Microbial Engineering Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India.
DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnologygrid.425195.e, New Delhi, India.
Appl Environ Microbiol. 2021 Nov 10;87(23):e0185521. doi: 10.1128/AEM.01855-21. Epub 2021 Sep 29.
Furfural is a common furan inhibitor formed due to dehydration of pentose sugars, like xylose, and acts as an inhibitor of microbial metabolism. Overexpression of NADH-specific FucO and deletion of NADPH-specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in Escherichia coli, which highlights the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified the gene as an overexpression target to confer tolerance against furfural. YghA preferably used NADH as a cofactor and had an apparent value of 0.03 mM against furfural. In the presence of 1 g liter furfural and 10% xylose (wt/vol), overexpression in an ethanologenic E. coli strain SSK42 resulted in an ethanol efficiency of ∼97%, with a 5.3-fold increase in ethanol titers compared to the control. YghA also exhibited activity against the less toxic inhibitor 5-hydroxymethyl furfural, which is formed due to dehydration of hexose sugars, and thus is a formidable target for overexpression in ethanologenic strain for fermentation of sugars in biomass hydrolysate. Lignocellulosic biomass represents an inexhaustible source of carbon for second-generation biofuels. Thermo-acidic pretreatment of biomass is performed to loosen the lignocellulosic fibers and make the carbon bioavailable for microbial metabolism. The pretreatment process also results in the formation of inhibitors that inhibit microbial metabolism and increase production costs. Furfural is a potent furan inhibitor that increases the toxicity of other inhibitors present in the hydrolysate. Thus, it is desirable to engineer furfural tolerance in E. coli for efficient fermentation of hydrolysate sugars.
糠醛是戊糖（如木糖）脱水形成的常见呋喃抑制剂，作为微生物代谢的抑制剂。过去，过表达 NADH 特异性 FucO 和缺失 NADPH 特异性 YqhD 是赋予大肠杆菌对糠醛耐受性的成功策略，这突出了氧化还原酶在赋予糠醛耐受性方面的重要性。在一个包含各种氧化还原酶、脱氢酶和还原酶的筛选中，我们确定了基因是赋予对糠醛耐受性的过表达靶标。YghA 更喜欢 NADH 作为辅助因子，对糠醛的表观值为 0.03 mM。在 1 g/L 糠醛和 10%木糖（wt/vol）存在下，在产乙醇大肠杆菌菌株 SSK42 中过表达，导致乙醇效率约为 97%，与对照相比，乙醇浓度提高了 5.3 倍。YghA 还对毒性较小的抑制剂 5-羟甲基糠醛表现出活性，5-羟甲基糠醛是由己糖脱水形成的，因此是在产乙醇菌株中过表达以发酵生物质水解液中糖的有吸引力的目标。木质纤维素生物质代表了第二代生物燃料的取之不尽的碳源。对生物质进行热酸性预处理是为了疏松木质纤维素纤维，并使碳可用于微生物代谢。预处理过程还会形成抑制剂，抑制微生物代谢并增加生产成本。糠醛是一种有效的呋喃抑制剂，会增加水解液中其他抑制剂的毒性。因此，在大肠杆菌中进行工程改造以获得糠醛耐受性，从而有效地发酵水解液糖是可取的。
