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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0476-9) contains supplementary material, which is available to authorized users.

Microbial cycling of isoprene, the most abundantly produced biological volatile organic compound on Earth

“…Isoprene synthase was partially purified from Bacillus subtilis , but proved to be labile, thus allowing only initial investigation, e.g., demonstrating a lower divalent cation requirement and lower pH optimum than isoprene synthases from the chloroplasts of plants [ 16 ]. An alternative route to isoprene production was proposed by Ge et al [ 22 ], who found that IspH, the final enzyme of the MEP pathway, converts HMBPP to isoprene, thus bypassing DMAPP [HMBPP or (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate is an intermediate metabolite in the transformation of 2- C -methyl- d -erythritol-2,4-cyclodiphosphate to IPP and DMAPP]. However, IspH gene expression was not correlated with higher isoprene production in the transcriptome-based study of [ 19 ].…”

Positioning Bacillus subtilis as terpenoid cell factory

“…Isoprene is generated from HMBDP, while two isoamylenes are directed from DMADP and IDP (Ge et al, 2016). Yet, whether this activity is also found in B. subtilis 168 IspH still requires more exploration.…”

“…N16‐5 evidently possessed the isoprene and isoamylene synthase activity. Isoprene is generated from HMBDP, while two isoamylenes are directed from DMADP and IDP (Ge et al 2016…”

Isoprene production by Escherichia coli through the exogenous mevalonate pathway with reduced formation of fermentation byproducts

“…Bacillus sp. are known to natively produce isoprene, and endogenous IspH from the MEP pathway can catalyze isoprene formation from HMBPP [16]. The Bacillus sp.…”