Phospholipase A1(PLA1)is a kind of specific phospholipid hydrolase widely used in food,medical,textile.However,limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-...Phospholipase A1(PLA1)is a kind of specific phospholipid hydrolase widely used in food,medical,textile.However,limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS.In this study,we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation.Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells,resulting in improved cell growth and increased protein solubility of the protein.The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS(ΔN27 PlaS),highlighting their binding capabilities.Furthermore,in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 andΔN27 PlaS,exhibiting high affinity.Molecular docking analysis provides insights into the hydrogen bond interactions betweenΔN27 PlaS and PlaA1,identifying key amino acid residues crucial for their binding.Finally,the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test.Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1,offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.展开更多
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 c...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.展开更多
Corynebacterium glutamicum is a safe strain with great potential for industrial applications,but more research is needed on secretory expression systems.Here,we constructed a non-inducible secretory expression system ...Corynebacterium glutamicum is a safe strain with great potential for industrial applications,but more research is needed on secretory expression systems.Here,we constructed a non-inducible secretory expression system of the strain.By building a signal peptide library,we screened several Sce-type signal peptides and analyzed the relationship between their constitutive properties and secretory efficiency.To further meet the safety requirements in industrial applications,fifteen constitutive promoters were screened,and protein expression was optimized by promoter tandem strategy.In the WYJ1,WYJ2,WYJ3,and WYJ4 engineering strains,we confirmed that the modification of cell permeability favored protein secretion.The engineering strains WYJ2P35SP35 and WYJ4P35SP35 were scaled up for culture,and their extracellular enzyme activities and proteins reached 26.42 U/mL and 19.65 mg/L,and 23.97 U/mL and 13.84 mg/L,respectively.This secretory expression system increases the potential of industrial applications of Corynebacterium glutamicum and lays the foundation for applications.展开更多
基金financially supported by National Natural Science Foundations of China(No.31471615)Scientific Research Start-up Fund for Introduced Talents of Anhui Polytechnic University(2022YOO068)Natural Science Research Project of Colleges and Universities in Anhui Province(Grant 2022AH050971).
文摘Phospholipase A1(PLA1)is a kind of specific phospholipid hydrolase widely used in food,medical,textile.However,limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS.In this study,we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation.Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells,resulting in improved cell growth and increased protein solubility of the protein.The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS(ΔN27 PlaS),highlighting their binding capabilities.Furthermore,in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 andΔN27 PlaS,exhibiting high affinity.Molecular docking analysis provides insights into the hydrogen bond interactions betweenΔN27 PlaS and PlaA1,identifying key amino acid residues crucial for their binding.Finally,the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test.Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1,offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.
基金supported by the Scientific Research Foundation of the Higher Education Institutions of Anhui Province,China(Grant No.2022AH050971)Key R&D and Achievement Transformation Projects(2023yf096)the Scientific Research Start-up Fund for Talents of Anhui Polytechnic University(2022YQQ064).
文摘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.
基金supported by the National Key Research and Development Program of China(No.2021YFC2100900)the National Natural Science Foundation of China(No.32171471)+1 种基金the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX24_2586)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘Corynebacterium glutamicum is a safe strain with great potential for industrial applications,but more research is needed on secretory expression systems.Here,we constructed a non-inducible secretory expression system of the strain.By building a signal peptide library,we screened several Sce-type signal peptides and analyzed the relationship between their constitutive properties and secretory efficiency.To further meet the safety requirements in industrial applications,fifteen constitutive promoters were screened,and protein expression was optimized by promoter tandem strategy.In the WYJ1,WYJ2,WYJ3,and WYJ4 engineering strains,we confirmed that the modification of cell permeability favored protein secretion.The engineering strains WYJ2P35SP35 and WYJ4P35SP35 were scaled up for culture,and their extracellular enzyme activities and proteins reached 26.42 U/mL and 19.65 mg/L,and 23.97 U/mL and 13.84 mg/L,respectively.This secretory expression system increases the potential of industrial applications of Corynebacterium glutamicum and lays the foundation for applications.
