Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and ß-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.

[Microbial Community Succession in Industrial Composting with Livestock Manure and Peach Branches and Relations with Environmental Factors].

This study sets out to understand the evolution of the microbial community structure in industrial composting with livestock manure and peach branches. Pig manure, peach branches, and decomposed organic fertilizer were used as materials for composting. Changes in physical and chemical indicators and the evolution in the structure of the compost microbial community, determined by high-throughput sequencing, were analyzed. The results of physical and chemical parameters show that the pile reached the high-temperature stage on day 2, and the thermophilic period lasted for 30 days. The changes in total carbon were volatile, and there was an overall decline in the amount of TOC in the whole process of composting; The final content of TN was 20.58 g·kg-1, which was 5.90% lower compared to the initial compost. Alpha analysis indicated that a different microbial community diversity existed at different times during aerobic composting periods. At the bacterial phyla level, Firmicutes and Actinobacteria were the dominant phyla, and the proportion of relative abundance were 79.31%-95.09% and 2.98%-19.70%, respectively, in the entire compost. The relative abundance of Firmicutes and Actinobacteria were 87.36% and 9.66%, respectively, and their respective relative abundances were 79.38% and 19.70% at the end of composting. At the bacterial genus level, the dominant group changed from Clostridium_sensu_stricto_1, Terrisporobacter, and Bacillus to norank_f_Bacillaceae, Bacillus, Oceanbacillus, and Pseudogracilibacillus; Regarding the fungus phyla, the Ascomycota was the dominant phylum. For the fungus genus, the relative abundance of norank_c_Sordariomycetes gradually increased during composting, and finally was predominant group. The redundancy analysis (RDA) showed that the correlation rank between environmental factors and microbial community structure was:pH > NH4+-N > T > TOC > TN, where pH had the greatest impact on the microbial community composition. norank_c_Sordariomycetes, norank_o_Sordariales, and norank_c_Agaricomycetes may be related to the volatilization of ammonium nitrogen.

Purification, characterization, and application of a high activity 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum DSM 1381.

Δ1-Dehydrogenation is one of the most important reactions for steroid drug modification. Numerous 3-ketosteroid-Δ1-dehydrogenases (KstDs) catalyzing this reaction were observed in various organisms. However, only a few have been characterized and used for substrate conversion. In this study, a promising enzyme (KstD2) from Mycobacterium neoaurum DSM 1381 was purified and characterized. Interestingly, KstD2 displayed a high activity on a range of substrates, including 17α-hydroxypregn-4-ene-3,20-dione (17α-OH-P); androsta-4,9(11)-diene-3,17-dione (NSC 44826); and 4-androstene-3,17-dione (AD). These reactions were performed under optimal conditions at 40 °C and pH 8.0. Noteworthy, both the activity and stability of the enzyme were sensitive to various metal ions. After optimizing the expression and biocatalyst conditions, up to 1586 U mg-1 intracellular KstD activity on AD could be produced. Furthermore, the associated conversion rate was 99% with 30 g L-1 AD after 8 h. On the other hand, we obtained 99%, 90%, and over 80% of conversion with 20 g L-1 NSC 44826; 10 g L-1 16,17α-epoxyprogesterone; and 20 g L-1 17α-OH-P or canrenone, respectively, after 24 h. Sequence homology and structural analyses indicated that the residue R178 located in a unique short loop among cluster 2 is crucial for substrate recognition which was confirmed by mutagenesis. In summary, this study reports on the first purification and characterization of a KstD from cluster 2. Its remarkable properties deserve more attention to potentially lead to further industrial applications.