PMID- 31550146 OWN - NLM STAT- MEDLINE DCOM- 20200707 LR - 20200707 IS - 2161-5063 (Electronic) IS - 2161-5063 (Linking) VI - 8 IP - 10 DP - 2019 Oct 18 TI - Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2'-Fucosyllactose Synthesis in Bacillus subtilis. PG - 2418-2427 LID - 10.1021/acssynbio.9b00314 [doi] AB - 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 5'-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, ykfN, 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. FAU - Deng, Jieying AU - Deng J AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Gu, Liuyan AU - Gu L AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Chen, Taichi AU - Chen T AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Huang, Hao AU - Huang H AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Yin, Xiaoqiang AU - Yin X AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Lv, Xueqin AU - Lv X AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Liu, Yanfeng AU - Liu Y AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Li, Nan AU - Li N AD - State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute , Bright Dairy & Food Co, Ltd. , Shanghai 200436 , China. FAU - Liu, Zhenmin AU - Liu Z AD - State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute , Bright Dairy & Food Co, Ltd. , Shanghai 200436 , China. FAU - Li, Jianghua AU - Li J AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Du, Guocheng AU - Du G AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. FAU - Liu, Long AU - Liu L AUID- ORCID: 0000-0002-9679-9130 AD - Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. AD - Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20191003 PL - United States TA - ACS Synth Biol JT - ACS synthetic biology JID - 101575075 RN - 0 (Oligosaccharides) RN - 0 (Trisaccharides) RN - 146-91-8 (Guanosine Diphosphate) RN - 28RYY2IV3F (Fucose) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 2.4.1.- (Fucosyltransferases) RN - J2B2A4N98G (Lactose) RN - XO2533XO8R (2'-fucosyllactose) SB - IM MH - Bacillus subtilis/*genetics MH - Fermentation/genetics MH - Fucose/genetics MH - Fucosyltransferases/genetics MH - Guanosine Diphosphate/genetics MH - Guanosine Triphosphate/genetics MH - Helicobacter pylori/genetics MH - Lactose/genetics MH - Metabolic Engineering/methods MH - Milk, Human/metabolism MH - Oligosaccharides/genetics MH - Regeneration/*genetics MH - Trisaccharides/*genetics OTO - NOTNLM OT - 2'-fucosyllactose OT - Bacillus subtilis OT - fucosyltransferase OT - guanosine 5'-triphosphate OT - lactose permease EDAT- 2019/09/25 06:00 MHDA- 2020/07/08 06:00 CRDT- 2019/09/25 06:00 PHST- 2019/09/25 06:00 [pubmed] PHST- 2020/07/08 06:00 [medline] PHST- 2019/09/25 06:00 [entrez] AID - 10.1021/acssynbio.9b00314 [doi] PST - ppublish SO - ACS Synth Biol. 2019 Oct 18;8(10):2418-2427. doi: 10.1021/acssynbio.9b00314. Epub 2019 Oct 3.