EP1558743A2 - VERFAHREN ZUR HERSTELLUNG VON BERNSTEINSûURE AUS ROHHYDROLYSATEN - Google Patents

VERFAHREN ZUR HERSTELLUNG VON BERNSTEINSûURE AUS ROHHYDROLYSATEN

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Publication number
EP1558743A2
EP1558743A2 EP02778771A EP02778771A EP1558743A2 EP 1558743 A2 EP1558743 A2 EP 1558743A2 EP 02778771 A EP02778771 A EP 02778771A EP 02778771 A EP02778771 A EP 02778771A EP 1558743 A2 EP1558743 A2 EP 1558743A2
Authority
EP
European Patent Office
Prior art keywords
recited
succinic acid
mutant
organism
hydrolysate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02778771A
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English (en)
French (fr)
Other versions
EP1558743A4 (de
Inventor
Nhuan Phu Nghiem
Mark Donnelly
Cynthia Y. Sanville-Millard
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UT Battelle LLC
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UT Battelle LLC
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Publication date
Application filed by UT Battelle LLC filed Critical UT Battelle LLC
Priority claimed from PCT/US2002/035761 external-priority patent/WO2004043881A2/en
Publication of EP1558743A2 publication Critical patent/EP1558743A2/de
Publication of EP1558743A4 publication Critical patent/EP1558743A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

Definitions

  • This invention relates to a fermentation method to produce succinic acid, and more particularly this invention relates to a method for creating a bacterial strain capable of utilizing a myriad of sugars to produce succinic acid as a major fermentation product.
  • succinic acid can serve as a feedstock for such plastic precursors as 1,4 butanediol
  • Anaerobic rumen bacteria such as Bacteroides ruminicola and Bacteroides a ylophilus also produce succinate.
  • rumen organisms are characteristically unstable in fermentation processes. It has been long been known that a mixture of acids are produced from E. coli fermentation, as elaborated in Stokes, J.L. 1949 "Fermentation of glucose by suspensions of Escherichia coli" J. Bacteriol. 57:147-158. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to the desired product.
  • A. succiniciproducens utilized in fermentation processes as outlined in U.S. Patent No. 5,143,834 to Glassner et al., naturally produce succinic acid in moderate liters up to only about 35-40 grams per liter (g/L).
  • the A. succiniciproducens host strain has been shown to be not highly osmotolerant in that it does not tolerate high concentrations of salts and is further inhibited by moderate concentrations of product.
  • A. succiniciproducens presents handling in that as an obligate anaerobe, procedures using the organism must be done in the absence of oxygen. Also, medium preparation for the inoculum requires the addition of tryptophan.
  • the method should be enabled by any organism having a particular, and easily determined, genotype.
  • the method should be able to be performed in relatively inert conditions using robust organisms (i.e., those having high feed back inhibition thresholds), and also so as to obviate the need for sophisticated environmental control measures.
  • the method should produce superior results utilizing mixtures of sugars derived from hydrolysis of lignocellulosic materials, inasmuch as these substrates offer a cheaper source of sugars, and as such, their use could reduce production costs for succinic acid.
  • a feature of the invention is the utilization of bacterial genomes containing a plurality of mutant genes to enable the method.
  • An advantage of the invention is that bacteria can be readily manipulated to produce the plurality of mutants.
  • bacteria already containing the plurality of mutations can be utilized without further manipulation.
  • Still another object of the present invention is to provide a process for manipulating bacteria to produce large amounts of succinic acid.
  • a feature of the invention is the disruption of the normal regulation of sugar metabolism in the bacteria.
  • An advantage of the invention is the ability to manipulate a variety of bacteria to facilitate relatively high product- to-growth substrate ratios (i.e., at or above 1 :1) in fermentation processes for producing succinic acid.
  • Another advantage of the invention is the ability to utilize bacteria which become glucose metabolisers and non-glucose metabolisers.
  • Yet another object of the present invention is to produce succinic acid fermentatively.
  • a feature of the invention is the utilization of bacteria containing altered phosphotransferase (pts) systems, pyruvate formate lyase (pfl) systems, and lactate dehydrogenase (ldh) systems.
  • An advantage of the invention is that the bacteria can be derived from many genera which use these enzyme systems for sugar fermentation.
  • a method of producing succinic acid from industrial-grade hydrolysates comprising: supplying an organism that contains mutations for the genes ptsG, pflB, and IdhA; allowing said organism to accumulate biomass; and allowing said organism to metabolize the hydrolysate.
  • a bacteria mutant characterized in that it produces succinic acid from substrate contained in industrial-grade hydrolysate in a ratio of between 0.6:1 and 1.3:1 succinic acid to substrate (e.g., between 0.6 and 1.3 grams succinic acid per gram of total sugar consumed).
  • FIG. 1 is a graph depicting an enhanced production of succinic acid after transformation of a bacteria with a mutant gene, in accordance with features of the present invention
  • FIG. 2 is a graph depicting fermentation of industrial hydrolysate via a triple mutant organism, in accordance with features of the present invention
  • FIG. 3 is a graph depicting fermentation of synthetic sugar via a triple mutant organism, in accordance with features of the present invention.
  • the inventors have developed a method for fermentatively producing high yields of succinic acid.
  • the method exploits altered catabolite repression mechanisms of selected organisms so as to allow the organisms to produce succinic acid using mixtures of glucose and non-glucose feedstocks.
  • the resulting mutants and protocols result in a succinate to feedstock ratio of up to 1.3:1, and typically 0.9:1.
  • Succinate accumulations of between 60 g/L and 75 g/L are achieved.
  • Typical protocol durations are more than 70 hours, and usually between 120 and 170 hours.
  • yields of 70 g/L are obtained after 160 hours.
  • the process is viable at from between approximately 25 °C and 45 °C, with a preferable range of approximately 30 to 39 °C.
  • a pH of between 5 and 9 is suitable, with a more preferable range of approximately 6.1 and 7.2.
  • the invented mutants are especially viable components of the fermentative protocol inasmuch as they have increased tolerance to fermentative products. For example, concentrations of 72 g/L for succinate, 22 g/L for acetate, 14 g/L for ethanol, and 8 g/L for lactate are achievable without inducing feedback inhibition.
  • Feedstock Detail A salient feature of the invented method and mutant is the direct utilization of industrial feedstocks.
  • feedstocks can be utilized, including, but not limited to light steep water, lignocellulosic hydrolysate produced by various methods of hydrolysis, corn-derived sugar solutions (such as corn steep liquor), lactose from whey, and other industrial-grade sugars.
  • lignocellulosic hydrolysate produced by concentrated acid hydrolysis, or dilute acid hydrolysis, enzyme hydrolysis or hydrolysates produced by a combination of these processes are all suitable.
  • Corn-derived sugar solutions are also suitable.
  • Industrial feedstocks generally are mixtures of glucose and other sugars, the most common non-glucose sugar being xylose.
  • FIG. 2 depicts the utilization of glucose and xylose by one of the invented mutants.
  • any feedstocks containing glucose and/or non-glucose sugars are suitable.
  • feedstocks containing glucose, sorbitol, xylose, arabi-nose, mannose, lactose, glucuronic acid, galactose, fructose, and combinations thereof are appropriate.
  • the invented method utilizes organisms containing alterations in the catabolite repression system of the organisms.
  • the inventors have found that when alterations exist to the phosphotransferase (pts) system, pyruvate formate lyase (pfl) system, and lactate dehydrogenase (ldh) system of bacteria, these bacteria are suitable for use in the invented succinic acid producing process.
  • pts phosphotransferase
  • pfl pyruvate formate lyase
  • lactate dehydrogenase lactate dehydrogenase
  • the only limitation on the type of organism utilized in the invented fermentative process is that the organism originally must have these systems.
  • An organism naturally containing alterations in these systems i.e., spontaneous mutants), or organisms which are specifically altered, can be utilized.
  • fermentative bacteria having no or low succinic acid product yields i.e., less than 0.5 moles per one mole of fed growth substrate
  • bacteria having high succinic acid product yields i.e., greater than or equal to 1 mole of succinic acid per one more of fed growth substrate.
  • Any bacteria able to make any succinic acid fermentatively are particularly suitable transduction candidates, including but not limited to gram-negative and gram-positive fermentative bacteria.
  • suitable strains include but are not limited to E.coli, Klebsiella, Erwinia, and Lactobacillus.
  • Organisms to be altered to include the three knockouts are modified by serial transduction using bacteriophage P 1. Standard P 1 transduction protocols were utilized, an exemplary protocol disclosed in J. H. Miller, ed. Experiments in Molecular Genetics 1972 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and incorporated herein by reference. Using this method, wild-type or near wild-type strains of bacteria (e.g., the C600 strain of E.coli; ATTC accession no. 23724) can be used to create mutant substrains that lack one, two, or three functional genes selected from pfl, ldh, ptsG.
  • Standard P 1 transduction protocols were utilized, an exemplary protocol disclosed in J. H. Miller, ed. Experiments in Molecular Genetics 1972 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and incorporated herein by reference.
  • wild-type or near wild-type strains of bacteria e.g., the C600 strain of E.coli; ATTC acces
  • AFP 184 has the pfl deletion, ldh knockout, and the different mutant form of ptsG deliberately inserted into a near wild-type strain of E. coli.
  • Another strain called AFP 415 might also be used.
  • AFP 415 differs from AFP 184 only in having the knockout of ptsG. It performs similarly to AFP 184.
  • Table 1 provides a comparison of succinic acid production by AFP 184 and a W1485 derivative (AFP 111). It is noteworthy that while the W1485 derivative utilized fairly refined feedstocks, AFP 184 still provided higher values with industrial grade hydrolysates.
  • a mutation containing all three knockouts also can be generated using a bacterium already containing one or two of the genetic anomalies, and then inducing the remainder knockout(s).
  • a viable starting organism is W1485, ATCC Accession Number 12435.
  • AFP 400 is a deliberately-made triple knockout. It contains the pfl deletion by
  • FMJ123 is produced pursuant to the protocol found in P.K. Bunch et al. (1997) Microbiology 143, 187-195, and incorporated herein by reference.
  • AFP 400 also contains the IdhA knockout, also made by Clark and inserted into FMJ123 to produce DC1327.
  • DC1327 is produced pursuant to the protocol found in Chatterjee et al, Appl. Environ. Microbiol. 67, pp 148- 154, and incorporated by reference.
  • AFP 400 contains the ptsG knockout, as described in the Chatterjee reference.
  • a triple knockout AFP404 was also constructed by introduction of three knockouts into strain C600.
  • AFP404 is similar to AFP 184 but has a knockout of ptsG rather than a point mutation of the gene. It also produces succinic acid in a yield of approximately 1 mol/mol glucose.
  • the native ptsG gene of E. coli was cloned by PCR from genomic DNA prepared from W1485 using primers targeting the N- and C- termini of the protein with no additional genomic sequences amplified.
  • the gene was cloned in the vector pFJl 18EH to give pJFptsG.
  • the gene was disrupted by insertion of the kanamycin resistance cassette of pUC-4K
  • NZN 111 already includes a kanamycin resistance marker, an equivalent stain was constructed by transducing TnlO-inactivated IdhA gene from stain SE1752 into FMJ123. The resulting strain, DC1327, was indistinguishable in its physiology from NZN 111.
  • the disrupted ptsG gene was transferred in DC 1327 by transforming the cells with pTSGK, growing the cells for approximately 30 generations in the presence of kanamycin and absence of ampicillin, then plating the culture on LB plates containing glucose and incubating anaerobically. Colonies that were able to grow fermentatively were purified and screened for their sensitivity to the two antibiotics.
  • AFP400 was isolated as a stable kanamycin resistant, ampicillin sensitive strain that fermented glucose to succinate, acetate, and ethanol. Proper integration of the disrupted ptsG gene was confirmed by PCR. The disrupted gene was amplified from AFP400 DNA using primers that matched flanking sequences approximately 110 base pairs outside the coding region of the gene. These sequences were not present in the integration vector. The resulting product was 3.0 kb in size, as predicted from the known sequence ptsG, its flanking regions, and the Kanamycin insert.
  • the product was digested with Clal (site in the kanamycin cassette) and Agel (site in ptsG), and generated the fragments expected for insertion of the cassette into the Mfel site of ptsG (1.95 and 1.05 kb for Clal, and 2.3 and 0.7 kb for Agel).
  • Yet another strain containing the three knock outs, AFP 404, is also derived from C600, a near wild-type E. coli K12 strain, using the same protocol above.
  • the knockouts are introduced by putting a copy of the knock-out gene, having a resistance marker, into the cells. Homologous recombination is allowed to occur, as facilitated by host enzymes. The chromosome containing the marker is then selected. The ptsG knockout was introduced this way. Proof of its insertion, via PCR, is detailed in Chatterjee, et al., previously incorporated by reference. Growth Detail
  • the triple mutant organisms produced by the inventors are not obligate anaerobes. As such, initial accumulation of biomass can occur aerobically, after which fermentative conditions are established.
  • the advantages of this two-stage process (i.e., aerobic-then anaerobic) protocol are illustrated in FIG. 2 wherein the rate of production of succinic acid is much larger compared to the single-stage anaerobic protocol growth curve of FIG. 1.
  • a fermenter In industrial protocols, a fermenter is charged with light steep water plus lignocellulosic hydrolysate'. Antibiotics were included as necessary at the following concentrations: 100 ⁇ g of carbenicillin per ml, 30 ⁇ g of kanamycin per ml, 10 ⁇ g of tetracycline per ml, and 30 ⁇ g of chloramphenicol per ml. Rich broth contained (per liter), 10 g of tryptone, 5 g of NaCl, and 1 g of yeast extract. Solid media for plates contained 1.5 percent (wt/vol) Difco Bacto-Agar. Minimal medium E was prepared as described in Vogel, HJ. 1956 Acetylornithinase in E. coli, . Biol.
  • hinocula for the anaerobic liquid cultures were prepared by growing the strains aerobically overnight in LB medium supplemented with antibiotic. A sample of the overnight culture was diluted 100-fold in fresh media and allowed to grow aerobically to an A600 of approximately 1 ; the anaerobic growth media was inoculated with 1 ml of the innocula. Samples were removed anoxically from the sealed tubes at appropriate times for analysis of levels of glucose (or alternate sugar substrates) remaining and fermentation products formed. For anaerobic growth on solid media, agar plates were incubated at 37°C in an anaerobic jar under an H2-CO2 atmosphere generated by use of a Gas-Pak.
  • LB or Medium E-agar are two of several mediums which can be utilized.
  • Medium E-agar is a minimum-nutrient medium commonly used, and discussed in Vogel, H.J., 1956 Acetylornithase in E. coli, J. Bio/Chem 218:97-103 and incorporated herein by reference, hi exemplary protocols, LB or Medium E-agar is supplemented with 4 g/L of glucose, 4 g/L of lactose, 3 mg/L of 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside (X-gal), and antibiotics.
  • X-gal/glucose agar These media are hereinafter referred to as X-gal/glucose agar.
  • the inventors also have devised a method for utilizing the mutant in a continuous process. Repetitive experiments were conducted in which after the culture had produced approximately 50 g/L succinic acid, one milliliter of the mixture was added to a fresh enclosure containing LB media, glucose and MgCO3. This new innoculum continued to produce succinic acid effectively. This process was repeated 3-4 times, in each case resulting in efficient production of succinic acid.
  • AFP 184 was placed in a fermenter with true hydrolysate, from rice straw.
  • An exemplary hydrolysate is that commercially prepared and made available from Arkenol Inc., of Mission Viejo, CA, via its concentrated acid hydrolysis process.
  • the rice straw medium contains approximately 600 g/L glucose and 169 g/L xylose as the two main sugar components, plus minor quantities of other sugars.
  • the experimental data are found in Table 2 and in FIG. 2.
  • the fermentation medium contained the following components: Difco yeast extract 5 g/L, tryptone 10 g/L, (NH 4 )2SO 4 2 g/L, MgSO 4 -7H 2 O 0.2 g/L, NaCl 10 g/L, K 2 HPO 4 7 g/L, KH 2 PO 4 3 g/L, Arkenol's hydrolysate 16.5 mg/L, and kanamycin 30 mg/L.
  • the industrial hydrolysate contained 607 g/L glucose and 169 g/L xylose as the two main sugar components plus minor quantities of other sugars.
  • the medium with all of the components except the antibiotic was autoclaved at 121°C for 20 minutes. Kanamycin then was added upon cooling.
  • This fermentation medium was used for both the inoculum flasks and the one-liter fermenter.
  • 50 mg medium was placed in a 250-mg flask and inoculated with 0.2 mg of the AFP184 stock culture which was maintained in 30% glycerol and at -70°C.
  • the flask was incubated in a incubator shaker at 37°C and 250 rpm overnight (about 16 hours). The entire flask contents then were used to inoculate the fermenter which was maintained at 37°C.
  • the medium in the fermenter was aerated to allow fast growth of the organism.
  • a fermentation protocol was developed utilizing AFP 184 in combination with a synthetic sugar feedstock. As can be noted on FIG. 3, succinate production was rapid up to
  • the fermentation medium contained the following components: Difco yeast extract 5 g/L, tryptone 10 g/L, (NH 4 )2SO 4 2 g/L, MgSO 4 -7H 2 O 0.2 g/L, NaCl 10 g/L, K 2 HPO 4 7 g/L, KH 2 PO 4 3 g/L, glucose 7.6 g/L, xylose 1.85 g/L, and kanamycin 30 mg/L.
  • the medium with all of the components except the antibiotic was autoclaved at 121°C for 20 minutes.
  • Kanamycin then was added upon cooling.
  • This fermentation medium was used for both the inoculum flasks and the one-liter fermenter.
  • 50 mg medium was placed in a 250-mg flask and inoculated with 0.2 mg of the AFP 184 stock culture which was maintained in 30% glycerol and at -70°C.
  • the flask was incubated in a incubator shaker at
  • the medium in the fermenter was aerated to allow fast growth of the organism. After six hours when the required cell mass was achieved, the following actions were taken: 1. Air was turned off to exert anaerobic conditions, which would initiate production of succinic acid; 2. Carbon dioxide gas was sparged into the medium at a rate of 0.03 mg per minute; and
  • Table 3, infra, and FIG. 3 illustrate the succinic acid production resulting from the utilization of the synthetic sugar mixture.

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EP02778771A 2002-11-07 2002-11-07 VERFAHREN ZUR HERSTELLUNG VON BERNSTEINSûURE AUS ROHHYDROLYSATEN Withdrawn EP1558743A4 (de)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2002/035761 WO2004043881A2 (en) 2001-03-30 2002-11-07 A method to produce succinic acid from raw hydrolysates

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EP1558743A2 true EP1558743A2 (de) 2005-08-03
EP1558743A4 EP1558743A4 (de) 2007-11-14

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EP (1) EP1558743A4 (de)
JP (1) JP2006505276A (de)
CN (1) CN1886516A (de)
AU (1) AU2002340410A1 (de)
BR (1) BR0215933A (de)
CA (1) CA2505423A1 (de)
HU (1) HUP0500960A2 (de)
MX (1) MXPA05004865A (de)

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KR101581504B1 (ko) * 2013-03-18 2015-12-31 지에스칼텍스 주식회사 2,3-부탄디올의 생성능이 증강된 재조합 미생물 및 이를 이용한 2,3-부탄디올의 생산 방법

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Abstracts of Papers, American Chemical Society; 221st National Meeting of the ACS; San Diego, California, USA; April 01-05, 2001 Nghiem et al: Production of succinic acid from lignocellulosic materials XP008084505 *
CHATTERJEE ET AL: "Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 67, 2001, pages 148-154, XP000996310 *
HONG ET AL: "Metabolic flux analysis for succinic acid production by recombinant Escherichia coli with amplified malic enzyme activity" BIOTECHNOLOGY AND BIOENGINEERING, vol. 74, 2001, pages 89-95, XP003009624 *
See also references of WO2004043881A2 *
VEMURI ET AL: "Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, April 2002 (2002-04), pages 1715-1727, XP002987293 *
VEMURI ET AL: "Succinate production in dual-phase Escherischia coli fermentations depends on the time of transition from aerobic to anaerobic conditions" JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, vol. 28, June 2002 (2002-06), pages 325-332, XP003009348 *

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BR0215933A (pt) 2005-09-27
EP1558743A4 (de) 2007-11-14
AU2002340410A1 (en) 2004-06-03
HUP0500960A2 (en) 2006-01-30
MXPA05004865A (es) 2005-07-22
CA2505423A1 (en) 2004-05-27
JP2006505276A (ja) 2006-02-16
CN1886516A (zh) 2006-12-27

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