Improving microbial biogasoline production in Escherichia coli using tolerance engineering.
Improving microbial biogasoline production in Escherichia coli using tolerance engineering.
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DOI：
10.1128/mbio.01932-14
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发表时间：
2014-11-04
期刊：
影响因子：
6.4
通讯作者：
Mukhopadhyay A
中科院分区：
文献类型：
--
作者：
Foo JL;Jensen HM;Dahl RH;George K;Keasling JD;Lee TS;Leong S;Mukhopadhyay A
工程化微生物宿主用于生产可替代燃料需要减轻对生产能力造成的限制。一个这样的限制来自于溶剂样生物燃料化合物对生产菌株（例如大肠杆菌）的固有毒性。在这里，我们通过研究外源异戊烯醇上调基因的过表达来显示宿主工程对短链醇生产的重要性。使用系统生物学数据，我们选择了40个基因，这些基因在异戊烯醇暴露后上调，随后在大肠杆菌中过表达。这些候选人中的几个的过表达提高了对外源添加的异戊烯醇的耐受性。赋予异戊烯醇耐受性表型的基因属于不同的功能组，如氧化应激反应（soxS、fpr和nrdH）、一般应激反应（metR、yqhD和gidB）、热休克相关反应（ibpA）和运输（mdlB）。为了确定这些基因是否也可以提高异戊烯醇的生产，我们单独与异戊烯醇生产途径共表达的耐受性增强基因。我们的数据显示，8种候选物中的6种的表达提高了大肠杆菌中异戊烯醇的生产，其中甲硫氨酸生物合成调节剂MetR将异戊烯醇生产的滴度提高了55%。此外，MdlB，ABC转运蛋白的表达，促进了12%的异戊烯醇生产的改善。据我们所知，MdlB是可用于改善短链醇生产的转运蛋白的第一个例子，并为乙酰胆碱生产中的宿主工程提供了有价值的新途径。利用微生物宿主平台生产大宗商品，如化学品和燃料，现在是许多生物技术工作的重点。这些化合物中的许多对宿主微生物具有固有的毒性，这反过来限制了生产，尽管努力优化生物转化途径。为了达到经济上可行的生产水平，还需要工程化生产菌株，使其对这些化合物具有改进的耐受性。我们证明，使用转录组学数据的微生物耐受性工程也可以确定提高产量的目标。我们的研究结果包括一个出口商和甲硫氨酸生物合成调节剂，提高异戊烯醇生产，提供了一个起点，以进一步工程化的主机生产乙酰胆碱。
Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.