Abstract
BackgroundBiohydrogen 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Biohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness
Metabolic pathways for microbial hydrogen synthesis vary with microbial species, but can be classified into three types: (1) the mixed-acid fermentation pathway of facultative anaerobes represented by Enterobacter; (2) the butyric acid fermentation pathway of obligate anaerobic bacteria represented by Clostridium; and (3) the NADH regeneration pathway [17,18,19,20,21], the schematic diagram of the three metabolic pathways for fermentative hydrogen production from glucose is represented as Additional file 1: Fig. S1
Analysis and overexpression of the formate hydrogen lyase activator (fhlA) gene in E. cloacae WL1318 The full-length sequence of fhlA (2058 bp) was successfully cloned using the primer pair, fhlA-fw and fhlA-rv, which encoded a peptide of 685 amino acids with a calculated molecular mass of ~ 77 kDa and a predicted pI of 5.78, the nucleotide sequence and amino acid sequence are illustrated in Additional file 1: Figs
Summary
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. Little work has been reported in terms of cloning and expressing the fhlA gene in E. cloacae for lignocellulose-based hydrogen production. Biohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness [10,11,12,13]. Research on the FHL complex in other species of the Enterobacter genus is still limited; only a few studies have reported fhlA gene expression in Enterobacter aerogenes [24, 25], Enterobacter sp. CN1 [26] and Klebsiella HQ-3 [27], whereas the fhlA gene of Enterobacter cloacae and its expression have not yet been reported
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