Microbial Bioprocess for Extracellular Squalene Production and Formulation of Nanoemulsions

Sustainable production of natural resources requires a clean manufacturing process with clean raw materials due to the risk of environmental contaminations. Microbial cell factories have enabled the production of natural products including hydrophobic chemicals. However, the microbial cells limit the production titer because of the limited intracellular storage capacity and no transport across the cell membrane. Herein, we engineered Corynebacterium glutamicum to produce hydrophobic squalene as a model product. Notably, squalene was secreted in the defined culture medium without in situ extractions. As a result, the secreted squalene was related to the glycolipid association with synthesized squalene in the cell envelope and a facilitated secretion occurred during cell division. Protein engineering and pathway engineering of C. glutamicum enhanced the total squalene (tSQ) production, where fed-batch fermentation resulted in 1406 ± 16 mg/L tSQ production (32% tSQ in the medium). Promoting extraction with dodecane overlay, bio-squalene nanoemulsions were formulated and the creaming effects were analyzed for cosmetic application. Combining metabolic pathway engineering and facilitated secretion in microbial cells will be a good strategy to produce sustainable hydrophobic chemicals.

鉴于环境污染风险，自然资源的可持续生产需依托洁净的制造流程与清洁原料。微生物细胞工厂（microbial cell factories）已可实现包括疏水化学品在内的天然产物合成，但受限于胞内储存能力不足且无法跨细胞膜转运产物，其生产滴度受到制约。本研究以疏水角鲨烯作为模式产物，对谷氨酸棒杆菌（Corynebacterium glutamicum）开展工程化改造。值得注意的是，角鲨烯可直接分泌至限定培养基中，无需进行原位提取。进一步分析显示，胞外分泌的角鲨烯与细胞包膜内合成角鲨烯所结合的糖脂相关，且细胞分裂过程可促进该分泌过程。对谷氨酸棒杆菌实施蛋白质工程与途径工程改造后，总角鲨烯（total squalene, tSQ）产量得到显著提升；采用补料分批发酵工艺时，总角鲨烯产量可达1406 ± 16 mg/L，其中培养基中总角鲨烯占比达32%。通过十二烷（dodecane）覆盖层强化提取后，可制备生物源角鲨烯纳米乳液，并针对化妆品应用场景分析了其乳析效应（creaming effect）。综上，将代谢途径工程与微生物细胞促分泌策略相结合，将是可持续生产疏水化学品的有效路径。