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首页> 外文期刊>ACS Synthetic Biology >Enhancing Tolerance to Short-Chain Alcohols by Engineering the Escherichia coil AcrB Efflux Pump to Secrete the Non-native Substrate n-Butanol
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Enhancing Tolerance to Short-Chain Alcohols by Engineering the Escherichia coil AcrB Efflux Pump to Secrete the Non-native Substrate n-Butanol

机译：通过设计大肠杆菌盘管AcrB外排泵以分泌非天然底物正丁醇来提高对短链醇的耐受性

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摘要

The microbial conversion of sugars to fuels is a promising technology, but the byproducts of biomass pretreatment processes and the fuels themselves are often toxic at industrially relevant levels. One promising solution to these problems is to engineer efflux pumps to secrete fuels and inhibitory chemicals from the cell, increasing microbial tolerance and enabling higher fuel titer. Toward that end, we used a directed evolution strategy to generate variants of the Escherichia coli AcrB efflux pump that act on the non-native substrate n-butanol, enhancing growth rates of E. coli in the presence of this biofuel by up to 25%. Furthermore, these variants confer improved tolerance to isobutanol and straight-chain alcohols up to n-heptanol. Single amino acid changes in AcrB responsible for this phenotype were identified. We have also shown that both the chemical and genetic inactivation of pump activity eliminate the tolerance conferred by AcrB pump variants, supporting our assertion that the variants secrete the non-native substrates. This strategy can be applied to create an array of efflux pumps that modulate the intracellular concentrations of small molecules of interest to microbial fuel and chemical production.
机译：从糖类到燃料的微生物转化是一种有前途的技术，但是生物量预处理过程的副产品和燃料本身在工业上通常具有毒性。解决这些问题的一种有希望的解决方案是设计外排泵，以从电池中分泌燃料和抑制性化学物质，从而提高微生物的耐受性并实现更高的燃料滴度。为此，我们使用了定向进化策略来生成作用于非天然底物正丁醇的大肠杆菌AcrB外排泵的变体，从而在存在这种生物燃料的情况下将大肠杆菌的生长速率提高了25％ 。此外，这些变体提高了对异丁醇和直链醇直至正庚醇的耐受性。鉴定了导致该表型的AcrB中的单个氨基酸变化。我们还表明，泵活动的化学和遗传失活都消除了AcrB泵变异体赋予的耐受性，支持了我们的观点，即变异体分泌非天然底物。可以应用此策略来创建外排泵阵列，这些外排泵可调节感兴趣的小分子在细胞内的浓度，以产生微生物燃料和化学物质。

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