Improved furfural tolerance in Escherichia coli mediated by heterologous NADH-dependent benzyl alcohol dehydrogenases.
Improved furfural tolerance in Escherichia coli mediated by heterologous NADH-dependent benzyl alcohol dehydrogenases.
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DOI：
10.1042/bcj20210811
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发表时间：
2022-05-27
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虽然木质纤维素是用于微生物发酵的可再生糖的有前途的来源，但是典型的木质纤维素原料中抑制性化合物（例如糠醛）的存在阻碍了它们的利用。在大肠杆菌中，糠醛毒性的主要途径是NADPH池的消耗，这是由于其被YqhD酶用作底物，将糠醛还原为其毒性较低的醇形式。在这里，我们研究了利用苯甲醇脱氢酶作为替代手段来提供相同的催化功能的潜力，但使用更丰富的还原剂NADH，作为增加糠醛去除能力的策略。我们确定了三个这些酶的生化特性，从恶臭假单胞菌，醋酸钙不动杆菌，和伯克霍尔德氏菌ambifaria，这都表明糠醛还原酶活性。此外，我们还发现恶臭假单胞菌和B. ambifaria酶能够通过允许更快速地转化为糠醇并恢复生长而提供体内糠醛耐受性的显著增加。该研究表明，寻找替代辅因子依赖性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。
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.