Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.
Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.
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DOI：
10.3389/fmicb.2015.00847
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发表时间：
2015
影响因子：
5.2
通讯作者：
Atomi H
中科院分区：
文献类型：
--
作者：
Kanai T;Simons JR;Tsukamoto R;Nakajima A;Omori Y;Matsuoka R;Beppu H;Imanaka T;Atomi H
极端嗜热古菌Thermococcus kodakarensis可以利用糖或丙酮酸盐生长。在不存在元素硫的情况下，通过这些底物的氧化的电子被质子接受，产生分子氢（H2）。负责该反应的氢化酶是膜结合[NiFe]-氢化酶（Mbh）。在这项研究中，我们已经研究了几种可能性，以增加蛋白水平的Mbh在T。通过基因工程的方法来控制柯达卡热病。当整个mbh操纵子的启动子（TK 2080-TK 2093）被交换为来自谷氨酸脱氢酶基因（TK 1431）（菌株MHG 1）的强组成型启动子时，实现了最高水平的细胞内Mbh水平。当在连续培养条件下使用基于柠檬酸盐的培养基培养MHG 1时，在0.31 h−1的稀释率下观察到比产氢速率（SHPR）高出近25%，为35.3 mmol H2 g-dcw−1 h− 1。我们还结合mbh过表达使用一个更强的组成型启动子从细胞表面糖蛋白基因（TK 0895）与破坏的基因编码的胞质氢化酶（Hyh）和丙氨酸氨基转移酶（AlaAT），这两者都参与氢消耗（株MAH 1）。在0.30 h-1的稀释率下，SHPR为36.2 mmol H2 g-dcw-1 h-1，与宿主T相比增加了28%。科达卡菌株将稀释速率增加到0.83 h−1或1.07 h−1导致SHPR为120 mmol H2 g-dcw−1 h−1，这是微生物发酵中观察到的最高生产速率之一。
The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw−1 h−1 was observed at a dilution rate of 0.31 h−1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h−1, the SHPR was 36.2 mmol H2 g-dcw−1 h−1, corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h−1 or 1.07 h−1 resulted in a SHPR of 120 mmol H2 g-dcw−1 h−1, which is one of the highest production rates observed in microbial fermentation.