Mining And Engineering Of Biological Parts And Their Application In Gene Expression Regulation System

Synthetic biology greatly promotes the development of life science by constructing artificial biological systems with new functions and characteristics.Biological parts such as promoters and regulators are the basis for artificial biological systems.With the rapid development of synthetic biology,the artificial biological system is more and more complex,and higher requirements are put forward for biological parts.The development of high-performance biological parts with more precise,more stable and new functions has become a research hotspot in synthetic biology.Therefore,this study deeply mined gene expression regulators,developed novel strategies for promoter engineering,and constructed novel promoters.Highly efficient expression system and heterogenous quorum-sensing system were developed through the adaptation of promoters and other regulators,which provided theoretical basis and technical support for the development of synthetic biology.The main results obtained in this study are as follows:（1）Development of semi-rational engineering strategy（Stepwise Evolution Targeting the Spacer Region of the Core Promoter,SETarSCoP）for the rapid engineering of promoter.The conservative difference of the sequences at the upstream of-10 box betweenσ^A dependent strong and weak promoters in Bacillus subtilis was revealed through mining and analyzing transcriptomics data.This indicated that the sequences at the upstream of-10 box could regulate the promoter activity.Accordingly,the SETarSCoP was developed through stepwise directed evolution of“3 bp+4 bp”sequences at the upstream of-10 box in the promoter PsrfA.In this way,the mutant BH4 with higher activity and robustness was obtained.The expression levels of GFP,glucuronidase GusA and nattokinase mediated by BH4 were 2.8,14.8and 1.3 times than that by wild type P_srfA,respectively.Furthermore,SETarSCoP was also successfully applied to engineer strong constitutive promoter PylbP and xylose induced promoter P_xylA,which confirmed its universality.（2）Improvement of the strength and stability of gene expression by constructing multipleσs recognizable artificial promoter.To break through the limitation of promoter performance in different growth phases caused by unique sigma factors（σs）,the multipleσs recognizable artificial hybrid promoter（AHP）was constructed in this study.First,the strong natural minimal promoter（NMP）PrpoB（σ^A dependent）,P_spoVG（σ^H dependent）and P_sigW（σ^W dependent）were identified by mining transcriptomics data.Then,the multipleσs recognizable AHP were constructed based on the permutation and combination of NMP.From the verification on both population level and single-cell level,AHP exhibited high activity in different growth phases and had higher compatibility with RBS and transcription factors.Further research indicated that the functional complementation among NMP was a crucial reason why AHP had higher activity.Based on these findings,inducible AHPs were developed for precise regulation of heterologous genes and complex genetic circuits.（3）Construction of cobalt-induced promoter and its application in the highly efficient production of nitrile hydratase.A cobalt-induced promoter was constructed based on the biological parts of the Co²⁺responsive rcn operon from Escherichia coli.And a cobalt-induced expression system（CIES）was further developed.After optimization,CIES could express the Heterologous gene with a low leaky level and high induction ratio.When GFP was employed as the reporter,500μM Co²⁺induced the highest gene expression level.By engineering the Co²⁺influx and efflux system,the utilization of Co²⁺was further enhanced,resulting in higher gene expression levels at the lower concentrations of Co²⁺.When CIES was used to express NHase,Co²⁺functioned as both inducer and metal-ligand of NHase,in which NHase was produced with high efficiency.These results indicated CIES had huge potential in industrial applications.（4）Construction of a heterogenous quorum-sensing system for cell growth-dependent dynamic regulation of gene expression in B.subtilis.A heterogenous quorum-sensing system（HQSS）was developed in B.subtilis through modular design and reconstruction of the AIP synthesis gene（agrD and agrB）,response gene（agrC and agrA）and effect promoter P3 of agr quorum-sensing system from Staphylococcus aureus.The HQSS could be tuned at both levels of turn-on time and expression strength by modular regulation.Besides,HQSS that sensitively responded 0.01 nM～1 nM AIP could be employed as the AIP biosensor.Series of AIP mutants were screened to finely tune the signal transduction,which expanded the dynamic regulation range of HQSS.Furthermore,the results of RNA-seq confirmed that HQSS had less crosstalk with endogenous genes and could play a stable role in B.subtilis.