3-Ketosteroid-Δ1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modifications of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identified in Mycobacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplified their potential application for industrial steroid transformation. The recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-dione (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specific activities of 22.40 and 19.19 U mg−1, respectively. However, the specific activities of recombinant KstD2 from Escherichia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had significantly lower specific activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) from 8 g L−1 AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the substrate, while the mRNA levels of kstD2 and kstD3 were bearly affected by the phytosterols. Therefore, we attempted to create a 4HP producing strain without kstD1, which could covert 20 g L−1 phytosterols to 14.18 g L−1 4HP. In vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the findings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ΔkstD1 strain for 4HP production using phytosterols as the substrate.

The present study investigated the effects of bedding material (BM) waste on physicochemical properties, organic matter (OM) degradation, microbial community structure and metabolic function during composting. The results showed that bedding material (CK-0, S1-40%, S2-25%) optimized the composting conditions for lignocellulose and OM biodegradation. The highest OM degradation and humic substance (HS) synthesis rates were observed in the 40% BM addition group. Firmicutes was more abundant in the bedding material addition groups, whereas Proteobacteria was more abundant in the group without bedding material. Functional prediction showed higher carbohydrate and amino acid metabolism in the BM groups than that in control group. Animal and plant pathogens were almost eliminated, and saprotrophs were the dominant fungal trophic modes after 40% BM addition composting. Cellulose, hemicellulose, and organic matter had strong associations with microbial communities, such as Lysinibacillus and Corynebacterium (bacteria), compared to the associations of Aspergillus, Candida, and Sordariomycetes (fungi) (p value < 0.05). Network analysis revealed closer microbial community interactions in 40% BM addition group than in other groups. These findings provide detailed information about the coupling of material conversion, of bacterial and fungal succession during composting, and that bedding materials waste can also be used as an effective compost amendment.