Glycolipids are target molecules in biotechnology and biomedicine as biosurfactants, biomaterials and bioactive molecules. An engineered E. coli strain for the production of glycoglycerolipids (GGL) used the MG517 glycolipid synthase from M. genitalium for glucosyl transfer from UDPGlc to diacylglycerol acceptor (Mora-Buyé et al., 2012). The intracellular diacylglycerol pool proved to be the limiting factor for GGL production. Here we designed different metabolic engineering strategies to enhance the availability of precursor substrates for the glycolipid synthase by modulating fatty acids, acyl donor and phosphatidic acid biosynthesis. Knockouts of tesA, fadE and fabR genes involved in fatty acids degradation, overexpression of the transcriptional regulator FadR, the acyltransferases PlsB and C, and the pyrophosphatase Cdh for phosphatidic acid biosynthesis, as well as the phosphatase PgpB for conversion to diacylglycerol were explored with the aim of improving GGL titers. Among the different engineered strains, the ΔtesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Attempts to boost UDPGlc availability by overexpressing the uridyltransferase GalU or knocking out the UDP-sugar diphosphatase encoding gene ushA did not further improve GGL titers. Most of the strains produced GGL containing a variable number of glucosyl units from mono-to tetra-saccharides. Interestingly, the strains co-expressing Cdh showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs) whereas the strains with a fadR knockout presented a higher amount of unsaturated acyl chains. In all cases, GGL production altered the lipidic composition of the E. coli membrane, observing that GGL replace phosphatidylethanolamine to maintain the overall membrane charge balance.

糖脂是生物技术和生物医学中的目标分子，作为生物表面活性剂、生物材料和生物活性分子。用于生产甘油糖脂 (GGL)的工程大肠杆菌菌株使用来自生殖器支原体的 MG517 糖脂合酶将葡萄糖基从 UDPGlc 转移到甘油二酯受体（Mora-Buyé 等，2012）。细胞内二酰基甘油池被证明是 GGL 产生的限制因素。在这里，我们设计了不同的代谢工程策略，通过调节脂肪酸、酰基供体和磷脂酸的生物合成来提高糖脂合酶前体底物的可用性。tes A、fad E 和fab 的淘汰赛研究了参与脂肪酸降解、转录调节因子 FadR 过表达、酰基转移酶 PlsB 和 C 以及用于磷脂酸生物合成的焦磷酸酶 Cdh 以及用于转化为二酰基甘油的磷酸酶 PgpB 的 R 基因，目的是提高 GGL 滴度。之间的不同的工程化菌株中，Δ TES的菌株共表达MG517和融合蛋白PlsCxPgpB是最好的生产商，GGL滴度相比增加表达MG517单独亲本株的350％。尝试通过过度表达尿苷转移酶 GalU 或敲除 UDP-糖二磷酸酶编码基因ush来提高 UDPGlc 的可用性A 没有进一步提高 GGL 滴度。大多数菌株产生的 GGL 含有从单糖到四糖的可变数量的葡萄糖基单元。有趣的是，共表达 Cdh 的菌株显示出 GGL 谱向二糖基化脂质（高达总 GGL 的 80%）的转变，而具有时尚R 基因敲除的菌株则呈现出更多的不饱和酰基链。在所有情况下，GGL 的产生都改变了大肠杆菌膜的脂质组成，观察到 GGL 取代了磷脂酰乙醇胺以维持整个膜电荷平衡。