Green manufacture of steroid precursors from diosgenin by microbial replacing multistep chemical synthesis has been elusive. It is currently limited by the lack of strain and degradation mechanisms. Here, we demonstrated the feasibility of this process using a novel strain Mycolicibacterium sp. HK-90 with efficiency in diosgenin degradation. Diosgenin degradation by strain HK-90 involves the selective removal of 5,6-spiroketal structure, followed by the oxygenolytic cleavage of steroid nuclei. Bioinformatic analyses revealed the presence of two complete steroid catabolic gene clusters, SCG-1 and SCG-2, in the genome of strain HK-90. SCG-1 cluster was found to be involved in classic phytosterols or cholesterol catabolic pathway through the deletion of key kstD1 gene, which promoted the mutant m-∆kstD1 converting phytosterols to intermediate 9α-hydroxyandrostenedione (9-OHAD). Most impressively, global transcriptomics and characterization of key genes suggested SCG-2 as a potential gene cluster encoding diosgenin degradation. The gene inactivation of kstD2 in SCG-2 resulted in the conversion of diosgenin to 9-OHAD and 9,16-dihydroxy-pregn-4-ene-3,20-dione (9,16-(OH) 2 -PG) in mutant m-ΔkstD2 . Moreover, the engineered strain mHust-ΔkstD1,2,3 with a triple deletion of kstDs was constructed, which can stably accumulate 9-OHAD by metabolizing phytosterols, and accumulate 9-OHAD and 9,16-(OH) 2 -PG from diosgenin. Diosgenin catabolism in strain mHust-ΔkstD1,2,3 was revealed as a progression through diosgenone, 9,16-(OH) 2 -PG, and 9-OHAD to 9α-hydroxytestosterone (9-OHTS). So far, this work is the first report on genetically engineered strain metabolizing diosgenin to produce 21-carbon and 19-carbon steroids. This study presents a promising biosynthetic platform for the green production of steroid precursors, and provide insights into the complex biochemical mechanism of diosgenin catabolism.
Abstract Water–oil interface of bacterial cell-stabilized Pickering emulsions is an exceptional habitat for microbial assimilation of both hydrophobic nutrients solubilized in oil phase and hydrophilic ones solubilized in water phase. Crystal substrate inhibition, i.e., decreasing phytosterol degradation with the increase loading of crystal phytosterols, is always observed during microbial transformation of phytosterols into steroid synthons in Mycolicibacterium sp (China Center of Industrial Culture Collection, CICC 21,097) cell-stabilized Pickering emulsions. In the present work, we confirmed that crystal substrate inhibition was attributed to the interaction between M. neoaurum and phytosterol crystals that led to the detachment of bacterial cells from the oil–water interfaces in bacterial cell-stabilized Pickering emulsions. Under the selected operation condition (25 ml BEHP per 40 ml water, 60 g/L glucose, 25 g/L phytosterols), the product androst-4-ene-3, 17-dione (AD) and androsta-1, 4-dien-3, 17-dione (ADD) concentration increased linearly with the progress of microbial transformation and reached almost 6 g/L at the 11 th day. This is a paradigm for microbial transformation of crystal substrates as well as in the presence of other surface active additives (such as chitosan and nonionic surfactants) in bacterial cell-stabilized Pickering emulsions. Key points • Microbial transformation of crystal phytosterols in Pickering emulsions • Crystal substrate inhibition occurring during microbial transformation • Interaction between phytosterol crystals and bacterial cells leading to demulsification
危险品化学品经营许可证（带存储）