian'gang Chen; Zhixia Ji; Yangyang Zhan; Xin Ma; Junyong Chen; Dongbo Cai; Shouwen Chen

doi:10.1016/j.synbio.2023.05.002

Metabolic engineering of Bacillus amyloliquefaciens for efficient production of α-glucosidase inhibitor1-deoxynojirimycin

Synth Syst Biotechnol. 2023 Jun 7;8(3):378-385. doi: 10.1016/j.synbio.2023.05.002. eCollection 2023 Sep.

Authors

Xujie Li¹, Meng Zhang¹, Yu Lu², Ningyang Wu¹, Jian'gang Chen³, Zhixia Ji², Yangyang Zhan¹, Xin Ma¹, Junyong Chen⁴, Dongbo Cai¹, Shouwen Chen¹

Affiliations

¹ State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, 430062, China.
² State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China.
³ Wuhan Jun'an Biotechnology Co., Ltd., Wuhan, 430070, China.
⁴ Department of Urology, Zhuhai People's Hospital (Zhuhai Hospital Affiliated to Jinan University), Zhuhai, 519000, China.

Abstract

Owing to the feature of strong α-glucosidase inhibitory activity, 1-deoxynojirimycin (1-DNJ) has broad application prospects in areas of functional food, biomedicine, etc., and this research wants to construct an efficient strain for 1-DNJ production, basing on Bacillus amyloliquefaciens HZ-12. Firstly, using the temperature-sensitive shuttle plasmid T2 (2)-Ori, gene ptsG in phosphotransferase system (PTS) was weakened by homologous recombination, and non-PTS pathway was strengthened by deleting its repressor gene iolR, and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold, reached 110.72 mg/L. Then, to increase precursor fructose-6-phosphate (F-6-P) supply, phosphofructokinase was weaken, fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement, moreover, regulator gene nanR was deleted, 1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold. Subsequently, promoter of 1-DNJ synthetase cluster was optimized, as well as 5'-UTRs of downstream genes in synthetase cluster, and 1-DNJ produced by the final strain reached 478.62 mg/L. Last but not the least, 1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter, which was the highest yield of 1-DNJ reported to date. Taken together, our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis, this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

Keywords: 1-DNJ synthetase Gene cluster; 1-Deoxynojirimycin; Bacillus amyloliquefaciens; Fructose 6-phosphate; Glucose transport pathway; Metabolic engineering.