Natural promoters for gene expression and metabolic monitoring in bacillus species

Abstract

Genes have been identified in the Bacillus genome that are responsive to various metabolic conditions and growth cycle changes. The new responsiveness of these genes allows for their use in regulated gene expression in Bacillus sp. and for the monitoring of bioreactor health.

What is claimed is:  
     
         1 . A method for the expression of a coding region of interest in a Bacillus sp comprising: 
 a) providing a transformed Bacillus sp cell having a chimeric gene comprising a nucleic acid fragment comprising the promoter region of a Bacillus gene operably linked to a coding region of interest expressible in a Bacillus sp, wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of narGHJI, csn, yncM, yvyD, yvaWXY, ydjL, sunA, and yolIJK and homologues thereof; and    b) growing the transformed Bacillus sp cell of step (a) in the absence of oxygen wherein the chimeric gene of step (a) is expressed.    
     
     
         2 . A method for the expression of a coding region of interest in a Bacillus sp comprising: 
 a) providing a transformed Bacillus sp cell having a chimeric gene comprising a nucleic acid fragmnent comprising the promoter region of a Bacillus gene operably linked to a coding region of interest expressible in a Bacillus sp, wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of narGHJI, csn, yncM, yvyD, yvaWXY, ydjL, sunA, and yolIJK and homologues thereof;    b) growing the transformed Bacillus sp. cell of step (a) in the presence of oxygen whereby the cell density is increased; and    c) removing oxygen form the transformed Bacillus sp. cell or step (b) whereby the chimeric gene is expressed.    
     
     
         3 . A method according to  claim 2  wherein after step (c) oxygen is re-supplied to the transformed Bacillus sp. cell.  
     
     
         4 . A method according to either of claims  1  or  2  wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of SEQ ID NOs: 1-15.  
     
     
         5 . A method for the expression of a coding region of interest in a Bacillus sp comprising: 
 a) providing a transformed Bacillus sp cell having a chimeric gene comprising a nucleic acid fragment comprising the promoter region of a Bacillus gene operably linked to a coding region of interest expressible in a Bacillus sp, wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of feuABC, ykuNOP, and dhbABC, and homologues thereof; and    b) growing the transformed Bacillus sp cell of step (a) in the absence of oxygen and in the presence of nitrite wherein the chimeric gene of step (a) is expressed.    
     
     
         6 . A method according to  claim 5  wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of SEQ ID NOs: 16-24.  
     
     
         7 . A method according to  claim 6  wherein the concentration of nitrite is from about 1 mM to about 10 mM.  
     
     
         8 . A method for the expression of a coding region of interest in a Bacillus sp comprising: 
 a) providing a transformed Bacillus sp cell having a chimeric gene comprising a nucleic acid fragment comprising the promoter region of a Bacillus gene operably linked to a coding region of interest expressible in a Bacillus sp, wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of ycgMN, dhaS rapF, rapG, rapH, rapK, yqhIJ, yveKLMNOPQST, yhfSTUV, csn, yncM, yvyD, yvaWXY, ydjL, sunA, and yolIJK, and homologues thereof; and    b) growing the transformed Bacillus sp cell of step (a) in the presence of oxygen until the cell reaches about T0 of the stationary phase wherein the chimeric gene of step (a) is expressed.    
     
     
         9 . A method according to  claim 8  wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of SEQ ID NOs: 75, 76, 25-49, and 5-15.  
     
     
         10 . A method for the expression of a coding region of interest in a Bacillus sp comprising: 
 a) providing a transformed Bacillus sp cell having a chimeric gene comprising a nucleic acid fragment comprising the promoter region of a Bacillus gene operably linked to a coding region of interest expressible in a Bacillus sp, wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of acoABCL, and glvAC, and homologues thereof; and    b) growing the transformed Bacillus sp cell of step (a) in the presence of oxygen until the cell reaches about T1 of the stationary phase wherein the chimeric gene of step (a) is expressed.    
     
     
         11 . A method according to  claim 10  wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of SEQ ID NOs: 41-44 and 50-51.  
     
     
         12 . A method for the expression of a coding region of interest in a Bacillus sp comprising: 
 a) providing a transformed Bacillus sp cell having a chimeric gene comprising a nucleic acid fragment comprising the promoter region of a Bacillus gene operably linked to a coding region of interest expressible in a Bacillus sp, wherein the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of yxjCDEF, yngEFGHI, yjmCDEFG, ykfABCD, and yodOPRST; and homologues thereof; and    b) growing the transformed Bacillus sp cell of step (a) in the presence of oxygen until the cell reaches about T3 of the stationary phase wherein the chimeric gene of step (a) is expressed.    
     
     
         13 . A method according to  claim 12  the nucleic acid fragment comprising the promoter region of a Bacillus gene is selected from the group consisting of SEQ ID NOs: 52-74.  
     
     
         14 . A method according to any of claims  1 ,  2  or  3  wherein the expression of the chimeric gene is down-regulated at T0 of the stationary phase.  
     
     
         15 . A method according to any one of claims  1 ,  2 ,  3 ,  4 ,  8 ,  10  and  12  wherein the Bacillus sp. cell is selected from the species consisting of  Bacillus subtillus, Bacillus thuringiensis, Bacillus anthracis, Bacillus cereus, Bacillus brevis, Bacillus megaterium, Bacillus intermedius, Bacillus thermoamyloliquefaciens, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus licheniformis, Bacillus macerans, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus laterosporus, Bacillus acidocaldarius, Bacillus pumilus,  and  Bacillus pseudofirmus.    
     
     
         16 . The method according to any one of claims  1 ,  2 ,  3 ,  4 ,  8 ,  10  and  12 , wherein the coding region of interest is selected from the group consisting of crtE crtB, pds, crtD, crtL, crtZ, crtX crtO, phaC, phaE, efe, pdc, adh, genes encoding limonene synthase, pinene synthase, bomyl synthase, phellandrene synthase, cineole synthase, sabinene synthase, and taxadiene synthase.  
     
     
         17 . A method for monitoring the state of the cell metabolism of a Bacillus sp. culture comprising: 
 a) providing a culture of actively growing Bacillus sp. cells; and    b) measuring the expression levels of a pool of genes isolated from the Bacillus cells of step (a), the pool of genes comprising narGHJI, feuABC, ykuNOP, dhbABC, ydjL, sunA, yolIJK, csn, yncM, yvyD, yvaWXY, yhfRSTUV, yveKLMNOPQST, dhaS, rapF, rapG, rapH, rapK, ycgMN, yqhIJ, glvAC, acoABCL, yxjCDEF, yngEFGHI yjmCDEFG, ykfABCD, yodOPRST, alsT, and yxeKLMN, and homologues thereof.    
     
     
         18 . A method according to  claim 17  wherein a pool of genes isolated from the Bacillus cells is selected from the group consisting of SEQ ID NOs: 1-81.  
     
     
         19 . A method according to  claim 17  wherein the measuring of gene expression levels is accomplished using a format selected from the group consisting of northern blots, nuclease protection assay or primer extension assays.  
     
     
         20 . A method according to  claim 19  wherein the measuring of gene expression levels is accomplished using a nucleic acid microarray having the genes narGHJI, feuABC, ykuNOP, dhbABC, ydjL, sunA, yolIJK, csn, yncM, yvyD, yvaWXY, yhfRSTUV, yveKLMNOPQST, dhaS, rapF, rapG, rapH, rapK, yqhIJ, glvAC, acoABCL, yxjCDEF, yngEFGHI yjmCDEFG, ykfABCD, yodOPRST, alsT, and yxeKLMN, and homologues thereof, contained therein.  
     
     
         21 . A method according to  claim 17  wherein the Bacillus sp. cell is. selected from the species consisting of  Bacillus subtillus, Bacillus thuringiensis, Bacillus anthracis, Bacillus cereus, Bacillus brevis, Bacillus megaterium, Bacillus intermedius, Bacillus thermoamyloliquefaciens, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus licheniformis, Bacillus macerans, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus laterosporus, Bacillus acidocaldarius, Bacillus pumilus,  and  Bacillus pseudofirmus.    
     
     
         22 . A method according to  claim 17  wherein the actively growing culture is grown in the absence of oxygen and the expression of genes narGHJI, ydjL, sunA, yolIJK, csn, yncM, yvyD, and yvaWXY are up-regulated in the log phase.  
     
     
         23 . A method according to  claim 17  wherein the actively growing culture is grown in the absence of oxygen and in the presence of nitrite and the expression of genes feuABC, ykuNOP, and dhbABC are up-regulated in the log phase.  
     
     
         24 . A method according to either of claims  22  or  23  wherein the expression of genes narGHJI is down-regulated at about T0 of the stationary phase.  
     
     
         25 . A method according to  claim 17  wherein the actively growing culture is grown in the presence of oxygen and the expression of genes ycgMN, yqhIJ, ydjL, sunA, yolIJK, csn, yncM, yvyD, yvaWXY, yhfRSTUV, yveKLMNOPQST, dhaS, rapF, rapG, rapH, rapK, are up-regulated at about T0 of the stationary phase.  
     
     
         26 . A method according to  claim 17  wherein the actively growing culture is grown in the presence of oxygen and the expression of genes, acoABCL and glvAC are up-regulated at about T1 of the stationary phase.  
     
     
         27 . A method according to  claim 17  wherein the actively growing culture is grown in the presence of oxygen and the expression of genes, yxjCDEF, yngEFGHI yjmCDEFG, ykfABCD, and yodOPRST are up-regulated at about T3 of the stationary phase.  
     
     
         28 . A method according to  claim 17  wherein the actively growing culture is grown in the presence of oxygen and the expression of genes, alsT and yxeKLMN are down-regulated at stationary phase or under nutrient-limiting conditions.