Corynebacterium stationis,a high-GC Gram-positive bacterium with significant industrial potential,has faced limitations due to the lack of efficient genetic tools.In this study,we developed a CRISPR/Cas9-based genome ...Corynebacterium stationis,a high-GC Gram-positive bacterium with significant industrial potential,has faced limitations due to the lack of efficient genetic tools.In this study,we developed a CRISPR/Cas9-based genome editing platform specifically tailored for C.stationis.First,electroporation efficiency was optimized to 1.81±0.16×10^(5) CFU(colony forming units)/μg plasmid DNA through medium selection,pulse parameter adjustments(2.5 kV,2 pulses),and concentration optimization of cell wall-weakening agents(3.0%glycine,0.25%isoni-azid).Three functional shuttle vectors(p99E-pCG1,p19-Kan,p19-Spe)were constructed,enabling stable het-erologous gene expression.By engineering a tightly regulated Cas9 expression system(Plac promoter with dual LacO*operators),we achieved high-efficiency genome editing,with deletion efficiencies of 81.2-98.6%for 1.7-50 kb fragments and insertion efficiencies of 27.5-65.2%for 1-5 kb fragments.CRISPR/Cas9-assisted ssDNA recombineering facilitated single/triple nucleotide changes with>90%efficiency.Applying this toolbox,we engineered C.stationis for hypoxanthine biosynthesis by combining purA deletion with integration of heterologous feedback-resistant prs^(D128A) and endogenous purF deregulation(purF^(K334Q)),achieving a titer of 0.047 g/L.This study establishes a robust genetic platform for C.stationis,accelerating its industrial application in the production of biochemicals and biofuels.展开更多
The concept of building up a“well-connected fully-integrated,enviomentfriendly,passenger-oriented,economic-efficient,culturally-rich,intelligent and A convenient”station has been being applied to practice in recent ...The concept of building up a“well-connected fully-integrated,enviomentfriendly,passenger-oriented,economic-efficient,culturally-rich,intelligent and A convenient”station has been being applied to practice in recent years.China State Railway Group Co.Ltd .(hereinafier referred to as CHINA RAILWAY)and the Academyof Arts&Design,Tsinghua University have jointly initiated a series of study projects around the topic of“cultural and artistic expression”aiming to optimize design concepts and explore the design aesthetics of China's railway passenger stations in practice.These projects have achieved some positive results and this paper aims to provide a better analysis of the design concept of railway passenger stations in the new era through ananalysis of those stations' design aesthetic.展开更多
基金support.This work was supported by Guangdong S&T Program(2024B1111150001).
文摘Corynebacterium stationis,a high-GC Gram-positive bacterium with significant industrial potential,has faced limitations due to the lack of efficient genetic tools.In this study,we developed a CRISPR/Cas9-based genome editing platform specifically tailored for C.stationis.First,electroporation efficiency was optimized to 1.81±0.16×10^(5) CFU(colony forming units)/μg plasmid DNA through medium selection,pulse parameter adjustments(2.5 kV,2 pulses),and concentration optimization of cell wall-weakening agents(3.0%glycine,0.25%isoni-azid).Three functional shuttle vectors(p99E-pCG1,p19-Kan,p19-Spe)were constructed,enabling stable het-erologous gene expression.By engineering a tightly regulated Cas9 expression system(Plac promoter with dual LacO*operators),we achieved high-efficiency genome editing,with deletion efficiencies of 81.2-98.6%for 1.7-50 kb fragments and insertion efficiencies of 27.5-65.2%for 1-5 kb fragments.CRISPR/Cas9-assisted ssDNA recombineering facilitated single/triple nucleotide changes with>90%efficiency.Applying this toolbox,we engineered C.stationis for hypoxanthine biosynthesis by combining purA deletion with integration of heterologous feedback-resistant prs^(D128A) and endogenous purF deregulation(purF^(K334Q)),achieving a titer of 0.047 g/L.This study establishes a robust genetic platform for C.stationis,accelerating its industrial application in the production of biochemicals and biofuels.
文摘The concept of building up a“well-connected fully-integrated,enviomentfriendly,passenger-oriented,economic-efficient,culturally-rich,intelligent and A convenient”station has been being applied to practice in recent years.China State Railway Group Co.Ltd .(hereinafier referred to as CHINA RAILWAY)and the Academyof Arts&Design,Tsinghua University have jointly initiated a series of study projects around the topic of“cultural and artistic expression”aiming to optimize design concepts and explore the design aesthetics of China's railway passenger stations in practice.These projects have achieved some positive results and this paper aims to provide a better analysis of the design concept of railway passenger stations in the new era through ananalysis of those stations' design aesthetic.
