Method of producing succinic acid and other chemiclas using facilitated diffusion for sugar import

Abstract

This invention relates to the production of succinic acid and other chemicals derived from phosphoenolpyruvate (PEP) by fermentation with a microorganism in which the fermentation medium contains one or more sugars, and in which one or more of the sugars is imported into the cell by facilitated diffusion. As a specific example, succinic acid is produced from a glucose-containing renewable feedstock through fermentation using a biocatalyst. Examples of such a biocatalyst include microorganisms that have been enhanced in their ability to utilize glucose as a carbon and energy source. The biocatalysts of the present invention are derived from the genetic manipulation of parental strains that were originally constructed with the goal to produce one or more chemicals (for example succinic acid and/or a salt of succinic acid) at a commercial scale using feedstocks that include, for example, glucose, fructose, or sucrose. The genetic manipulations of the present invention involve the introduction of exogenous genes involved in the transport and metabolism of glucose or fructose into the parental strains. The genes involved in the transport and metabolism of glucose or fructose can also be introduced into a microorganism prior to developing the organism to produce a particular chemical. The genes involved in the transport and metabolism of sucrose can also be used to augment or improve the efficiency of sugar transport and metabolism by strains already known to have some ability for glucose utilization in biological fermentations.

What is claimed is: 
     
         1 . A bacterium producing more than 30 grams per liter of a desired chemical, wherein one of the biosynthetic intermediates for said desired chemical is phosphoenolpyruvate, and said bacterium contains at least one exogenous gene that encodes a protein that functions in the facilitated diffusion of a sugar. 
     
     
         2 . The bacterium of  claim 1 , wherein the bacterium is selected from a group consisting of  Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus  sp. CCM825,  Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus  sp. ATCC 15592,  Rhodococcus  sp. ATCC 19070 , Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, Bacillus subtilis, Bacillus licheniformis, Bacillus  amylolliquefaciens and  Xanthomonas citri.    
     
     
         3 . The bacterium of  claim 1 , wherein the bacterium is selected from a group consisting of  Escherichia coli, Corynebacterium glutamicum Brevibacteium flavum, Mannhemia succiniproducens  and  Anaerobiospirilum succiniproducens.    
     
     
         4 . The bacterium according to  claim 1 , wherein said desired chemical is selected from a group consisting of succinic acid, fumaric acid, glucaric acid, malonic acid, maleic acid, 2,5-furan dicarboxylic acid, propionic acid, 3-hydroxypropionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutryolactone, and butanediols such as 1,4 butnaediol, 1,3-butanediol and 2,3-butanediol. 
     
     
         5 . The bacterium according to  claim 1 , wherein said desired chemical is succinate. 
     
     
         6 . The bacterium according to  claim 1 , wherein said desired chemical is cis, cis-muconic acid. 
     
     
         7 . The bacterium according to  claim 1 , wherein said desired chemical is an aromatic biochemical. 
     
     
         8 . A bacterium according to  claim 1 , wherein the bacterium is a PTS −  bacterial strain. 
     
     
         9 . A bacterium according to  claim 1 , wherein the bacterium has a reduced level of phosphotransferase activity compared to a related wild type strain. 
     
     
         10 . A bacterium of  claim 1  further comprising a mutation or deletion in one or more genes that encode proteins that functions in a phosphotransferase system for sugar import, said mutated or deleted gene being other than a crr gene. 
     
     
         11 . A Bacterium of  claim 10  further comprising a deletion in a gene that encodes a sugar importer that functions using proton symport. 
     
     
         12 . A bacterium of  claim 10  wherein said mutated or deleted gene is a ptsH gene or a homolog thereof. 
     
     
         13 . A bacterium of  claim 10  wherein said mutated or deleted gene ptsI gene or a homolog thereof. 
     
     
         14 . A bacterium of  claim 10  wherein said mutated or deleted genes are selected from a group consisting of ptsH, ptsI, homolog of ptsH and homolog of ptsI. 
     
     
         15 . A bacterium according to  claim 1 , wherein the bacterium is a galP −  bacterial stain. 
     
     
         16 . The bacterium of  claim 1 , wherein said exogenous gene is contained on a replicating plasmid. 
     
     
         17 . The bacterium of  claim 1 , wherein said exogenous gene is integrated into the host chromosome. 
     
     
         18 . The bacterium of  claim 1  wherein said exogenous gene is a glf gene. 
     
     
         19 . The bacterium of  claim 1  wherein said exogenous genes are a glf and a glk genes. 
     
     
         20 . The bacterium of  claim 1  in which said exogenous genes are a glf gene and a frk gene. 
     
     
         21 . The bacterium of  claim 1  in which said exogenous gene is derived from a yeast. 
     
     
         22 . The bacterium of  claim 1  in which said exogenous gene or genes are derived from a strain of  Zymomonas mobilis.    
     
     
         23 . A bacterium of  claim 1 , wherein said bacterium is grown in minimal medium. 
     
     
         24 . A bacterium of  claim 1  wherein said bacterium produces more than 64 grams per liter of a desired chemical. 
     
     
         25 . A bacterium of  claim 1  wherein said bacterium produces more than 83 grams per liter of a desired chemical. 
     
     
         26 . A bacterium containing two or more copies of a functional crr gene or a functional homolog of a crr gene. 
     
     
         27 . A method for producing a desired chemical comprising the steps of:
 growing a bacterium of  claim 1  in a minimal fermentation medium; and   optionally purifying said chemical from the fermentation medium.   
     
     
         28 . A method for producing succinic acid comprising the steps of:
 growing a bacterium comprising at least one exogenous gene that encodes a protein that function in the facilitated diffusion of sugar and producing at least 60 grams of succinate per liter and less than 4.2 grams of acetate per liter in a minimal fermentation medium; and   
       optionally purifying succinic acid from the fermentation medium. 
     
     
         29 . A method for improving the titer and yield of a desired chemical produced by a bacterial strain that has been engineered to use facilitated diffusion for import of a sugar, comprising the steps of:
 subjecting a parent strain to serial transfers into fresh liquid medium;   plating the resulting culture for single colonies on a petri plate; and   choosing a single colony.   
     
     
         30 . A bacterial strain engineered to use facilitated diffusion for import of a sugar comprising a glf-glk operon, wherein said bacterial strain has been evolved for improved titer and yield for succinate production, and further comprising one or more mutations in the glf-glk operon. 
     
     
         31 . A bacterial strain of  claim 30  in which said one or more mutations alter bases in the DNA sequence that corresponds to the 5′ untranslated leader region of an mRNA that encodes either of said glf or glk gene. 
     
     
         32 . A bacterial strain engineered to use facilitated diffusion for import of a sugar comprising a glf gene, wherein said bacterial strain has been evolved for improved titer and yield for succinate production, and said glf gene comprises one or more mutations. 
     
     
         33 . A bacterial strain engineered to use facilitated diffusion for import of a sugar comprising a glk gene, wherein said bacterial strain has been evolved for improved titer and yield for succinate production, and said glk gene comprises one or more mutations.