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首页> 外文期刊>Applied Microbiology >Plasmidic Expression of nemA and yafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate
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Plasmidic Expression of nemA and yafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

机译：NemA和yafC *的质粒表达提高了产乙醇的大肠杆菌LY180对稀酸处理甘蔗渣和人工水解产物的非挥发性副产物的抗性

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Hydrolysate-resistant Escherichia coli SL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA: nemA ( N -ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region, yafC *. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region of nemR ( nemRA operon) in SL100. This mutation increased nemA activity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increased nemA mRNA levels >200-fold. Addition of hydrolysates induced nemA expression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressing nemA or yafC* was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affected furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction of N -ethylmaleimide by NemA while also serving as substrates. Expression of the nemA or yafC* plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate.
机译：在含有甘蔗渣稀酸水解产物的AM1培养基中依次转移后，先从产乙醇的LY180中分离出耐水解物的大肠杆菌SL100，并用作抗性基因的来源。先前已经描述了许多影响糠醛耐受性的基因，糠醛是最丰富的抑制剂。为了鉴定与糠醛以外的抑制剂相关的基因，在经过真空处理以去除糠醛的人工水解物中选择质粒克隆。从SL100基因组DNA的Sau3A1文库中发现了两个新的抗性基因：nemA（N-乙基马来酰亚胺还原酶）和一个推定的调控基因，在编码区yafC *中含有突变。通过测序证实了SL100中这些突变的存在。在SL100的nemR（nemRA操纵子）的上游调控区中发现了一个突变。在没有水解产物的情况下，这种突变使nemA活性比AM1培养基中的亲代生物（LY180）提高了20倍，并且使nemA mRNA水平提高了200倍以上。与转录控制一致，添加水解产物诱导的nemA表达（mRNA和活性）。 NemA活性在细胞提取物中稳定（9 h，37°C），从而消除了蛋白酶在调节中的作用。与具有空载体对照的LY180相比，具有表达nemA或yafC *的质粒的LY180对真空处理的甘蔗渣水解物和真空处理的人工水解物具有更高的抗性。这两个基因均不影响糠醛耐受性。真空处理的水解产物抑制了NemA对N-乙基马来酰亚胺的还原，同时还用作底物。 neLY或yafC *质粒在LY180中的表达使真空处理的甘蔗渣水解产物的乙醇生产速率提高了一倍。

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