Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2′-Fucosyllactose Synthesis in Bacillus subtilis
Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2′-Fucosyllactose Synthesis in Bacillus subtilis
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DOI：
10.1021/acssynbio.9b00314
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发表时间：
2019-10-01
影响因子：
4.7
通讯作者：
Liu, Long
中科院分区：
文献类型：
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作者：
Deng, Jieying;Gu, Liuyan;Liu, Long
人乳低聚糖（HMO）已被证明对婴儿的肠道健康和免疫系统有益。2 '-岩藻糖基乳糖（2'-FL）是最丰富和研究最深入的HMO，已被批准作为婴儿配方奶粉的添加剂。如何构建高效、安全的微生物细胞工厂生产2 '-FL越来越受到人们的关注。在这项工作中，我们通过改造底物转运和辅因子鸟苷S '-三磷酸（GTP）再生系统，将枯草芽孢杆菌改造为高效的2'-FL生产菌。首先，我们通过引入脆弱拟杆菌补救途径基因fkp构建了2 '-FL前体鸟苷5'-二磷酸-L-岩藻糖（GDP-L-岩藻糖）的合成途径，并通过过量表达转运蛋白来改善岩藻糖的输入。通过引入不同来源的岩藻糖基转移酶构建了2 '-FL合成的完整途径，发现幽门螺杆菌（Helicobacter pylori）的岩藻糖基转移酶基因是2'-FL合成的最佳基因。我们还通过引入异源乳糖渗透酶和消除内源性β-半乳糖苷酶（yesZ）来阻断乳糖降解来改善底物乳糖输入。接下来，通过微调辅因子鸟苷5 '-三磷酸再生模块基因gmd、ndk、guaA、guaC、ykf N、deoD和xpt的表达来改善2'-FL和GDP-L-岩藻糖的产生。最后进行3L流加发酵，2 '-FL效价最高可达5.01g/L，岩藻糖产量可达0.85mol/mol。我们优化了B中2 '-FL的合成模块。本研究为进一步提高2 '-FL产量的代谢工程提供了一个良好的起点。
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.