期刊
ACS SYNTHETIC BIOLOGY
卷 8, 期 10, 页码 2418-2427出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.9b00314
关键词
2 '-fucosyllactose; Bacillus subtilis; guanosine 5 '-triphosphate; lactose permease; fucosyltransferase
资金
- National Natural Science Foundation of China [31871784, 31870069, 21676119, 31671845]
- National Key Research and Development Program of China [2018YFA0900300]
- Postgraduate Research & Practice Innovation Program of Jiangsu Province [KYCX19_1838]
Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants' intestinal health and immune systems. 2'-Fucosyllactose (2'-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2'-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2'-FL producer by engineering the substrate transport and cofactor guanosine S'-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2'-FL precursor guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2'-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2'-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous beta-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2'-FL and GDP-L-fucose was improved by fine-tuning the expression of cofactor guanosine 5'-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykf N, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2'-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2'-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2'-FL production in the future.
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