| Bacillus subtilis is widely used in basic research and applied research because of its robust secretory capacity,ability to produce stress-resistance spore and food-grade status.Therefore,in order to facilitate the genetically modification of B.subtilis,the previous researchers have developed a number of practical gene regulatory expression tools.However,with the rapid development of synthetic biology,these existing tools have been unable to meet the needs of the researchers.Therefore,targeted gene regulation and expression systems were constructed in this study and applied in fermentation engineering.Major results achieved in this work are highlighted below:?1?Construction of endogenous growth phase-dependent promoter library in B.subtilisBy analyzing the relationship between 114 B.subtilis endogenous promoters stength and the growth periods,3 exponential phase-dependent promoters,31 middle-log and stationary phases-dependent promoters,74 sustained promoters and 3 stationary phase-dependent promoters were obtained.The effects of different temperature conditions on the strength of endogenous promoters were analyzed,and low temperature repressible promoters PyrxA and Pykn W,low temperature inducible promoters PbltD,PydaD,PgerBC and PylnF,and temperature-independent promoters PmenB,Pgap,PyoxA,PthrS and PydfK were identified.In addition,by comparing the changes in the activities of the promoters at pH 5.6,6.0,7.0,8.0 and 8.3,acid and alkali repressible promoters PsrfAA,PytvI,PspoVB,PodhA,Phbs,PrapI,PtrxA,PspoIVCA,PyqfD and PmmgA,and promoters PabrB,PyraD,PgsiB,PywnJ,PydfK,PgerBC,PrelA and PyitG,which exhibit highest activity at pH 6.0,were identified.The expression levels of esterase,keratinase and alkaline pectinase were increased by the use of different growth promoters.?2?Construction of broad-spectrum promoters and plasmidsBy engineering S.cerevisiae promoter Pmin,an E.coli,B.subtilis and S.cerevisiae universal promoter Pbs was obtained.Then E.coli-B.subtilis-S.cerevisiae shuttle vector pEBS?5.8 kb?,E.coli-B.subtilis shuttle vector pEB?4.0 kb?,E.coli-S.cerevisiae shuttle vector pES?4.2 kb?were constructed by employing Pbs-kan R as the sole screening marker.pEBS was absolutely stable in E.coli,B.subtilis and S.cerevisiae after 100 generations.We also obatined the other broad-spectrum promoters Pbs1,Pbs2 and Pbs3 by semi-rational design of Pbs.?3?Construction of post-transcriptional gene expression regulation system in B.subtilisBy engineering Type I toxin-antitoxin system bsrG/sr4,the MS-DOS?modulation via the sRNA-dependent operation system?gene expression regulation system was constructed in B.subtilis.The effectiveness of MS-DOS gene regulation system was confirmed by regulating the expression of FtsZ and GFP.The reversible and precise control of the expression of target gene in the range of 8.1%to 82.0%were achieved by using IPTG-inducible promoter PgroE to drive the transcription of RNA Sr4.By analysing the inhibitory effect of truncated Sr4 on the expression of GFP,the 33 bp which located at 3'end of Sr4 was proved to be the core region.The expression of neutral protease,alkaline protease and serine protease were simultaneous regulated,and the results suggested that the MS-DOS system could regulate multiple gene expression by single sRNA.By quantitatively analysing the mRNA levels of different regions of target genes,the mechanism of the post-transcriptional regulatory system was deduced:Sr4and gene-opr mRNA were paired and formed a complex,leading to degradation of the target gene mRNA from 3'to 5'and the reduced expression of the target gene.?4?Construction of food-grade expression system in B.subtilisBy engineering Type II toxin-antitoxin system ydcD/ydcE,a food-grade expression system was constructed in B.subtilis.The critical concentrations of xylose were 2.0 g×L-1 and 5.0 g×L-1 under the condition of no other carbon source and high concentration of carbon source,respectively.This expression system was absolutely stable after 100 generations.Compared with traditional antibiotic-dependent expression systems,this novel expression system resulted in greater biomass and higher titers of desired products.It is truly a food-grade expression system as all the used genetic elements are derived from the food-grade host B.subtilis.?5?Optimization of hyaluronic acid synthesis pathway in B.subtilis by using the constructed gene regulation and expression toolsThe production of hyaluronic acid with a yield of 0.95 g×L-1 was achieved by expressing S.zooepidemicus hasA in B.subtilis food-grade system.The expression of key genes in hyaluronic acid synthesis competition pathway were regulated by MS-DOS regulatory system,and the expression of zwf,pfkA and galE were identified to impair hyaluronic acid production.The simultaneous down-regulation of zwf,pfkA and galE incresed hyaluronic acid production by 94.7%.The expression of tuaD,gtaB and glmU in the hyaluronic acid synthesis pathway was optimized by employing the endogenous promoters,and the hyaluronic acid yields were increased by 49.5%,54.7%and 26.8%,respectively.By down-regulating the expression of zwf,pfkA and galE as well as overexpressing tuaD,gtaB and glmU,the yield of hyaluronic acid reached 2.65 g×L-1. |
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