盐单胞菌中利用廉价碳源合成3-羟基丙酸代谢通路的构建及定向进化

The three-carbon 3-hydroxypropionic acid (3HP) is an important platform compound. Petrochemical synthesis of 3-HP is not suitable for industrial production due to the high cost of the precursors and processes, as well as environmental concerns. This research is devoted to reducing the cost of the biotechnological production of 3-HP through metabolic engineering, synthetic biology and directed evolution. Taking Halomonas TD as the production platform bacterium, which is suitable for continuous fermentation processes without the need for sterilization, the metabolic pathway for 3-hydroxypropionic acid production from glucose as a sole carbon source will be constructed. At the same time, genomic integration of expression cassettes with different intensities will be carried out to optimize the 3-HP synthesis pathway. Biosensor circuits and directed evolution methods based on CRISPR-guided DNA polymerases will be established. Directed evolution of rate-limiting enzymes in the synthetic pathway and the whole genome will be carried out to increase 3-HP production. The resulting engineered Halomonas TD strain will have the potential to be used as a platform strain for low-cost industrial production of 3-HP. Taking 3HP production as an example, the methods established in this study have significance as a reference for the low-cost production of other kinds of bio based chemicals.

3-羟基丙酸（3-hydroxypropionic acid，3-HP）是一种重要的平台化合物，化学法合成3-HP的效率不高，并且污染环境，不适用于工业化生产。本研究致力于通过代谢工程、合成生物学和定向进化的方法降低3-HP的生产成本，以适用于无需灭菌的连续发酵工艺的盐单胞菌TD作为生产平台菌，构建以葡萄糖为单一碳源生产3-羟基丙酸的代谢通路，并利用合成生物学方法优化3-HP合成通路，然后将不同强度的代谢通路整合到基因组中；构建3-HP生物传感器元件，并建立基于CRISPR-guided DNA polymerases的EvolvR定向进化方法，通过对合成通路中限速酶以及全基因规模的定向进化两个层面提高3-HP的产量，得到的重组盐单胞菌TD可作为3-HP低成本工业化生产的底盘菌。本研究以3-羟基丙酸作为入手点，同时对其他种类生物基化学品的合成途径构建、低成本生产都将具有指导意义。

聚羟基脂肪酸酯是一类具有生物可降解性的生物基塑料，其中聚3-羟基丙酸酯（P3HP）具有较大应用价值，其单体3-羟基丙酸（3-hydroxypropionic acid，3HP）是一种重要的平台化合物，位列美国能源部列举的12种最具价值的平台化合物之一。其生物合成较多的是在大肠杆菌等通用底盘菌株中构建异源表达的代谢通路，但是大肠杆菌在工业生产过程中有易染菌的缺点。而本研究利用的盐单胞菌可以进行连续开放发酵，发酵全程不用灭菌处理，并且不易染菌等特点，可以大幅度提高工业生物技术的竞争性。本研究旨在盐单胞菌中构建3HP及其共聚物的合成途径。我们解析了盐单胞菌中3-羟基丙酸的降解途径，鉴定了盐单胞菌内源的催化PDO到3-羟基丙酸生产的醇脱氢酶AdhP和醛脱氢酶AldDTD，并通过合成生物学调控代谢途径的表达，结合调节氧化还原平衡获得了3-羟基丙酸高产工业化生产底盘菌株，最终发酵罐生产实现154 g/L的产量，是目前已经报道的最高产量，得到的重组盐单胞菌可作为3-HP及其共聚物低成本生产的平台菌株，对工业生产3HP及其共聚物具有重要意义，主要成果发表于Nature Communications。