Wang Jian, Wu Yifei, Sun Xinxiao, Yuan Qipeng, Yan Yajun
College of Engineering, The University of Georgia , Athens, Georgia 30602, United States.
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology , Beijing 100029, China.
ACS Synth Biol. 2017 Oct 20;6(10):1922-1930. doi: 10.1021/acssynbio.7b00136. Epub 2017 Jun 23.
Microbial based bioplastics are promising alternatives to petroleum based synthetic plastics due to their renewability and economic feasibility. Glutarate is one of the most potential building blocks for bioplastics. The recent biosynthetic routes for glutarate were mostly based on the l-lysine degradation pathway from Pseudomonas putida that required lysine either by feeding or lysine overproduction via genetic manipulations. Herein, we established a novel glutarate biosynthetic pathway by incorporation of a "+1" carbon chain extension pathway from α-ketoglutarate (α-KG) in combination with α-keto acid decarboxylation pathway in Escherichia coli. Introduction of homocitrate synthase (HCS), homoaconitase (HA) and homoisocitrate dehydrogenase (HICDH) from Saccharomyces cerevisiae into E. coli enabled "+1" carbon extension from α-KG to α-ketoadipate (α-KA), which was subsequently converted into glutarate by a promiscuous α-keto acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD). The recombinant E. coli coexpressing all five genes produced 0.3 g/L glutarate from glucose. To further improve the titers, α-KG was rechanneled into carbon chain extension pathway via the clustered regularly interspersed palindromic repeats system mediated interference (CRISPRi) of essential genes sucA and sucB in tricarboxylic acid (TCA) cycle. The final strain could produce 0.42 g/L glutarate, which was increased by 40% compared with the parental strain.
基于微生物的生物塑料因其可再生性和经济可行性，是石油基合成塑料的有前景的替代品。戊二酸是生物塑料最具潜力的构建模块之一。最近戊二酸的生物合成途径大多基于恶臭假单胞菌的L-赖氨酸降解途径，该途径需要通过喂食赖氨酸或通过基因操作过量生产赖氨酸来获取赖氨酸。在此，我们通过在大肠杆菌中引入来自α-酮戊二酸（α-KG）的“+1”碳链延伸途径并结合α-酮酸脱羧途径，建立了一种新的戊二酸生物合成途径。将酿酒酵母的同柠檬酸合酶（HCS）、同乌头酸酶（HA）和同异柠檬酸脱氢酶（HICDH）引入大肠杆菌，实现了从α-KG到α-酮己二酸（α-KA）的“+1”碳延伸，随后α-KA由一种混杂的α-酮酸脱羧酶（KivD）和琥珀酸半醛脱氢酶（GabD）转化为戊二酸。共表达所有五个基因的重组大肠杆菌从葡萄糖中产生了0.3 g/L的戊二酸。为了进一步提高产量，通过三羧酸（TCA）循环中必需基因sucA和sucB的成簇规律间隔短回文重复序列系统介导的干扰（CRISPRi），将α-KG重新导向碳链延伸途径。最终菌株能够产生0.42 g/L的戊二酸，与亲本菌株相比提高了40%。
