Construction And Application Of Second Generation Protease-deficient Hosts Of Bacillus Subtilis

Bacillus subtilis has been used as an important recombinant protein expression host because of its host safety,efficient protein secretion,simple genetic manipulation system,and easy fermentation culture.However,there are some bottlenecks limiting the production level of target protein in B.subtilis,such as plasmid unstability and extracellular proteases.Knocking out the protease genes is an effective way to solve the problem of proteases,the first generation protease-deficient strains such as B.subtilis 1A751,B.subtilis WB600,B.subtilis WB800 were constructed to have lower extracellular protease activity,which were used as expression hosts for heterologous proteins production.However,there is still no selection criteria for use of various protease-deficient hosts.At the same time,not all eight extracellular proteases are harmful to all recombinant proteins.A protease may be beneficial to protein A,which is unfavorable for protein B.The first generation defective host cannot meet the individualized needs of the target protein.In addition,some wild-type B.subtilis have unmatched growth ability and protein secretion ability,and how to develop wild-type strains as expression hosts remains a problem.In this paper,B.subtilis 168 was used as the research object,the transformation and screening of host strains were systematically studied,meanwhile,it was applied to wild type B.subtilis and the following results were obtained.1.Construction of the best protease-deficient strains of heterologous proteins.Although one of the major factors limiting the application of B.subtilis as an expression host has been its production of at least eight extracellular proteases,researchers have also noticed that some proteases benefited the secretion of foreign proteins at times.Therefore,to maximize the yield of a foreign protein,the proteases should be selectively inactivated.This raises a new question that how to identify the favorable and unfavorable proteases for a target protein.Here,an evaluation system containing nine mutant strains of B.subtilis 168 was developed to address this question.The mutant strain PD8 has all the eight protease genes(nprE,aprE,epr,bpr,mpr,nprB,vpr,wprA)were inactivated while each of the other eight mutant strains(PD8CnprE,PD8CaprE,PD8Cepr,PD8Cbpr,PD8Cmpr,PD8CnprB,PD8Cvpr,PD7)expresses only one kind of these eight proteases.The target protein is secreted in these nine mutant strains;if the production of target protein in a mutant strain is higher than that in strain PD8,the corresponding protease is regarded as favorable.Accordingly,the optimal protease-deficient host is constructed through inactivating the unfavorable proteases.Identification of the optimal protease-deficient expression host for MPH is PD8 through above evaluation system.Meanwhile,the secretion level of Chd in mutant strain PD9(?nprE,?aprE,?nprB,?vpr,?wprA)was highest,which was 1.7 fold of that in mutant strain PD8.AmyM also got its best extracellular production in its optimal host PD3(?nprE,?aprE,?epr).The highest extracellular activity of AmyM in mutant strain PD3 was 1.6 fold of that in mutant strain PD8.Meanwhile,the integrative vectors of pDnprE,pDaprE,pDepr,pDbpr,pDmpr,pDnprB,pDvpr and pDwprA were construed,which can easily realize the unmarked knockout of the protease genes and the construction of multi-copy expression host.2.Screening and transformation of wild type B.subtilis expression host Screening wild-type expression hosts with high protein secretion capacity can be an effective strategy to improve the production of heterologous proteins.According to the characteristics of AmyM against proteases,which used as a reporter protein to evaluate the potential of wild-type strains.Four strains(PAA5,M2,Za,24-DA-2)were isolated and identified as wild-type B.subtilis.The strain of PAA5 had the highest productivity of AmyM in them.So,host modification of PAA5 resulted in efficient secretion of AmyM in PAA5-03.The highest ?-amylase activity was 1960 U/mL,which is the optimal expression host for AmyM in this study.3.Stable and efficient expression of MPH in B.subtilisThe host of high-yield AmyM is not suitable for the secretion of MPH,further modication of the strain PD8,the yield of MPH obtained by DegUH12L,?dltABCD were increased by 22%and 14%,respectively,compared with the strain PD8,so,the production level of the target protein was further improved in the host transformation.In order to further improve the production of MPH,the promoter of the PNBP3510-mpd expression cassette was replaced by the Pac promoter,and the Pac-mpd expression cassette was successfully constructed.The highest MPH activity of the recombinant strain under shaking flask culture condition was 380.7 U/mL,which is the highest MPH yield in the current study.In summary,this study nine mutant strains of B.subtilis were constructed in the model strain B.subtilis 168,and the effects of each protease on the secretion of heterologous proteins were evaluated in detail;proteases that are not conducive to the production of the target protein were inactived,and the heterologous protein secretion level was improved by constructing the optimal protease-deleting host in B.subtilis.At the same time,the study in the model strain was applied to the wild-type B.subtilis strain,and the wild strain was excavated to develop as an efficent expression host which has a high-yield of interest protein.Finally,the strain of PD8 was further enhanced by host modication,and the efficient and stable production of the target protein is achieved by a single copy of chromosome integration.