Kim Jung-Hun, Wang Chonglong, Jang Hui-Jung, Cha Myeong-Seok, Park Ju-Eon, Jo Seon-Yeong, Choi Eui-Sung, Kim Seon-Won
Division of Applied Life Science (BK21 Plus), PMBBRC, Institute of Agricultural and Life Science, Gyeongsang National University, Jinju, 52828, South Korea.
Research Center for Industrial Chemical Biotechnology, KRICT, Ulsan, 44468, South Korea.
Microb Cell Fact. 2016 Dec 23;15(1):214. doi: 10.1186/s12934-016-0612-6.
Isoprene, a volatile C5 hydrocarbon, is an important platform chemical used in the manufacturing of synthetic rubber for tires and various other applications, such as elastomers and adhesives.
In this study, Escherichia coli MG1655 harboring Populus trichocarpa isoprene synthase (PtispS) and the exogenous mevalonate (MVA) pathway produced 80 mg/L isoprene. Codon optimization and optimal expression of the ispS gene via adjustment of the RBS strength and inducer concentration increased isoprene production to 199 and 337 mg/L, respectively. To augment expression of MVA pathway genes, the MVA pathway was cloned on a high-copy plasmid (pBR322 origin) with a strong promoter (P), which resulted in an additional increase in isoprene production up to 956 mg/L. To reduce the formation of byproducts derived from acetyl-CoA (an initial substrate of the MVA pathway), nine relevant genes were deleted to generate the E. coli AceCo strain (E. coli MG1655 ΔackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA). The AceCo strain harboring the ispS gene and MVA pathway showed enhanced isoprene production of 1832 mg/L in flask culture with reduced accumulation of byproducts.
We achieved a 23-fold increase in isoprene production by codon optimization of PtispS, augmentation of the MVA pathway, and deletion of genes involved in byproduct formation.
异戊二烯是一种挥发性C5烃，是一种重要的平台化学品，用于制造轮胎用合成橡胶以及各种其他应用，如弹性体和粘合剂。
在本研究中，携带毛果杨异戊二烯合酶（PtispS）和外源甲羟戊酸（MVA）途径的大肠杆菌MG1655产生了80mg/L的异戊二烯。通过调整核糖体结合位点（RBS）强度和诱导剂浓度对ispS基因进行密码子优化和最佳表达，使异戊二烯产量分别提高到199mg/L和337mg/L。为了增强MVA途径基因的表达，将MVA途径克隆到具有强启动子（P）的高拷贝质粒（pBR322来源）上，这导致异戊二烯产量进一步提高至956mg/L。为了减少源自乙酰辅酶A（MVA途径的初始底物）的副产物的形成，删除了九个相关基因以产生大肠杆菌AceCo菌株（大肠杆菌MG1655ΔackA-pta、poxB、ldhA、dld、adhE、pps和atoDA）。携带ispS基因和MVA途径的AceCo菌株在摇瓶培养中显示出增强的异戊二烯产量，为1832mg/L，同时副产物积累减少。
我们通过对PtispS进行密码子优化、增强MVA途径以及删除参与副产物形成的基因，使异戊二烯产量提高了23倍。
