嗜碱芽孢杆菌铁硫蛋白IspH非特异性酶活力催化机制及其异戊二烯合成研究

Microorganism can produce isoprene. However, the enzyme responsible for isoprene synthesis has not been identified and the molecular mechanism for microbial isoprene synthesis is unclear. The iro-sulfur protein IspH is the last enzyme in the MEP pathway which catalyzes the formation of DMAPP and IPP from HMBPP. The IspH protein from Bacillus sp. N16-5 was previously found to have other non-specific activity for catalyzing the formation of isoprene from HMBPP and the conversion of DMAPP into isoamylene. So we speculate that IspH might be the nonspecific isoprene synthesis enzyme for microbial isoprene biosynthesis. However, whether this novel activity is universal for other IspH proteins is not clear. Also, the catalytic mechanism for this activity is still unknown. In this study, we want to reveal the molecular mechanism and the enzymatic basis of IspH for the synthesis of isoprene and isopentene by crystal structure resolving and sequence and mutagenesis analysis. Also, by enzymatic function analysis of other IspH proteins in vitro and vivo, we want to clarify the universality of this novel activity in microbial IspH. Furthermore, we want to find the relationship between IspH sequence and isoprene synthesis activity and the species specificity of IspH by the sequence alignment and mutagenesis analysis. These researches will be of great theoretical and practical value to reveal the molecular and physiological mechanism of biosynthesis of isoprene by microorganism and to promote the synthesis of bio-based isoprene.

微生物能合成异戊二烯，但至今没发现类似植物异戊二烯合成酶IspS功能的蛋白，微生物异戊二烯合成分子机制还不清楚。铁硫蛋白IspH是微生物MEP途径中最后一个酶，催化HMBPP合成异戊二烯前体物质DMAPP和IPP。我们前期研究发现嗜碱芽孢杆菌Bacillus sp.N16-5的IspH还具有直接催化HMBPP合成异戊二烯和催化DMAPP合成异戊烯的非特异性新活性，因此我们推测微生物合成异戊二烯也许并无特异性酶参与，而是IspH非特异催化活性的结果，而这种新活性是否具有普遍性及其催化机制还不清楚。本项目将通过IspH体内外催化功能研究和蛋白结构解析，结合序列和结构比对及突变分析等方法，揭示IspH催化生成异戊二烯和异戊烯反应的分子机制，明确IspH这种非特异催化活性的普适性和序列与活性的关联。本研究对于揭示微生物合成异戊二烯的分子及生理机制、推动生物基异戊二烯合成具有重要理论及应用价值。

嗜碱芽孢杆菌Bacillus sp. N16-5的IspH体外实验证实具有直接催化HMBPP合成异戊二烯和催化DMAPP合成异戊烯的非特异性新活性，这种非特异性新活性是否即为微生物合成异戊二烯的酶学基础以及其活性是否具有普遍性及其催化机制还不清楚。本项目将通过IspH体内外催化功能研究和蛋白结构和突变分析，来揭示其催化关键位点和模型以及其异戊二烯活性合成的酶学基础和种属特异性。我们首先通过表达和纯化步骤优化，获得了高浓度活性BspIspH蛋白，解决这一严格厌氧铁硫簇蛋白难以高浓度活性表达纯化问题；进一步通过不同来源IspH体外催化活性研究，揭示其异戊二烯合成活性不具有普遍性，仅存在于某些如芽孢杆菌属中，具有一定种属特异性；建立了菌株基于CRISPR-Cas9高效基因编辑技术，进一步通过BspIspH替代敲除体内实验，明确了BspIspH即为菌株异戊二烯合成的酶学基础，即微生物可能并没有专一异戊二烯合酶，而是由IspH进化出了催化HMBPP合成异戊二烯的非特异性活性，从而实现异戊二烯的合成；通过与小分子底物HMBPP共结晶，初步获得了其与底物复合物的晶体培养条件，并通过突变分析获得多个与异戊二烯和异戊烯合成相关的关键位点，揭示两种催化活性之间具有一定关联，获得了2个专一性催化HMBPP生成异戊二烯的合成酶，并显著提高了菌株的异戊二烯产量，为细菌基于MEP途径的高效异戊二烯合成工程菌构建提供了酶元件；基于研究推测了BspIspH催化合成异戊二烯和异戊烯的5步反应模型，其中焦磷酸基团是关键，它可以作为一个路易斯酸来参与反应，并吸取烯碳中间体的一个电子，导致碳氧键断裂，从而使焦磷酸基团离开HMBPP，为其催化机制的深入理解提供了理论模型。研究结果对于揭示微生物合成异戊二烯的分子及生理机制、推动生物基异戊二烯的合成具有重要理论及应用意义。