Mutant Strains of Pseudomonas Fluorescens And Variants Thereof, Methods For Their Production, And Uses Thereof In Alginate Production

Abstract

It is described biologically pure bacterial cultures of mutant strains of Pseudomonas fluorescens , which produces large amounts of alginate. The alginate may contain a certain determined content of mannuronate and guluronate residues, possible presence and determined level of acetyl groups in the alginate, and a desired molecular weight of the alginate. Also high yielding mutants with regulation of alginate production, is described. The invention further provides methods for producing new mutant strains of Pseudomonas fluorescens and variants thereof, and use of the resulting strains in alginate production.

1 - 26 . (canceled) 
     
     
         27 . A mutant strain of  P. fluorescens  which comprises a mutant algG gene and produces alginate having a defined guluronate residue (G)-content between 0 and 30%, wherein said algG gene is inactivated or encodes an enzyme having reduced epimerase activity. 
     
     
         28 . A mutant strain of  P. fluorescens  which produces alginate, wherein in said mutant strain the alginate biosynthetic operon has been placed under the control of an inducible promoter which replaces the native promoter of said operon. 
     
     
         29 . The mutant strain of  P. fluorescens  of  claim 28 , wherein said mutant strain produces at least 10 g alginate per liter medium. 
     
     
         30 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the inducible promoter is a Pm promoter or a mutant thereof. 
     
     
         31 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the inducible promoter is a Pm promoter, and the mutant strain further comprises an xylS gene. 
     
     
         32 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the inducible promoter is a Pm promoter from the  Pseudomonas putida  TOL plasmid. 
     
     
         33 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the said mutant produces an alginate consisting of mannuronate residues only. 
     
     
         34 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the said mutant produces alginate having a defined guluronate residue (G)-content between 0 and 30%. 
     
     
         35 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the said mutant produces alginate without, or with a reduced number of O-acetyl groups. 
     
     
         36 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the said mutant produces alginate with a molecular weight of between 50,000 and 3,000,000 Daltons. 
     
     
         37 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the mutant strain further comprises a mutant gene selected from the group consisting: a mutant algG gene, a mutant algI gene, a mutant algJ gene, a mutant algL gene, and a mutant algF gene. 
     
     
         38 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the mutant strain further comprises a mutant algG gene which encodes an epimerase enzyme having reduced epimerase activity. 
     
     
         39 . The mutant strain of  P. fluorescens  of  claim 28 , wherein the mutant strain further comprises a mutant algG gene which is inactivated. 
     
     
         40 . A biologically pure bacterial culture of the mutant strain of  P. fluorescens  of  claim 28 . 
     
     
         41 . A mutant strain of  P. fluorescens , wherein said strain produces at least 10 g alginate per liter medium. 
     
     
         42 . The mutant strain of  claim 41  which has a mutation corresponding to the mutation in  Pseudomonas fluorescens  mutant strain Pf201 and which is stable over at least 60 generations. 
     
     
         43 . The mutant strain of  claim 41 , wherein the mutant strain further comprises a mutant gene selected from the group consisting of: a mutant algG gene, a mutant algI gene, a mutant algJ gene, a mutant algL gene and a mutant algF gene. 
     
     
         44 . A method of producing a mutant strain of  P. fluorescens  of  claim 41 , wherein:
 a) a wild-type strain of  P. fluorescens  is contacted with a mutagenic agent, and   b) the treated  P. fluorescens  of step (a) are grown in the presence of one or more antibiotics, and   c) antibiotic resistant mucoid mutants are isolated by selection, and   d) the alginate production properties of the isolated mucoid mutants of step (c) are determined.   
     
     
         45 . The method according to  claim 44 , wherein the mutagenic agent is nitrosoguanidine. 
     
     
         46 . The method according to  claim 44 , wherein the treated  P. fluorescens  of step (a) are grown in the presence of a β-lactam or aminoglycoside antibiotic. 
     
     
         47 . The method according to  claim 44 , wherein the treated  P. fluorescens  of step (a) are grown in the presence of carbenicillin. 
     
     
         48 . A method of producing a mutant strain of  P. fluorescens  of  claim 28 , wherein (i) the alginate biosynthetic operon promoter of a wild type strain of  P. fluorescens  is exchanged by an inducible promoter by homologous recombination, and (ii) optional effector genes are introduced into the bacterium of (i) by homologous recombination, transposon mutagenesis or by means of a plasmid, and (iii) mutants are grown and then isolated by selection, and (iv) the alginate production properties of the isolated mutants of (iii) are determined. 
     
     
         49 . The method according to  claim 48 , herein the inducible promoter is Pm from  P. putida  Tol-plasmid, or a mutated Pm promoter. 
     
     
         50 . A method of producing a mutant strain of  P. fluorescens  of  claim 27 , wherein
 a) the wild type algG-gene, encoding the C-5 epimerase is cloned in a plasmid or minitransposon and mutagenized by chemical mutagenesis or PCR,   b) a derivative of an alginate-producing strain of  P. fluorescens , which lacks the algG gene (ΔalgG-strain), is constructed, and   c) the library of mutagenized algG of step (a) is transferred to the ΔalgG-strain of  P. fluorescens , and the plasmid or transposon-containing strains are identified and assayed for alginate-production and epimerase-activity, and   d) the plasmid or transposon-containing strains of a mutant algG encoding an epimerase that provides alginate with a guluronic acid residue content between 0 and 30% are identified by the assay in step (c), and   e) the mutant algG gene is cloned into a gene-replacement vector, and   f) the gene-replacement vector of step (e) is then transferred to an alginate-producing strain of  P. fluorescens  in order to replace its algG gene with the mutant algG gene, and making it capable of expressing the mutant gene.   
     
     
         51 . A method of producing a mutant strain of  P. fluorescens  of  claim 27 ,
 a) one or more amino acids, which are identified by mutagenesis and subsequent screening to be important for epimerization, are exchanged, at the gene-level, by site-specific mutagenesis to amino acids different from the ones occurring both in the mutant and the wild-type AlgG-protein, and   b) the mutant gene is cloned into a gene-replacement vector and this vector is transferred to an alginate-producing strain of  P. fluorescens  where it replaces the wild-type algG gene and is capable of being expressed.   
     
     
         52 . A method of producing alginate comprising culturing at least one mutant strain of  P. fluorescens  of  claim 27  under conditions sufficient to produce alginate. 
     
     
         53 . A method of large scale fermentor production of alginate comprising culturing at least one mutant strain of  P. fluorescens  of  claim 27  under conditions sufficient for large scale fermentor production of alginate. 
     
     
         54 . A method of producing alginate comprising culturing at least one mutant strain of  P. fluorescens  of  claim 28  under conditions sufficient to produce alginate. 
     
     
         55 . A method of large scale fermentor production of alginate comprising culturing at least one mutant strain of  P. fluorescens  of  claim 28  under conditions sufficient for large scale fermentor production of alginate. 
     
     
         56 . A composition comprising the alginate produced by the mutant strain of  P. fluorescens  of  claim 33 .