摘要
中链脂肪酸(medium-chain fatty acids,MCFAs)及其衍生化学品在能源、医药、化工等诸多领域具有重要的应用价值.微生物发酵生产MCFAs是一条绿色、可持续的路线.大肠杆菌(Escherichia coli)可利用天然脂肪酸合成(fatty acid biosynthesis,FAB)路径和逆向β氧化(reversedβ-oxidation,RBO)路径合成MCFAs.MCFAs生产存在链长难控制、合成效率低、细胞损伤大等问题.近年来,工程大肠杆菌合成MCFAs取得了重大进展.通过链长控制蛋白的筛选与改造,MCFAs合成特异性得以提高;通过增加前体供应和强化产物释放,对合成路径进行“推-拉”,MCFAs合成效率得以改善;通过膜工程改造、应激响应调控及适应性进化,工程菌株对MCFAs的耐受性得以增强.本文基于大肠杆菌合成MCFAs的两种路径,系统综述了近年来控制产物链长、优化合成路径和增强细胞耐受的工程化策略,并展望了后续改善MCFAs合成的研究方向.
Medium-chain fatty acids(MCFAs), straight-chain fatty acids with 6–12 carbons, can be transformed into aliphatic hydrocarbons, triglycerides, and other oleochemicals. MCFAs and their derivatives show strong potential as useful materials in the energy, medicine, food and chemical industries. Compared with the conventional extraction methods based on petroleum or plant biomass, microbial fermentation is a green and sustainable alternative for MCFA production.Escherichia coli are frequently used as model microorganism for the analyses of metabolic mechanisms and industrial processes owing to the factors: rapid replication, clear genetics, ease of rearing in the laboratory, simple process for amplification and abundance of gene manipulation tools. In E. coli, MCFAs can be synthesised through the fatty acid biosynthesis(FAB) pathway and the reverse β-oxidation(RBO) pathway. However, there are still many challenges that severely limit MCFAs biosynthesis in E. coli, such as chain-length control, synthesis efficiency and cytotoxicity. With the development and improvement of metabolic engineering, synthetic biology and high-throughput sequencing technology,great progress has been made in recent years in the engineering of E. coli for MCFAs overproduction.In this review, we first introduce the MCFAs synthetic pathways in E. coli, including the FAB and RBO pathways, and compare these two pathways in terms of extension unit, rate-limiting step and cofactors. We then summarise the challenges,advances and representative studies in MCFAS production, with a focus on three aspects: chain-length control, synthetic pathway optimisation and tolerance improvement. Chain-length control is the key to the directed synthesis of MCFAs, as multiple rounds of cycles in the synthetic pathway tend to generate mixtures of various chain lengths. We discuss the catalysis mechanism of functional protein in the synthetic pathways to achieving chain-length control, including thioesterase, thiolase, and ketoacyl synthase. Protein and metabolic engineering of these key enzymes for targeted synthesis of MCFAs are also summarised in this review. Furthermore, synthetic pathway optimisation is an effective strategy for the enhancement of MCFAs biosynthesis. The “Pull-Push” of the FAB and RBO pathways, a process frequently used to increase precursor supply and improve product release, is reviewed here. Tolerance engineering is extremely critical for MCFAs overproduction, owing to the cell toxicity of MCFAs. We discuss in this review the injury mechanisms associated with MCFAs production and the strategies for improving cellular tolerance. Specifically, MCFAs production reduces intracellular p H and changes the composition of the cell membrane, leading to disruption of cytoplasmic homeostasis and destruction of membrane structure and properties. Membrane engineering, stress response regulation and adaptive evolution are generally employed for improving cellular tolerance of MCFAs.In summary, we cover in this review the recent advancements in the bio-engineering of E. coli for MCFAs production,based on the FAB and RBO biosynthetic pathways. Protein, metabolic and membrane engineering are generally applied to control the chain length, optimise the synthetic pathway and improve cellular tolerance. We also provide some perspectives for further strengthening MCFAs biosynthesis, including improvement of computer technology for protein assessment, indepth and full perspective analysis of MCFCs toxicity and tolerance mechanisms and the mining of useful targets from exogenous and endogenous species using high-throughput screening.
作者
房立霞
冯雪茹
刘夺
韩紫依
刘梦晓
郝雪雁
曹英秀
Lixia Fang;Xueru Feng;Duo Liu;Ziyi Han;Mengxiao Liu;Xueyan Hao;Yingxiu Cao(School of Chemical Engineringand Technology Tianjin Universiny,Tanyin 300072,China;Key Laboratory of Systems Bioengineering(Ministry of Education),Frontier Science Center for Synthetic Biology Tianjin Universiy,Tianjin 300072,China)
出处
《科学通报》
EI
CAS
CSCD
北大核心
2022年第25期3014-3023,共10页
Chinese Science Bulletin
基金
国家重点研发计划(2021YFC2104400)资助。
关键词
大肠杆菌
中链脂肪酸
代谢工程
链长
耐受
Escherichia coli
medium-chain fatty acids
metabolic engineering
chain-length
tolerance
