进化与CRISPR工程大肠杆菌过量合成对羟基苯乙酸及其代谢调控机制研究

4-Hydroxyphenylacetic acid is an important pharmaceutical, pesticide and fine chemical intermediate with a high added-value and extensive market prospect. We have successfully increased the 4-hydroxyphenylacetic acid production in engineered E. coli through metabolic engineering in our previous studies. The concentration and yield of 4-hydroxyphenylacetic acid are much higher than those reported in literatures. However, the production level of 4-hydroxyphenyl acetic acid is much lower than the theoretical yield because 4-hydroxyphenylacetic acid is highly toxic to E. coli and the limited availabilities of ATP and NADPH. In order to solve the key scientific problems of adaptation and regulation mechanism of anabolism, we will plan to apply biosensor-mediated evolutionary engineering, ATP and NADPH engineering technology based on CRISPRi and quorum-sensing metabolic switch to improve the fitness between the host bacteria and the heterologous synthetic pathway from genomic, energy and reductive levels, and to promote 4-hydroxyphenylacetic acid production and its tolerance. Finally, we will apply comparative genomics, transcriptional level analysis and CRISPRi/a regulation to explore the relationship between cell genotype and product synthesis, and to reveal the regulation mechanism of p-hydroxyphenylacetic acid synthesis. On the basis of the results of the omic analysises, the genetic modifications will be further carried out to obtain a 4-hydroxyphenylacetic acid hyper-producer E. coli. The research will play important scientific and practical roles in the development of 4-hydroxyphenylacetic acid bioproduction and synthetic biotechnology.

对羟基苯乙酸是一种附加值高、市场前景广的医药、农药和精细化工中间体。虽然前期研究已使大肠杆菌合成对羟基苯乙酸的产量和产率，远高于文献报道的最高值，但因其高毒性、能量和还原力不足引起的宿主菌与外源途径的不适配，导致与理论产率还有很大差距，从而阻碍了对羟基苯乙酸生物制造的发展。为此，为解决适配性和合成代谢调控机制的关键科学问题，我们拟采用生物传感器介导的进化工程、基于CRISPRi和群体感应代谢拨动开关的ATP和NADPH工程技术，依次从基因组、能量和还原力层面提高宿主菌与外源合成途径间的适配性，在提高耐受性的基础上促进对羟基苯乙酸的合成；最后，通过比较基因组学、转录水平分析结合CRISPR抑制与激活，探索细胞基因型与产物合成间的规律，阐明对羟基苯乙酸的合成代谢调控机制，对途径再优化、创建高产菌株。项目研究将对生物基对羟基苯乙酸生物制造和合成生物技术的发展具有重要科学意义和实用价值。

对羟基苯乙酸是一种附加值高、市场前景广的医药、农药和精细化工中间体。但其高毒性导致其无法实现产业化。因此，项目重点研究了外源途径与宿主间适配性机制，以解决其高毒性问题。创建了对羟基苯乙酸产量创记录的工程大肠杆菌，为其产业化奠定了基础。构建了一个对羟基苯乙酸(4HPAA)生物传感器；发展了一种生物传感器介导的ARTP诱变、适应性进化与基因组改组相结合的育种技术，从基因组层面提高了外源途径与宿主间的适配性；发展了一种基于CRISPRi筛选的辅因子工程技术(CECRiS)，从能量和还原力层面提高了外源途径与宿主间的适配性；最终创建了含群体感应激活、抑制双水平系统的、对羟基苯乙酸产量创记录的工程大肠杆菌，2L发酵罐后期补料发酵产28.57 g/L 4HPAA、摩尔产率达27.64%；最后利用比较基因组学、结合转录水平分析和CRISPRa/i功能分析，初步揭示了高效合成4HPAA的调控机制，并为逆向代谢工程提出了有意的靶目标。项目的研究为对羟基苯乙酸的产业化奠定了良好的基础，所发展的合成生物学技术也将应用于其它人工微生物的创建中。