Ming Zhao,Mengkai Hu,Rumeng Han,Chao Ye,Xiangfei Li,Tianwen Wang,Yan Liu,Zhenglian Xue,Kun Liu
The production of androst-4-ene-3,17-dione (AD) by the steroidal microbial cell factory requires transcription factors (TFs) to participate in metabolic regulation. However, microbial cell factory lacks effective TFs that can respond to AD in its metabolic pathway. Additionally, finding and obtaining natural TFs that specifically respond to AD is a complex and onerous task. In this study, we devised an artificial TF that responds to AD, termed AdT, based on structure-guided molecular dynamics (MD) simulation. According to MD analysis of the conformational changes of AdT after binding to AD, an LBD in which the N - and C -termini exhibited convergence tendencies was used as a microswitch to guide the assembly of a DNA-binding domain lexA, a linker (GGGGS) 2 , and a transcription activation domain B42 into an artificial TF. As a proof of design, a AD biosensor was designed and constructed in yeast on the basis of the ligand-binding domain (LBD) of hormone receptor. In addition, the transcription factor activity of AdT was increased by 1.44-fold for its variant F320Y. Overall, we created non-natural TF elements for AD microbial cell factory, and expected that the design TF strategy will be applied to running in parallel to the signaling machinery of the host cell.
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）