Ni Zhijian, Li Zhongkui, Wu Jinyong, Ge Yuanfei, Liao Yingxue, Yuan Lixia, Chen Xiangsong, Yao Jianming
Institute of Plasma Physics, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China.
University of Science and Technology of China, Hefei, China.
Front Bioeng Biotechnol. 2020 Dec 3;8:611900. doi: 10.3389/fbioe.2020.611900. eCollection 2020.
2'-fucosyllactose (2'-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a -mutant C41 (DE3)ΔZ to ulteriorly increase 2'-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2'-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2'-FL synthesis, deletion of chromosomal genes (, and ) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2'-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2'-FL production, ultimately a highest titer of 2'-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2'-FL was achieved with a yield of 0.89 mol 2'-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2'-FL. As a proof of concept, our preliminary 2'-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.
2'-岩藻糖基乳糖（2'-FL）是母乳中最简单但也是最丰富的低聚糖之一，已被证明对新生儿的健康发育有许多积极益处。然而，其高成本生产和有限的可得性限制了它在婴儿营养中的广泛应用以及对其潜在功能的进一步研究。在本研究中，基于先前的成果，我们通过使用一株突变体C41(DE3)ΔZ构建了一个高效的细胞工厂，通过添加廉价的甘油进一步提高2'-FL的产量。首先，我们通过不同基于质粒的表达组合在C41(DE3)ΔZ中共表达GDP-L-岩藻糖生物合成基因和异源α-唾液酸基转移酶，从功能上构建了一条2'-FL生物合成的优选途径。为了进一步提高从GDP-L-岩藻糖到2'-FL合成的碳通量，我们对参与前体GDP-L-岩藻糖和GDP-甘露糖降解的染色体基因（、和）进行了缺失。值得注意的是，共导入两个异源正调控因子RcsA和RcsB被证实更有利于GDP-L-岩藻糖的形成，进而有利于2'-FL的生产。进一步对α-唾液酸基转移酶基因的单拷贝进行基因组整合以及对发酵条件的初步优化，使所得工程菌株实现了高滴度和高产率。综合考虑细胞内乳糖利用、GDP-L-岩藻糖的可得性以及2'-FL生产的岩藻糖基化活性，最终在我们优化的条件下，2'-FL的最高滴度在分批发酵中达到6.86 g/L，乳糖产率为0.92 mol/mol。此外，在50-L补料分批发酵系统中证明了大规模生产的可行性，其中2'-FL的最大滴度达到66.80 g/L，2'-FL产率为0.89 mol 2'-FL/mol乳糖，2'-FL生产率约为0.95 g/L/h。作为概念验证，我们初步的2'-FL生产展示了卓越的生产性能，这将为进一步的工业化生产提供一个有前景的候选工艺。
