Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization
Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization
复制标题
DOI：
10.1016/j.ymben.2018.06.003
复制
发表时间：
2018-07-01
影响因子：
8.4
通讯作者：
Beckham, Gregg T.
中科院分区：
文献类型：
--
作者：
Franden, Mary Ann;Jayakody, Lahiru N.;Beckham, Gregg T.
乙二醇用作聚对苯二甲酸乙二醇酯生产的原材料，防冻剂，管道中的气体水合物抑制剂以及许多其他工业应用。通过C2代谢途径，通过高毒性中间体乙醇醛和乙醇酸盐，通过需氧微生物过程进行代谢。恶臭假单胞菌KT 2440，它已被改造为环境修复应用，鉴于其高毒性耐受性和广泛的底物特异性，是不能有效地代谢乙二醇，尽管窝藏推定的基因，用于这一目的。为了进一步扩大恶臭假单胞菌的代谢组合，我们阐明了通过乙醛酸碳连接酶（gcl）与其他基因组合的系统性过表达来实现乙二醇的代谢途径。定量逆转录聚合酶链反应表明，基因组邻近gcl的所有四个基因（hyi、glxR、ttuD和pykF）都被转录为操纵子。在仅两个基因（腺体glxR）的表达导致在乙二醇中生长的情况下，当表达整个gcl操纵子时观察到生长和乙二醇利用的改善。乙醇醛和乙二醛都抑制浓度高于50 mM的乙二醇的生长。为了克服这一瓶颈，乙醇酸氧化酶（glcDEF）操纵子的额外过表达消除了乙醇酸瓶颈并使这些有毒中间体的产生最小化，在摇瓶实验中允许在高达2 M（类似于124 g/L）的乙二醇中生长并完全消耗0.5M（31 g/L）的乙二醇。此外，工程菌株能够将乙二醇转化为中链长聚羟基烷酸酯（mcl-PHA）。总的来说，这项研究提供了一个强大的恶臭假单胞菌KT 2440菌株的乙二醇消费，这将作为一个基础菌株，进一步生物催化剂的发展，在废聚酯塑料和生物质衍生的废水流的修复应用。
Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida, we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gland glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (similar to 124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, this study provides a robust P. putida KT2440 strain for ethylene glycol consumption, which will serve as a foundational strain for further biocatalyst development for applications in the remediation of waste polyester plastics and biomass-derived wastewater streams.