Effects Of Terminators And 5' Non-coding Regions In Bacillus Subtilis On Exogenous Gene Expression

Bacillus subtilis is an important gram-positive bacterium that has been used as a model organism for many years.With the completion of complete genome sequence sequencing and the continuous development of multi-omics analysis,such as transcriptome and proteomics,B.subtilis has become a potential synthetic biological substrate for the construction of metabolic pathways,recombinant protein expression systems and complex genetic circuits.At present,the use of synthetic biology to construct expression modules in B.subtilis has become a research hotspot.Artificial gene expression component is the basis of designing expression module and constructing gene circuit.At present,in B.subtilis,a variety of different types of artificial promoters have been widely used in gene circuits,while many other types of components,such as terminators and regulatory sequences in non-coding regions,have not been fully explored and developed.The regulation of these components on exogenous genes in B.subtilis has not been systematically studied.These problems hinder the development of constructing large-scale and complex regulatory networks and greatly restrict the advancement of synthetic biotechnology of B.subtilis.Based on this,this paper respectively studied the effect of the terminators of the effects of 3'non-coding region and the regulatory sequences of the 5'non-coding region in B.subtilis on the expression of heterologous genes.Furthermore,we engineered these two kinds of regulatory sequences through synthetic biology technology to improve the efficiency of the expression of exogenous genes.Specific results obtained are as follows:?1?Identification and molecular modification of high efficiency terminators in B.subtilis.With GFP and mCherry as reporter genes,a platform for determination of terminator strength based on double fluorescence signal was constructed,and the termination efficiency of 10 terminators from B.subtilis and 5 terminators from bacteriophage SPO1 were analyzed.The results showed that the termination efficiency ranged from 0 to 0.99,with strong terminators TB4,TB5,TB6,TB7,TB9 and ST1,and the termination efficiency was higher than 0.95.TB5 and TB6 had the best inhibitory effect on transcription read-through.TB5 had a 1.17 times higher termination efficiency than rrnBT1,and TB5 had a certain up-regulation effect on upstream GFP expression?1.24 times higher than rrnBT1?,and a 5.54 times higher inhibition effect on mCherry expression than rrnBT1.The double and triple series terminators were constructed and the characteristics of the terminators after the series terminators were analyzed.The results show that the series of weak terminators can further improve the terminators efficiency.The termination efficiency of a weak terminator in series with a strong terminator generally depends on the strong terminator.Moreover,after the strong terminator was in series,its termination efficiency and the enhancement of GFP expression would not be increased,but the inhibition of downstream mCherry expression would be further enhanced.?2?Regulation and mechanism of 5'non-coding region of mRNA on expression of exogenous genes in B.subtilis.The effects of 5'non-coding regions and 5'coding regions on protein translation efficiency were confirmed,and a general fusion peptide was proposed to reduce the inhibition of translation by the secondary structure of 5'coding regions of a specific gene.With the L aspartate-?-decarboxylase?PanD?from Tribolium castaneum as the model protein,the phenomenon of translation inhibition was verified by the regulation of PanD with efficient expression elements.The results showed that the expression level of recombinant PanD was extremely low driven by the highly efficient expression element composed of combination of strong promoter P_spovG and P_sigW with strong RBS?RBS206?,displaying translation inhibition.The characteristics of changes of the mRNA secondary structures after deletion of the 5'end coding region were analyzed to reveal the mechanism of translation inhibition.When 9 nt?three amino acids?was deleted from the downstream of the initial codon,the protein expression level and enzyme activity level of the PanD mutant mediated by the truncated mRNA was increased compared to the wildtype.The effects of translation inhibition were investigated by fusing the sfgfp gene to the 5'end of the panD gene.Secondary structure analysis of mRNA showed that the hairpin formed between the 5'coding region and the 5'UTR of wild-type panD blocked the RBS sequence in different degrees,leading to weakened translation initiation efficiency.The length of the fused peptides to PanD were engineered to elevate the adaption of the fusion to the chassis.After truncation of 9 nt of the 5'coding region,the inhibitory hairpin structure blocking the RBS was impaired.The fusion of sfGFP and mCherry into the N-terminal of PanD could improve the expression level as well as the enzyme activity of the recombinant fused protein.The data also showed that retaining the 143 amino acids from the N termini of sfGFP as fused peptide was able to produce equivalent expression levels to the fusion of full length of sfGFP.This strategy that N-terminal peptide fusion is expected to be a kind of universal peptidyl insulator adapted to a variety of heterologous proteins,by which translation inhibition by cis-interaction within the mRNA molecules could be eliminated efficiently.This novel strategy might has a wide application potential in the field of synthetic biology.