Department of Biology, University of York, York YO10 5DD, U.K.
Technology Facility, Department of Biology, University of York, York YO10 5DD, U.K.
Biochem J. 2022 May 27;479(10):1045-1058. doi: 10.1042/BCJ20210811.
While lignocellulose is a promising source of renewable sugars for microbial fermentations, the presence of inhibitory compounds in typical lignocellulosic feedstocks, such as furfural, has hindered their utilisation. In Escherichia coli, a major route of furfural toxicity is the depletion of NADPH pools due to its use as a substrate by the YqhD enzyme that reduces furfural to its less toxic alcohol form. Here, we examine the potential of exploiting benzyl alcohol dehydrogenases as an alternative means to provide this same catalytic function but using the more abundant reductant NADH, as a strategy to increase the capacity for furfural removal. We determine the biochemical properties of three of these enzymes, from Pseudomonas putida, Acinetobacter calcoaceticus, and Burkholderia ambifaria, which all demonstrate furfural reductase activity. Furthermore, we show that the P. putida and B. ambifaria enzymes are able to provide substantial increases in furfural tolerance in vivo, by allowing more rapid conversion to furfuryl alcohol and resumption of growth. The study demonstrates that methods to seek alternative cofactor dependent enzymes can improve the intrinsic robustness of microbial chassis to feedstock inhibitors.
虽然木质纤维素是微生物发酵中可再生糖的有前途的来源，但在典型的木质纤维素饲料中存在抑制化合物，如糠醛，这阻碍了它们的利用。在大肠杆菌中，糠醛毒性的主要途径是由于 YqhD 酶将糠醛还原为毒性较小的醇形式而用作底物，导致 NADPH 池耗竭。在这里，我们研究了利用苯甲醇脱氢酶作为替代方法提供相同催化功能的潜力，但使用更丰富的还原剂 NADH，作为增加糠醛去除能力的策略。我们确定了来自假单胞菌、醋酸钙不动杆菌和伯克霍尔德菌的三种酶的生化特性，它们都表现出糠醛还原酶活性。此外，我们表明，通过允许更快速地转化为糠醇和恢复生长，来自假单胞菌和伯克霍尔德菌的酶能够在体内提供对糠醛的耐受性的显著提高。该研究表明，寻找替代辅因子依赖酶的方法可以提高微生物底盘对原料抑制剂的内在鲁棒性。
