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
Ke Xia,Cui Jia-Hao,Ren Qi-Jie,Zheng Tong,Wang Xin-Xin,Liu Zhi-Qiang,Zheng Yu-Guo
Abstract Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space–time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal . Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space–time yield of 4.22 g·L −1 ·day −1 . The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. Key points • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration.
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