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United States Patent 19 11 Patent Number: 6,159,738 Donnelly Et Al

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United States Patent 19 11 Patent Number: 6,159,738 Donnelly Et Al USOO6159738A United States Patent 19 11 Patent Number: 6,159,738 Donnelly et al. (45) Date of Patent: Dec. 12, 2000 54 METHOD FOR CONSTRUCTION OF 56) References Cited BACTERIAL STRAINS WITH INCREASED SUCCINIC ACID PRODUCTION PUBLICATIONS Postma et al., Phosphoenolpyruvate Carbohydrate Phospho transferase Systems of Bacteria, Micro. Rev. 57(3): (75) Inventors: Mark I. Donnelly, Warrenville; 543–594, Sep. 1993. Cynthia Sanville-Millard, Plainfield; Ranjini Chatterjee, Park Ridge, all of Primary Examiner Rebecca E. Prouty III. ASSistant Examiner-Richard Hutson Attorney, Agent, or Firm-Cherskov & Flaynik Assignee: University of Chicago, Chicago, Ill. 57 ABSTRACT A fermentation process for producing Succinic acid is pro Appl. No.: 09/067,931 Vided comprising Selecting a bacterial Strain that does not produce Succinic acid in high yield, disrupting the normal Filed: Apr. 28, 1998 regulation of Sugar metabolism of Said bacterial Strain, and combining the mutant bacterial Strain and Selected Sugar in Int. Cl." ............................... C12P 7/46; C12P 7/00; anaerobic conditions to facilitate production of Succinic C12N 15/74; C12N 15/00 acid. Also provided is a method for changing low yield U.S. Cl. ....................... 435/471; 435/140; 435/252.3; Succinic acid producing bacteria to high yield Succinic acid 435/252.31; 435/252.33; 435/252.9; 435/440; producing bacteria comprising Selecting a bacterial Strain 435/145; 435/472; 536/23.2; 536/23.1; having a phosphotransferase System and altering the phos 536/23.7 photransferase System So as to allow the bacterial Strain to 58 Field of Search ................................. 435/145, 252.3, Simultaneously metabolize different Sugars. 435/471, 140, 252.31, 252.33, 252.9, 440, 472; 536/23.2, 23.1, 23.7 9 Claims, 3 Drawing Sheets U.S. Patent Dec. 12, 2000 Sheet 1 of 3 6,159.738 LAC OPERON : OUTSIDE -GLU CELL 16 28 18 14 U.S. Patent Dec. 12, 2000 Sheet 2 of 3 6,159,738 Fermentation of glucose by NZN 111 and AFP310 O 15 TIME (H) circles -glucose Fl 2 Squares - Succinic acid 8 O diamonds - acetic acid X'S - ethanol U.S. Patent Dec. 12, 2000 Sheet 3 of 3 6,159,738 Metabolism of glucose and xylose to succinic acid by AFP111 -H Glucose -a-Xylose -- Succinic acid -%-ACetic acid 40 60 80 TIME (H) 6,159,738 1 2 METHOD FOR CONSTRUCTION OF addition of a solution of cysteine, HCl, and sodium sulfide BACTERIAL STRAINS WITH INCREASED which contains corrosive and toxic H.S. SUCCINIC ACID PRODUCTION Previous efforts by the inventors to produce succinic acid has resulted in the isolation and utilization of a mutant CONTRACTUAL ORIGIN OF THE INVENTION 5 bacterium. The mutant, available as ATCC accession num The U.S. Government has rights in this invention under ber 202021, is the Subject of U.S. patent application Ser. No. Contract No. W-31-109-ENG-38 between the U.S. Depart 08/556,805 to the instant Assignee. U.S. application Ser. No. ment of Energy and the University of Chicago representing 08/556,805, incorporated herein by reference, teaches a Argonne National Laboratory. Succinic acid-producing bacterial Strain (AFP111) which Spontaneously mutates from its precursor. The mutant is able BACKGROUND OF THE INVENTION to grow fermentatively on glucose to produce Succinic acid in high yields, while its precursor is unable to do So. 1. Field of the Invention However, an obvious drawback to utilizing this method of This invention relates to a fermentation method to pro Succinic acid production is its limitation to a single mutant duce Succinic acid, and more particularly this invention 15 organism. relates to a method for creating bacterial Strains capable of None of the prior attempts outlined Supra have resulted in utilizing a myriad of Sugars to produce Succinic acid as a a proceSS for feeding a myriad of feedstocks to Several major fermentation product. different metabolizing organisms to produce high quantities 2. Background of the Invention of Succinic acid. Such flexibility in types of nutrients and Carboxylic acids hold promise as potential precursors for organisms utilized would result in exploiting the lower costs numerous chemicals. For example, Succinic acid can Serve of many feedstocks, Such as corn Stover, logging Slash, and as a feedstock for Such plastic precursors as 1,4-butanediol other lignocellulosic feedstocks, and the higher product (BDO), tetrahydrofuran, and gamma-butyrolactone. New tolerances and robust metabolisms of certain organisms. products derived from Succinic acid are under development, A need exists in the art for a fermentation process with the most notable of these being polyester which is made 25 whereby a myriad of feedstocks can be fed to several by linking Succinic acid and BDO. Generally, esters of different metabolizing organisms to produce large quantities Succinic acids have the potential of being new, "green' of Succinic acid. The process should utilize low cost Solvents that can Supplant more harmful Solvents. In total, nutrients, and perhaps different organisms to facilitate fer Succinic acid could Serve as a precursor for millions of mentation in various environments. The process should also pounds of chemicals annually at a total market value of over yield molar ratioS of approximately 1:1 product-to-growth S1 billion. Along with Succinic acid, other 4-carbon dicar Substrate. boxylic acids, Such as malic acid, and fumaric acid also have feedstock potential. SUMMARY OF THE INVENTION The production of these carboxylic acids from renewable It is an object of the present invention to provide a method feedstocks (in this case through fermentation processes) is 35 for producing Succinic acid that overcomes many of the an avenue to Supplant the more energy intensive methods of disadvantages of the prior art. deriving Such acids from nonrenewable Sources. It is another object of the present invention to provide a Succinate is an intermediate for anaerobic fermentations fermentation process that produces high yields of Succinic by propionate-producing bacteria but those processes result acid. A feature of the invention is the utilization of a mutant in low yields and concentrations. 40 gene in otherwise-intact bacterial genomes. An advantage of Anaerobic rumen bacteria, Such as Bacteroides rumini the invention is the alteration of the genomes of bacteria, cola and Bacteroides amylophilus also produce Succinate. which do not produce Succinic acid in high yield, to create However, rumen organisms are characteristically unstable in bacteria which produce primarily Succinic acid, wherein a fermentation processes. 45 myriad of feedstocks are simultaneously utilized by the It has long been known that a mixture of acids are now-altered host bacteria. produced from E. coli fermentation, as elaborated in Stokes, Still another object of the present invention is to provide J. L. 1949 “Fermentation of glucose by Suspensions of a process for manipulating bacteria to produce large Escherichia coli' J. Bacteriol. 57:147-158. However, for amounts of Succinic acid. A feature of the invention is the each mole of glucose fermented, only 1.2 moles of formic 50 disruption of the normal regulation of Sugar metabolism in acid, 0.1-0.2 moles of lactic acid, and 0.3–0.4 moles of the bacteria. An advantage of the invention is the ability to Succinic acid are produced. AS Such, efforts to produce manipulate a variety of bacteria to facilitate relatively high carboxylic acids fermentatively have resulted in relatively product-to-growth Substrate ratios (i.e., at or above 1:1) in fermentation processes for producing Succinic acid. Another large amounts of growth Substrates, Such as glucose, not advantage of the invention is the ability to utilize bacteria being converted to desired product. 55 which are glucose metabolisers and non-glucose metabolis Some bacteria, Such as A. Succiniciproducens, utilized in CS. fermentation processes as outlined in U.S. Pat. No. 5,143, Yet another object of the present invention is the utiliza 834 to Glassner et al., naturally produce Succinic acid in tion of a mutant gene to shut down catabolite repression moderate titers up to only about 35-40 grams per liter (g/L). processes. A feature of the invention is the homologous The A. Succiniciproducens host strain has been shown to be 60 recombination of the mutant gene into the recipient chro not highly OSmotolerant in that it does not tolerate high moSome of a Selected bacteria. An advantage of the inven concentrations of salts and is further inhibited by moderate tion is a bacteria with its catabolite repression mechanism concentrations of product. Lastly, A. SucciniciproducenS turned off So that both glucose and non-glucose can be presents handling problems in that as an obligate anaerobe, utilized to produce Succinic acid. procedures using the organism must be done in the absence 65 Briefly, the invention provides a fermentation process for of oxygen. Also, medium preparation for the inoculum producing Succinic acid in at least a 1:1 product to Sugar requires the addition of tryptophan and also requires the ratio, comprising Selecting a bacterial Strain incapable pro 6,159,738 3 4 ducing Succinic acid in high yield; disrupting the normal gene 12 is a glucose uptake protein that, in the presence of regulation of Sugar metabolism of Said bacterial Strain; and glucose 14 Stops the use by the bacteria of non-glucose combining the mutant bacterial Strain and Selected Sugar in Sugars. The ptsG gene encodes the cell membrane 16 anaerobic conditions to facilitate production of Succinic component of the glucose-specific phosphenolpyruvate:glu acid. cose phosphotransferase System. An intact ptsG gene takes up and phosphorylates glucose, and causes phosphate to The invention also provides a method for changing fer cleave from the catabolite repression release (crr) protein 18. mentative bacteria which may produce no Succinic acid or At this juncture, crr-P converts to its deactivated low amounts of Succinic acid into ones which produce (dephosphorylated) form crr 24, thereby causing a deacti primarily Succinic acid comprising Selecting a bacterial Vation of the mechanism 20 for utilizing non-glucose Sugars.
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