新型生长快速需钠弧菌底盘细胞的构建及应用

It is always absolutely necessary to develop the next generation of chassis cell for synthetic biology. The bacterium Vibrio natriegens has the fastest growth rate of any known bacteria. Its nutritional versatility, rapid doubling time, easy to be transformed and the fact that it is nonpathogenic to humans have all encouraged the use of this strain in synthetic biology studies. In this study, we are going to develop the genetic or genomic tools and methods to engineer or modify V. natriegens and demonstrate the advantages of using these engineered strains in common biotech processes based on the previous reports and our preliminary studies. Firstly, constitutive promoters with different strength will be screened for regulating the expression levels of target genes, and PCR-based tandem repeat assisted genome editing tool with high efficiency and fidelity for gene deletion, insertion and replacement will be developed in V. natriegens. Then, based on the genome analysis, the non-essential genes or gene clusters on the genome of V. natriegens will be knock out through sequential rounds of chromosome recombination. The cell growth and metabolism of the strains with genome reduction as well as their transcriptomic profiles will be investigated to reveal the mechanism of physiological regulation. Finally, the construction of 3-dehydroshikimic acid-overproducing strain through metabolic engineering strategies will prove the feasibility of the fast-growing V. natriegens as chassis cell for synthetic biology. This study will be helpful to promote the development of synthetic biology and lay a solid foundation for constructing next-generation robust chassis cells.

寻找新的、更具优势的底盘细胞是合成生物学发展需要解决的重要问题。作为已报道生长速率最快的细菌，需钠弧菌还具有适应性强、易于遗传操作、对人体无致病性等优点，是一种比较理想的合成生物学底盘细胞。本项目在前期报道和生长速率检测的基础上，通过研究需钠弧菌遗传操作体系，发掘可实现不同表达强度的组成型启动子，建立高效的需钠弧菌基因编辑方法，实现基因的无痕敲除/插入/替换，定向操控需钠弧菌的基因表达调控。通过分析需钠弧菌基因组序列，对其基因组上的非必需基因或基因簇进行逐轮敲除，研究缩减其基因组后，底盘细胞的基因表达差异和生长与代谢等表型变化，揭示其生理代谢的调控机制。最后以构建高产3-脱氢莽草酸的底盘细胞为例，测试需钠弧菌作为新一代快速生长底盘细胞的可行性。本研究将有助于进一步推动合成生物学的发展，为新一代底盘细胞的建立奠定理论基础。

需钠弧菌作为一种新型生长快速底盘细胞，在合成生物学领域展现出良好的应用前景。但是其生理学和遗传学相关研究基础十分薄弱，无法满足合成生物学日益发展的需要。针对上述问题，本研究从6株需钠弧菌中选取生长快速、表型稳定的CICC10908菌株作为后续研究的宿主细胞。建立并优化自然转化方法后，将筛选标记基因cat-sacB或KanR整合到染色体上的同源重组效率分别达到4×10–5和4×10–4。随后，利用双向选择性筛选方式，建立了精准、高效的基因组无痕编辑体系。通过测试，基因敲除、回补、插入和替换这4种不同类型基因组编辑阳性率分别为93.8%、100%、95.7%和100%。为了实现基因的精细表达调控，人工设计合成标准化的调控元件文库，包括间隔区和-35/-10区启动子文库以及RBS区文库。以绿色荧光蛋白为筛选信号，获得大量具有不同表达强度的启动子元件和不同翻译强度的RBS元件，其表达强度范围跨度达5个数量级，并具有良好的表达稳定性。利用弱、中、强3种强度调控元件对葡萄糖转运系统基因galP-glk进行组合调控，显著恢复了磷酸转移酶系统缺陷型菌株的生长速率和葡萄糖利用速率。利用基因组编辑体系和不同强度的调控元件，依次对莽草酸途径、磷酸戊糖途径、糖酵解途径的关键基因进行多位点组合调控，初步构建产3-脱氢莽草酸底盘细胞。在摇瓶发酵条件下，工程菌株DHS26的3-脱氢莽草酸产量达0.18g/L，相对于葡萄糖的摩尔转化率为3.2%。丙酮酸是糖酵解途径的重要产物。利用需钠弧菌具有极快的生长速率和底物利用速率特性，通过弱化调控丙酮酸利用途径，阻断副产物合成途径，构建高产丙酮酸细胞工厂。通过分批补料发酵18h，工程菌株PYR15的丙酮酸产量达39.2g/L，合成速率达2.18g/L/h。该项目研究对推进需钠弧菌在合成生物学领域的应用研究具有重要的理论和现实意义。