Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together. A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose–xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~ 18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96 and 120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144 and 168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration. E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose–xylose mixture.

中文翻译：

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大肠杆菌中 pgi 基因的缺失增加了对含有葡萄糖-木糖混合物的培养基中的糠醛和 5-羟甲基糠醛的耐受性。


糠醛和 5-羟甲基糠醛 (5-HMF) 是关键的呋喃抑制剂，是由于木质纤维素糖在高温和酸性处理条件下分解而产生的。糠醛和 5-HMF 均以协同方式抑制微生物代谢，并且对两者的抗性是木质纤维素碳有效转化为乙醇的理想特性。针对增加细胞 NADPH 库的基因操作已成功赋予对糠醛和 5-HMF 的耐受性。在本研究中，在产乙醇大肠杆菌菌株 SSK101 中研究了删除 pgi 基因作为增加通过戊糖磷酸途径 (PPP) 碳流的策略，以赋予对糠醛或 5-HM 两种抑制剂的耐受性。在产乙醇大肠杆菌菌株 SSK42 中删除 EMP 途径的关键基因 pgi，产生菌株 SSK101。在基本 AM1 培养基中存在 1 g/L 糠醛的情况下，菌株 SSK101 的生物量形成率比亲本 SSK42 菌株高出 1.9 倍，并且能够在一半的时间内清除糠醛。对抑制剂的耐受性与作为碳源的葡萄糖而非木糖有关，并且 SSK101 的耐受性优势在 LB 培养基中被中和。生物反应器研究是在糠醛和 5-HMF（各 1 g/L）的二元应力和葡萄糖-木糖混合物中的不同葡萄糖浓度下进行的，最终糖浓度为 5.5%，模拟稀酸处理的生物质水解产物的主要成分。在具有6g/L和12g/L葡萄糖的混合物中，SSK101菌株分别产生～18g/L和20g/L乙醇。有趣的是，最大乙醇生产率在葡萄糖负荷较低时更好，为 0.46 g/(L.h) 96 至 120 小时之间，相比之下，144 至 168 小时之间葡萄糖负荷较高，为 0.33 g/(Lh)。重要的是，亲本菌株 SSK42 在类似的抑制剂负载和糖浓度条件下没有表现出显着的代谢活性。删除 pgi 的大肠杆菌菌株 SSK101 对糠醛和 5-HMF 的耐受性增强，这与培养基中葡萄糖的存在有关。在含有葡萄糖-木糖混合物的培养基中，在高渗以及糠醛和 5-HMF 的二元胁迫下，菌株 SSK101 也具有改善的发酵特性。