Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation

Abstract Background Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD+) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD+-dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Results Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD+/NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD+/NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD+/NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols. Conclusions The ratio of NAD+/NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production

Phylogenomic and Evolutionary Analyses Reveal Diversifications of SET-Domain Proteins in Fungi

In recent years, many publications have established histone lysine methylation as a central epigenetic modification in the regulation of chromatin and transcription. The histone lysine methyltransferases contain a conserved SET domain and are widely distributed in various organisms. However, a comprehensive study on the origin and diversification of the SET-domain-containing genes in fungi has not been conducted. In this study, a total of 3816 SET-domain-containing genes, which were identified and characterized using HmmSearch from 229 whole genomes sequenced fungal species, were used to ascertain their evolution and diversification in fungi. Using the CLANS program, all the SET-domain-containing genes were grouped into three main clusters, and each cluster contains several groups. Domain organization analysis showed that genes belonging to the same group have similar sequence structures. In contrast, different groups process domain organizations or locations differently, suggesting the SET-domain-containing genes belonging to different groups may have obtained distinctive regulatory mechanisms during their evolution. These genes that conduct the histone methylations (such as H3K4me, H3K9me, H3K27me, H4K20me, H3K36me) are mainly grouped into Cluster 1 while the other genes grouped into Clusters 2 and 3 are still functionally undetermined. Our results also showed that numerous gene duplication and loss events have happened during the evolution of those fungal SET-domain-containing proteins. Our results provide novel insights into the roles of SET-domain genes in fungal evolution and pave a fundamental path to further understanding the epigenetic basis of gene regulation in fungi