Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication syste...Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.展开更多
Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The op...Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame(ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB(plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.展开更多
基金financially supported by the National Science Fund for Excellent Young Scholars(32222069)National Natural Science Foundation of China(32172349)+1 种基金Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)National Key Research and Development Program of China(2020YFA0908300).
文摘Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.
文摘Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame(ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB(plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.
