The depletion of fossil fuels and the environmental problems caused by their use necessitate the urgent development of new and alternative environmentally friendly energy sources [1]. Standing out among new energy sources, hydrogen is a completely clean fuel because its combustion only produces water while offering high energy density [2, 3]. However, despite the enormous development potential of hydrogen energy, the production and utilization of hydrogen still faces many challenges. At present, hydrogen is mainly produced via the electrolysis of water and the high-temperature cracking of fossil fuels [4], both of which are not only energy-intensive and have low conversion rates, but also pollute the environment. In terms of economy and environmental protection, they are not suitable for long-term industrial hydrogen production [5]. Compared with traditional thermochemical hydrogen production technology, biotechnological hydrogen fermentation is a more promising hydrogen production process, which does not require harsh conditions and requires only inexpensive equipment [6, 7].
As a facultative anaerobe, Enterobacter aerogenes has strong adaptability to the environment and can produce hydrogen at a high conversion rate under strictly anaerobic conditions [8]. In addition, E. aerogenes also has the advantages of fast growth, wide substrate utilization range, easy cultivation and mature genetic modification methods. As a consequence, it is considered an ideal strain for large-scale biological hydrogen production. However, due to the incomplete metabolic process, the actual hydrogen production is far lower than the theoretical 4 mol H2/mol glucose. Therefore, understanding and optimizing the metabolic network of E. aerogenes is crucial for hydrogen production [9].
During dark fermentation for hydrogen production, E. aerogenes produces hydrogen through the formate pathway and the NADH pathway [7, 10]. In the formate pathway, pyruvate is converted by pyruvate formate lyase (pfl) to produce acetyl coenzyme A and formic acid. As the accumulation of formic acid leads to a decrease of extracellular pH, formic acid is oxidized to CO2 by formate dehydrogenase (FHL), and the generated electrons are transferred to hydrogenase, where they react with protons to form hydrogen. In theory, the maximum hydrogen conversion rate of the formic acid cleavage pathway is 2 mol H2/mol glucose [11]. In the NADH pathway, the hydrogen production process is mainly driven by the release of excess electrons or energy formed during anaerobic respiration. In this case, NADH mainly acts as an electron donor [12]. Therefore, if the distribution of NADH in E. aerogenes can be regulated, the hydrogen production level can be improved [13].
Redox reactions are the most common type of chemical reaction occurring in organisms, and it is also a common and extremely important reaction in biotransformations [14]. As intracellular cofactors, NADH and NAD+ not only affect the regulation of intracellular enzymes, but can also influence the rate of substrate conversion by organisms [15]. Cofactor engineering can effectively change the intracellular NADH or NAD+ status through the elimination of endogenous cofactor metabolic pathways, cofactor regeneration and competitive utilization, thus affecting the metabolic network, signal transduction and material transport.
As the key enzyme of hydrogen production in the NADH pathway, NADH dehydrogenase couples the oxidation of NADH with the generation of the proton motive force. A DNA probe prepared by PCR was used to identify its locus, and revealed that it was composed of 14 subunits, called nuoA-nuoN [16, 17]. If the L-shaped model is used to approximate Complex I, it can be found that it consists mainly of peripheral segments, connecting segments, and membrane-integrated segments. The peripheral segments encoded by the nuclear homologues nuoE, nuoF and nuoG can oxidize NADH to NAD+ [18, 19, 20]. Thus, if a single- or multigene knockout is performed on the peripheral segment, it is likely to destabilize the complete structure, thus changing the distribution of NADH.
Succinic acid as one of the main products of anaerobic metabolism in E. aerogenes. Its production will not only convert a large amount of NADH into NAD+ [21], but also cause the culture medium to gradually acidify, which ultimately affects the normal growth of the strain. In the dark fermentation process of E. aerogenes, succinic acid is mainly produced from fumaric acid, which in turn generated from malic acid. As the key gene of NAD+ dependent malic acid enzyme, the transcriptional level of maeA can effectively modulate the conversion of malate into pyruvate [22], thus reducing the production of succinic acid. In addition, the pyruvate generated by maeA can also be used for formic acid production, increasing the hydrogen production rate of the formate pathway. At the same time, a large amount of NADH will be generated during the conversion of malic acid into pyruvate [23], which also lays a solid foundation for improving the hydrogen yield of the NADH pathway.
In this study, E. aerogenes IAM1183 was used as the starting strain. The nuoG, nuoEF and nuoEFG genes were knocked out by λ-Red recombination to create single- and multigene mutants, so as to compare and explore the effect of the deletion of nuoG on the overall hydrogen production of the strain. The effect of nuoG deletion on the hydrogen production through the NADH pathway was assessed by knocking out the pfl gene encoding pyruvate formate lyase, a key enzyme of the formate pathway. In addition, to further regulate anaerobic metabolism and enhance hydrogen production, In addition, the maeA gene encoding NAD+-dependent malic enzyme from Escherichia coli was cloned and heterologously expressed in E. aerogenes IAM1183 and its mutants to regulate intracellular NADH/NAD+ and promote hydrogen production through both the formate and NADH pathways. The strain with the highest hydrogen production was then tested in bioreactor scale-up to assess its potential for industrial production. These results further validate the effect of the nuoG and maeA genes on hydrogen production, and how the regulation of the NADH/NAD+ pool affects the metabolism of hydrogen-producing bacteria.