Biohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness. Overexpression of the formate hydrogen lyase activator (fhlA) gene is a promising tactic for enhancement of hydrogen production in facultative anaerobic Enterobacter. As a species of Enterobacter, Enterobacter cloacae was reported as a highly efficient hydrogen-producing bacterium. However, little work has been reported in terms of cloning and expressing the fhlA gene in E. cloacae for lignocellulose-based hydrogen production. In this study, the formate hydrogen lyase activator (fhlA) gene was cloned and overexpressed in Enterobacter cloacae WL1318. We found that the recombinant strain significantly enhanced cumulative hydrogen production by 188% following fermentation of cotton stalk hydrolysate for 24 h, and maintained improved production above 30% throughout the fermentation process compared to the wild strain. Accordingly, overexpression of the fhlA gene resulted in an enhanced hydrogen production potential (P) and maximum hydrogen production rate (Rm), as well as a shortened lag phase time (λ) for the recombinant strain. Additionally, the recombinant strain also displayed improved glucose (12%) and xylose (3.4%) consumption and hydrogen yield Y(H2/S) (37.0%) compared to the wild strain. Moreover, the metabolites and specific enzyme profiles demonstrated that reduced flux in the competitive branch, including succinic, acetic, and lactic acids, and ethanol generation, coupled with increased flux in the pyruvate node and formate splitting branch, benefited hydrogen synthesis. The results conclusively prove that overexpression of fhlA gene in E. cloacae WL1318 can effectively enhance the hydrogen production from cotton stalk hydrolysate, and reduce the metabolic flux in the competitive branch. It is the first attempt to engineer the fhlA gene in the hydrogen-producing bacterium E. cloacae. This work provides a highly efficient engineered bacterium for biohydrogen production from fermentation of lignocellulosic hydrolysate in the future.

中文翻译：

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通过甲酸氢裂解酶激活基因的过表达增强了阴沟肠杆菌 WL1318 棉秆水解产物的生物氢产量。

木质纤维素制氢以其多样性、可再生性和廉价性等优点成为重要的制氢方法。甲酸氢裂解酶激活剂 (fhlA) 基因的过表达是增强兼性厌氧肠杆菌产氢的一种很有前景的策略。作为肠杆菌属的一种，阴沟肠杆菌据报道是一种高效产氢细菌。然而，关于在阴沟肠杆菌中克隆和表达 fhlA 基因以生产基于木质纤维素的氢气方面的工作报道很少。在这项研究中，甲酸氢裂解酶激活剂 (fhlA) 基因被克隆并在阴沟肠杆菌 WL1318 中过表达。我们发现重组菌株在棉秆水解物发酵 24 小时后，累积产氢量显着提高了 188%，在整个发酵过程中，与野生菌株相比，产量保持在 30% 以上。因此，fhlA 基因的过表达导致重组菌株的产氢潜力 (P) 和最大产氢速率 (Rm) 增强，以及滞后期时间 (λ) 缩短。此外，与野生菌株相比，重组菌株还显示出改善的葡萄糖 (12%) 和木糖 (3.4%) 消耗和氢气产量 Y(H2/S) (37.0%)。此外，代谢物和特定酶谱表明，竞争分支（包括琥珀酸、乙酸和乳酸）和乙醇生成的通量减少，加上丙酮酸节点和甲酸分裂分支的通量增加，有利于氢合成。结果最终证明了大肠杆菌中 fhlA 基因的过表达。cloacae WL1318能有效提高棉秆水解液的产氢量，降低竞争分支的代谢通量。这是在产氢细菌阴沟肠杆菌中改造 fhlA 基因的首次尝试。这项工作为未来从木质纤维素水解物发酵生产生物氢提供了一种高效的工程菌。