Abstract:

With the increasing consumption of fossil fuels and growing depravation of environment, development of substitutes for petroleum-derived compounds is becoming more and more important. In the past few years, bio-based production of chemicals, fuels, nutraceuticals and pharmaceuticals from renewable raw materials via metabolic engineering has gained significant attention. As a high-value fragrance with aromatic taste, 3-phenylpropanol has been widely used in the production of food additives, cosmetics and etc. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Because of its high efficiency in promoting bile secretion and mild antispasmodic function, 3-phenylpropanol is widely used in the treatment of cholecystitis, gallstones and biliary surgery syndrome. The current production method mainly relies on plant extraction and chemical synthesis, which, however, are challenging due to the high cost of catalyst, strict reaction condition and low product yield. Recently, engineering microorganisms has become an attractive alternative to efficient production of high-value compounds, such as flavors, fragrances, cosmetics, pharmaceuticals, solvents, biofuels and other chemicals. It is of great significance to construct a microbial cell factory to synthesize 3-phenylpropanol from renewable resources. In this study, we designed and constructed two different artificial 3-phenylpropanol biosynthetic pathways by establishing a connection between the target compound and the microorganism's own metabolic network. Especially, the pathway that relies on carboxylic acid reductase exhibited high efficiency in the production of 3-phenylpropanol. When using glycerol as the sole carbon source, the recombinant strain successfully generated 91 mg/L 3-phenylpropanol in shake flask experiments. By eliminating the rate-limiting steps, increasing the carbon flux towards the shikimate pathway and knocking out the competitive pathways, the titer of 3-phenylpropanol in the shake flask fermentation culture was finally increased to 841 mg/L, representing a 9.2-fold increase compared with the titer generated by the original strain. This work provides a green and sustainable approach for the production of 3-phenylpropanol.

Key words: phenylpropanol, metabolic engineering, the shikimate pathway, artificial pathway design

摘要：

3-苯丙醇是一种具有芳香味的高价值香料，在医药、化妆品、食品等领域有广泛用途，是生产多种药品和化学品的重要前体。其目前的生产方法主要依赖于植物提取和化学合成，存在产物得率低、生产周期长和环境不友好等缺点。为解决这些问题，构建微生物细胞工厂利用可再生资源合成3-苯丙醇具有重要的意义。本研究通过将目标化合物与微生物自身代谢网络建立联系，基于底物或中间体与产物的结构类似性以及化合物间的基团转移关系，设计并构建了两条不同的3-苯丙醇的人工生物合成途径。其中，依赖羧酸还原酶的苯丙醇生物合成途径具有较高的生产效率。在大肠杆菌中实现了以甘油为碳源，从头生物合成3-苯丙醇，产量达91 mg/L。通过消除限速步骤，增加莽草酸途径碳通量以及敲除竞争途径等代谢工程策略的实施，将苯丙醇的产量提高到了841 mg/L，较初始菌株产量提高了9.2倍，为苯丙醇的绿色、可持续、大规模生产提供了基础。

关键词: 3-苯丙醇, 代谢工程, 莽草酸途径, 人工途径设计