Development And Application Research Of Expression Systems In Bacillus Subtilis And Streptomyces

Bacillus subtilis and Streptomyces have become attractive hosts for gene expression owing to their GRAS status, clear genetic background and mature genetic operation techniques. It is highly possible to establish food-grade expression system in these two strains after Lactococcus lactic. Although some expression systems have been developed in these two strains, the abundance of regulatory elements and available tools for expression are far behind the researches in E. coli. A highly active secretory expression system was constructed via an engineered dual promoter and a highly efficient signal peptide in B. subtilis. And then, a novel auto-regulatory gene expression system coupled with cell density was constructed in B. subtilis. Moreover, It was previously reported that TGasewere extracellularly overexpressed in S. hygroscopicus WSH03-13. The nucleotide sequences of the 5’ flanking region of TGase was identified, and a putative promoter region(P_tg) and signal peptide sequence(SP_tg) were predicted. In this study, the predicted P_tg and SP_tg were researched and used to construct a novel expression system in Sreptomyces. The results were summarized as follows:（1）Construction of a highly active secretory expression system in B. subtilis. Using AP as the reporter protein, a series of expression plasmids were separately constructed containing promoters PHpa II, PyxiE, P43, PgsiB, Pluxs, PaprE and Pgrac. The strengths of these seven promoters varied greatly in driving AP expression by analyzing the AP activities and the expression patterns. The activities of PgsiB and PHpaII were higher than the other five promoters. The promoters PHpa II, PyxiE, P43, Pgsi B, Pluxs and PaprE were separately inserted downstream of PHpaII, and PHpaII-PgsiBgave the best performanceamong the single and dual promoters. Signal peptide YncM（SPYnc M） was screened from the library consisted of 20 signal peptides to cooperate with the PHpaII-PgsiB, yielding pBSG24-SPYncM. The capacity of pBSG24-SPYnc Mwas evaluated by fermentation in a 5 L fermentor, and the yields of AP could be as high as 1.7g/L.（2） Construction and development of an auto-inducible gene expression system in B. subtilis. The E. coli-B. subtilis shuttle plasmid pBSG01 was constructed to evaluate promoter PsrfA using the green fluorescent protein（GFP） as the reporter protein. The PsrfA activity displayed a cell-density-dependent pattern by detecting the GFP expression along with the cell growth. The effect of four different culture media on PsrfA-mediated expression was estimated, and LB medium was the most optimal medium for PsrfA-mediated expression. Meanwhile, the GFP expression controlled by PsrfA was higher than that of PHpa II, and PsrfA had a similar expression pattern in wild-type B. subtilis 168, B. subtilis WB600, B. subtilis WB800 and BSG1681（ΔPsrfA）. In addition, adding 1.5% glucose to the medium generated 1.2-fold and 4.6-fold enhancement in promoter activity and yields, respectively. By changing the sequences of-10 region and-35 region of PsrfA into the σA recognized consensus sequences, the expression level was approximately 2-fold stronger than that of the wild-type promoter. Finally, the yields of the system was 8-fold higher than that of the original system by adding 1.5% glucose to the medium. Heterologous protein of aminopeptidase was successfully overexpressed using the expression system.（3） Optimization of the auto-inducible expression system and application for high cell density fermentation in B. subtilis. A spore-deficient strain BSG1682（ΔσF） was constructed, and the PsrfA-mediated GFP expression in BSG1682 was 60% higher than that of B. subtilis 168. The fragment containing PsrfAand gfpwas integrated into the chromosome to perform chromosomal integration expression. The integrated fragment had little effect on cell growth and the GFP expression was slightly lower than that of plasmid-based system. The-10,-16, and-35 region of PsrfA were individually or concurrently changed into the corresponding consensus sequences. Compared with the wild-type promoter, promoter P11 with the consensus-35 hexamer showed the best performance among the mutant promoters, which was 1.5-fold stronger than PsrfA. PHpaII and PgsiB were separately linked with PsrfA. The activity of PsrfA-PHpa II（P17） was enhanced during the given time while the PsrfA-PgsiB（P16） showed weaker strength across the growth time. The core regions of the P17 were changed separately or in combination to the consensus sequences andthe P23 promoter showed the highest expression ofGFP. Nattokinase（NK） and AP were used for the heterologous proteins expression experiments. The industrial scale capacity of the expression system was determined by performing fermentation experiments in a 5 L fermentor. The biomass determined by OD600 was 30 which was 3.2 folds higher than that of the flask. Though the fluorescence intensity was only 85% of that the flask, the total fluorescence were 2.7-fold higher than that of the flask.The the cell density determined by OD600 could be 122 in the optimized HCDC.（4） Construction and development of a novel expression system of Streptomyces. The plasmid pSG01 was constructed using P_tg-SP_tg, and the multiple cloning sites（MCS） and a transcriptional terminator fd（fd-ter）were inserted just downstream of P_tg-SP_tg to facilitate gene cloning and to minimize transcriptional read-though to enhance transcription. TGase was overexpressed in S. lividans1326 by pSG01. The rare codon for leucine and signal peptidase I interaction site in the SP_tgcoding sequence were mutant, yielding plasmid pSG02. The secreted TGase was increased by 1-fold by pSG02. The first five amino acid residues at the N-terminus of TGase were determined and the residues were demonstrated to be identical to those of the wild-type. These results demonstrated that the expression elements could work functionally for TGase expression. Comparing with the TGase expression controlled by Perm E*, the TGase expression controlled by P_tgwas 2.1-fold higher than that of PermE* and P_tg was assessed as as a strong promoter which could be used for construction of expression system in Streptomyces. Plasmid pSG02 was efficient in expressing TGase in various Streptomyces strains.In addition, the heterologous proteins aminopeptidase（BSAP） and phenylalanine ammonialyase（PAL） were also expressed by pSG02 in various Streptomyces strains. Moreover, application of P_tgand SP_tg in E. coli was researched. TGase was secreted using SP_tg in E. coli. However, the expression of TGase controlled by P_tg could not be detected in E. coli.