Pet family of efflux proteins

Abstract

The invention provides methods of increasing the production of aromatic carboxylic acids from a host cell via manipulation of the yhcRQP operon encoding a family of efflux proteins. Up-regulation of all or a sub-set of the genes in the yhcRQP were additionally found to enhance tolerance to aromatic carboxylic acids toxicity.

1 . A method of increasing the yield of an aromatic carboxylic acid from a host cell producing said aromatic carboxylic acid comprising: 
 a) providing a host cell which: 
 i) produces an aromatic carboxylic acid; and  
 ii) comprises at least one yhcQ gene and at least one yhcP gene; and  
   b) up-regulating the expression of the at least one yhcQ gene and the at least one yhcP gene whereby the yield of aromatic carboxylic acid is increased.    
   
   
       2 . (canceled)  
   
   
       3 . A method according to  claim 1  wherein the at least one yhcQ gene and the at least one yhcP gene are endogenous to said host cell.  
   
   
       4 . A method according to  claim 1  wherein the at least one yhcQ gene and the at least one yhcP gene are heterologous to said host cell.  
   
   
       5 . A method according to  claim 1  wherein the host cell is selected from the group consisting of bacteria, yeast, fungi and plants.  
   
   
       6 . A method according to  claim 5  wherein the host cell is an enteric bacteria.  
   
   
       7 . A method according to  claim 5  wherein the host cell is selected from the group of genera consisting of  Escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus, Corynebacterium, Pseudomonas, Rhodobacter , and  Synechocystis.    
   
   
       8 . A method according to  claim 1  wherein the aromatic carboxylic acid is selected from the group consisting of para-hydroxybenzoic acid, para-hydroxycinnamic acid, cinnamic acid, salicylic acid, benzoic acid, and 1-napthoic acid.  
   
   
       9 . A method according to  claim 1  wherein the at least one yhcQ gene and the at least one yhcP gene comprise at least one suitable promoter operably linked to at least one isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1-4.  
   
   
       10 . A method according to  claim 1  wherein the at least one yhcP gene comprises a suitable promoter operably linked to an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:5.  
   
   
       11 . A method according to  claim 1  wherein the at least one yhcQ gene and the at least one yhcP gene are expressed on a multicopy plasmid.  
   
   
       12 . A method according to  claim 1  wherein the at least one yhcQ gene and the at least one yhcP gene are under the control of a strong promoter.  
   
   
       13 . A method according to  claim 12  wherein the strong promoter is selected from the group consisting of lac, trp, IP L , IP R , T7, tac, and trc.  
   
   
       14 . A chimeric gene encoding an efflux protein comprising: 
 a) an isolated nucleic acid molecule having a nucleic acid sequence selected from the group consisting of: 
 i) an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1-4; and  
 ii) an isolated nucleic acid molecule, wherein said isolated nucleic acid molecule hybridizes with (i) under the following conditions: 0.1×SSC, 0.1% SDS at 65° C. and washed with 2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS; and  
   b) a promoter;    wherein the promoter is heterologous to the isolated nucleic acid molecule.