Phenol-induced proteins of Thauera aromatica

Abstract

This invention pertains to genes coding for phenol-induced proteins Five phenol-induced proteins isolated from Thauera aromatica . Three dominant phenol-induced proteins called F1, F2, and F3 respecitively were purified and sequenced to obtain the enzyme(s) that catalyze the 14 CO 2 :4-hydroxybenzoate isotope exchange reaction and the carboxylation of phenylphosphate. The N-terminal amino acid sequences of these proteins as well as the N-terminus of the phenol-induced proteins (F4 and F5) were also determined.

We claim:  
     
         1 . A polypeptide encoded by DNA selected from the group consisting of: 
 (a) DNA having the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6;    (b) a degenerate nucleotide sequence of the DNA of (1); and    (c) DNA that hybridizes with the complement of the nucleotide sequence of (1) or analog thereof under hybridization conditions wherein 6× SSC (1 NaCl), 40 to 45% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55 to 60° C. wherein the polypeptide is further characterized by phosphorylase activity on phenol substrates.    
     
     
         2 . The polypeptide of  claim 1  having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.  
     
     
         3 . An isolated nucleic acid fragment encoding the polypeptide of  claim 1 , the nucleic acid fragment selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6;    (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6;    (c) an isolated nucleic acid molecule that hybridizes with the nucleic acid fragment of (a) under hybridization conditions wherein 6× SSC (1 NaCl), 40 to 45% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55 to 60° C.; and    (d) an isolated nucleic acid fragment that is complementary to (a), (b), or (c),    wherein the isolated nucleic acid is further characterized by phosphorlase activity on phenol substrates.    
     
     
         4 . The DNA fragment of  claim 3 , wherein the DNA fragment is isolated from  Thauera aromatica.    
     
     
         5 . An expression cassette comprising the DNA fragment of  claim 3  operably linked to suitable signal sequences for the expression of the DNA fragment in a host microorganism.  
     
     
         6 . An expression vector comprising the expression cassette of  claim 5  and regulatory sequences ensuring the stable maintenance of said expression vector.  
     
     
         7 . A microorganism stably transformed with the DNA fragment of  claim 3 .  
     
     
         8 . A transformed microorganism comprising the expression vector of  claim 6 .  
     
     
         9 . A transformed microorganism comprising the expression cassette of  claim 5 , wherein the signal sequences of the expression cassette are a ribosome binding site and a promoter sequence located upstream of the DNA fragment.  
     
     
         10 . The transformed microorganism of  claim 9  wherein the promoter is at least one of CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, AOX1, lac, trp, 1P L , IP R , T7, tac, and trc or at least one strong promoter of Corynebacterium, Comamonas, Rhodococcus or Pseudomonas.  
     
     
         11 . The transformed microorganism of  claim 9 , wherein the ribosome binding site is selected from the group consisting of ribosome binding sites from the genomes of  E. coli, P. pastoris , Comamonas, Pseudomonas, Rhodococcus, and Corynebacterium.  
     
     
         12 . The transformed microorganism of  claim 11 , wherein the host microorganism is selected from the group consisting of Comamonas sp., Corynebacterium sp., Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella s.p, Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Zygosaccharomyces sp, Pichia sp., Kluyveromyces sp., Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torylopsis sp., Methylobacteriasp., Bacillussp., Escherichia sp., Pseudomonas sp., Rhizobium sp., and Streptomyces sp.  
     
     
         13 . An isolated and purified DNA fragment having a nucleotide sequence SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.  
     
     
         14 . An isolated and purified 14.27 kb DNA fragment as shown in FIG. 11.  
     
     
         15 . A microorganism stably transformed with chimeric genes having at least one copy of one or more of nucleotide sequences selected from the group consisting of SEQ ID NOs:6, 12, 14, 4, 8, 2, 16, 10, 18, and 20.  
     
     
         16 . A microorganism stably transformed with a chimeric gene having at least one copy of the nucleic acid sequence of SEQ ID NO:23.