Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout
Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout
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DOI：
10.1128/jb.00497-09
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发表时间：
2009-10-15
影响因子：
3.2
通讯作者：
Lynd, Lee R.
中科院分区：
文献类型：
--
作者：
Shaw, A. Joe;Hogsett, David A.;Lynd, Lee R.
在遗传、mRNA、酶和表型水平上研究了热厌氧菌糖溶菌中三个假定的氢化酶系统。一个包含两个[FeFe]-氢化酶基因的四基因操纵子，暂时称为hfs（氢化酶- fe - s），被发现是产氢的主要酶促催化剂。hfsB可能是操纵子中最有趣的基因，它包含一个[FeFe]-氢化酶和一个PAS感觉结构域，并且在梭状菌的糖解菌、纤维素解菌和致病菌中有几个保守的同源物。第二个氢化酶基因簇hyd表现出甲基viologen-linked hydrogenase酶活性，但hyd基因敲除不影响封闭系统批量发酵培养物的产氢量。这一结果，结合hydB含有NAD(P)+和FMN结合位点的观察，表明hyd基因对电子从NAD(P) H转移到氢离子具有特异性。第三个基因簇，假定的[NiFe]-氢化酶与每一个基因同源，在任何测试条件下都没有表现出氢化酶活性。hfs和hydA基因的缺失导致甲基紫原连接的氢化酶活性的丧失。缺失hfs基因的菌株氢和乙酸的产量降低了95%。一株hfs和ldh缺失的菌株从消耗的碳水化合物中增加了乙醇产量，这代表了一种新的工程策略来提高糖酵母菌的乙醇产量。
Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P) H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum.