Two unexpected promiscuous activities of the iron–sulfur protein IspH in production of isoprene and isoamylene

Microbial Cell Factories

Research

Yanfen Xue¹  Yanhe Ma¹  Deyong Ge² 

[1] State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, People’s Republic of China;State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, People’s Republic of China;University of Chinese Academy of Sciences, 100049, Beijing, People’s Republic of China;College of Medicine, Anhui University of Science and Technology, Huainan, People’s Republic of China;

关键词: Bacillus;    Isoprene;    2-Methyl-2-butene;    3-Methyl-1-butene;    E;    Dimethylallyl diphosphate (DMAPP);    Isoprene synthase;    IspS;    IspH;   

DOI  :  10.1186/s12934-016-0476-9

 received in 2016-02-08, accepted in 2016-04-27,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundBacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron–sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene.ResultsBacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP+-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300 % compared with the wild-type strain.ConclusionsIspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

【 授权许可】

CC BY   
© Ge et al. 2016