Hydrogen with high energy content is considered to be a promising alternative clean energy source. Biohydrogen production through microbes provides a renewable and immense hydrogen supply by utilizing raw materials such as inexhaustible natural sunlight, water, and even organic waste, which is supposed to solve the two problems of “energy supply and environment protection” at the same time. Hydrogenases and nitrogenases are two classes of key enzymes involved in biohydrogen production and can be applied under different biological conditions. Both the research on enzymatic catalytic mechanisms and the innovations of enzymatic techniques are important and necessary for the application of biohydrogen production. In this review, we introduce the enzymatic structures related to biohydrogen production, summarize recent enzymatic and genetic engineering works to enhance hydrogen production, and describe the chemical efforts of novel synthetic artificial enzymes inspired by the two biocatalysts. Continual studies on the two types of enzymes in the future will further improve the efficiency of biohydrogen production and contribute to the economic feasibility of biohydrogen as an energy source.

Docosahexaenoic acid (DHA) is an essential ω-3 polyunsaturated fatty acid (PUFA) with high nutritional and pharmaceutical value. The marine protist Aurantiochytrium is a promising industrial DHA producer; however, its DHA biosynthesis via the PUFA synthase pathway co-produces ω-6 docosapentaenoic acid (DPA), limiting DHA purity. Here, we introduced an ω-3 desaturase from Phytophthora infestans (Pin-O3D) into Aurantiochytrium sp. SD116. Functional validation in an Escherichia coli system co-expressing the native PUFA synthase confirmed that Pin-O3D converts DPA to DHA, shifting the DHA/DPA ratio from 1:1 to 2:1. Pin-O3D was then integrated into the fatty acid synthase (FAS) locus, simultaneously attenuating FAS activity and enabling heterologous gene expression. The engineered strain ΔFAS-Pin-O3D exhibited significantly (p < 0.0001 in t-test) increased DHA content (55.2% of total fatty acids) and DHA/DPA ratio (5.91) in shake flasks, with no negative impact on biomass or lipid accumulation. Fed-batch fermentation confirmed the scalability of this strategy, achieving a >20% increase in DHA/DPA ratio. This study demonstrates that combining heterologous ω-3 desaturase expression with FAS attenuation is an effective approach for optimizing PUFA profiles in Aurantiochytrium.

In Clostridium thermocellum, there are nine RsgI factors responsible for sensing different types of substrates and regulating the transcription and expression of cellulosome genes. Within the signaling pathway of RsgI, the membrane protease RseP cleaves RsgI in its transmembrane helix, thus releasing the N-terminal fragment of RsgI from the membrane. This released RsgI N-terminal fragment is subsequently recognized and degraded by a cytoplasmic protease complex consisting of an AAA+ ATPase and ClpP protease. Previous research showed that the ClpXP complex, comprising ClpX and ClpP, is capable of recognizing and degrading the N-terminal fragment of RsgI6. However, due to the low conservation of the transmembrane helical region of RsgI, it remains unclear whether other RsgIs are similarly recognized and degraded by the same unfoldase. In this study, we employed in vitro protease assays to examine the recognition and degradation of the N-terminal fragment of each RsgI by various ClpP-unfoldase complexes. Results confirm that ClpXP is responsible for degrading the N-terminal fragments of all RsgI proteins in C. thermocellum, suggesting a degree of sequence promiscuity in substrate recognition by ClpXP. ClpXP can recognize multiple XAA sites in the transmembrane helix region of RsgI. Moreover, we unexpectedly discovered that the cytoplasmic domain influences the degradation of RsgI2-NF by ClpXP in our in vitro assay. This study provides new insights into understanding the complex regulatory mechanisms of cellulosome genes and the role of AAA+ proteases in C. thermocellum, thereby offering critical clues for unraveling the internal regulatory networks of bacteria.