EP0660875A1 - Procede d'augmentation de l'efficacite de la conversion du carbone dans des micro-organismes - Google Patents

Procede d'augmentation de l'efficacite de la conversion du carbone dans des micro-organismes

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Publication number
EP0660875A1
EP0660875A1 EP94918169A EP94918169A EP0660875A1 EP 0660875 A1 EP0660875 A1 EP 0660875A1 EP 94918169 A EP94918169 A EP 94918169A EP 94918169 A EP94918169 A EP 94918169A EP 0660875 A1 EP0660875 A1 EP 0660875A1
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EP
European Patent Office
Prior art keywords
microorganism
carbon
conversion efficiency
glyoxylate
carbon conversion
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Withdrawn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
EP94918169A
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German (de)
English (en)
Other versions
EP0660875A4 (fr
Inventor
Sandra J. Koehn
Thomas M. Evans
Richard A. Nelson
Paul P. Taylor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nutrasweet Co
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Nutrasweet Co
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Publication date
Application filed by Nutrasweet Co filed Critical Nutrasweet Co
Publication of EP0660875A1 publication Critical patent/EP0660875A1/fr
Publication of EP0660875A4 publication Critical patent/EP0660875A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine

Definitions

  • the present invention generally relates to methods for increasing the carbon conversion efficiency of microorganisms.
  • TCA cycle tricarboxylic acid cycle
  • bacterial cells and other microorganisms utilize the TCA cycle as a primary metabolic pathway.
  • the TCA cycle generates energy sources via the oxidation of acetyl groups which enter the cycle as acetyl CoA molecules.
  • the TCA cycle primarily produces energy and various four-carbon intermediates, such as oxaloacetate, which serve as precursors in the synthesis of essential cell components such as amino acids.
  • These four-carbon intermediates result from two decarboxylation reactions in the TCA cycle, both generating carbon dioxide (CO->).
  • the first decarboxylation reaction occurs when the six-carbon metabolit, isocitrate, is converted to the five-carbon compound, ⁇ -ketoglutarate, by the enzyme isocitrate dehydrogenase.
  • the second carbon dioxide-producing reaction occurs in the next step of the TCA cycle, wherein ⁇ -ketoglutarate is converted to the four-carbon intermediate, succinyl CoA.
  • carbon conversion efficiency generally refers to a ratio of the amount of substrate metabolized to the amount of biosynthetic products formed by a microorganism. The greater the loss of carbon during the process of conversion from substrate to biosynthetic products the greater the inefficiency of carbon conversion.
  • TCA cycle is a predominant metabolic pathway in a microorganism
  • carbon conversion is limited by the amount of carbon lost through the aforementioned carbon-dioxide-evolving steps.
  • the present invention provides methods for increasing carbon conversion efficiency under such conditions.
  • the present invention provides methods for increasing carbon conversion efficiency in a microorganism.
  • methods for increasing carbon conversion efficiency are provided, wherein DNA encoding isocitrate lyase, alate synthase, and isocitrate dehydrogenase kinase/phosphatase is incorporated into the microorganism which is then cultivated under appropriate conditions in the presence of a substrate metabolized by the TCA cycle and wherein biosynthetic product may be isolated.
  • the DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase comprises a portion of the glyoxylate operon.
  • a microorganism according to the present invention may be any microorganism which normally utilizes the TCA cycle, but may preferably be a bacterial cell, and most preferably an Escherichia coli cell.
  • the DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase may be incorporated in the microorganism in an appropriate vector, such as a plasmid. Also in a preferred embodiment of the invention, the method further comprises calculating the carbon conversion efficiency of a microorganism.
  • a two-carbon compound such as acetate
  • the two-carbon compound may be utilized in the TCA cycle to produce energy.
  • both carbon atoms are lost as CO 2 , thus depleting carbon available for formation of biosynthetic products.
  • Microorganisms adjust to this situation by channeling some isocitrate into the glyoxylate cycle.
  • the interrelation of the TCA cycle and the glyoxylate cycle is depicted in Figure 1.
  • Carbon flow to the glyoxylate cycle is controlled by the bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (hereinafter referred to as "IDH k/p").
  • the glyoxylate cycle is repressed when glucose is available as a source of carbon.
  • IDH k/p reduces activity of isocitrate dehydrogenase, thus allowing the enzyme isocitrate lyase of the glyoxylate cycle to act upon isocitrate with reduced competition from isocitrate dehydrogenase.
  • the enzymes of the glyoxylate cycle in Escherichia coli are encoded by genes comprising the glyoxylate operon.
  • the known structural organization of the operon comprise the aceB gene which encodes malate synthase, the aceA gene which encodes isocitrate lyase, and the aceK gene which encodes IDH k/p. Transcription of the operon in its native sequence is under the negative regulation of the iclR gene and, to a lesser extent, X &fadR gene which maps outside the glyoxylate operon in E. coli. Cortay et al , Embo J. , 10:675- 679 (1991).
  • Two enzymes function in the glyoxylate cycle to convert isocitrate and acetyl CoA to succinate and malate. These are isocitrate lyase, which converts isocitrate to succinate and glyoxylate, and malate synthase, which catalyzes the conversion of acetyl CoA and glyoxylate to malate.
  • the net effect of the glyoxylate cycle is to avoid the carbon dioxide-evolving steps of the TCA cycle, thus generating four-carbon compounds from two-carbon precursors for use in the formation of cellular components.
  • the present invention provides methods for increasing the carbon conversion efficiency of microorganisms, thus resulting in numerous benefits to industrial processes utilizing microorganisms.
  • increasing carbon conversion efficiency allows the use of less substrate to manufacture a given amount of biosynthetic product as compared to cells not treated according to methods of the present invention.
  • a given amount of metabolized substrate such as glucose
  • Increased carbon conversion efficiency may also be utilized to effect increases in the production of a particular amino acid for use in industrial applications.
  • Microorganisms which display increased carbon conversion efficiency may also display increase efficiency in biomass production generally.
  • Carbon conversion efficiency is a measure of the extent to which carbon from substrates, such as glucose, are incorporated into the intermediates and products of cellular biosynthesis. Carbon conversion efficiency may be measured based on the accumulation of any product or products of cellular biosynthesis and its (their) precursors or on the basis of an increase in biomass with a fixed amount of substrate or on the basis of production of a given amount of biomass utilizing less substrate. Examples of products which may be used to measure carbon conversion efficiency include biomass generally, amino acids, nucleic acids, various structural proteins, enzymes, secondary metabolites and their precursors.
  • Example 1 relates to the use of exemplary plasmids comprising glyoxylate operon genes and the incorporation of such plasmids into exemplary host cells.
  • Example 2 provides results of enzymatic assays to confirm proper incorporation and function of a portion of the glyoxylate genes in host cells according to the invention.
  • Example 3 provides culture and fermentation conditions under which carbon conversion efficiency may be measured.
  • Example 4 relates to an exemplary means of calculating carbon conversion efficiency according to the present invention.
  • Escherichia coli, strain ATCC 13281, a tyrosine auxotroph was used as an exemplary host cell to demonstrate methods according to the present invention.
  • the skilled artisan realizes, however, that numerous host microorganisms may be used in the practice of the invention. Representative host microorganisms are reported in Kornberg et al., Advan. Enzymol., 23:401-470 (1961), incorporated by reference herein.
  • Plasmids pCL8 and pCLlOOO were used as the source of genes encoding glyoxylate enzymes. These plasmids contain the aceA, aceB, and aceK genes of the glyoxylate operon in a pBR322 vector and have been described in
  • Plasmids pCL8 and pCLlOOO were incorporated into E. coli host cells by electroporation. Prior to electroporation, a single colony of ATCC 13281
  • E. coli cells was inoculated into a 500 ml baffled flask containing 100 mis Luria Broth (Difco, Detroit, MI). The culture was grown on a rotary incubator/ shaker
  • plasmids contain an ampicillin resistance marker. Additional transformation procedures are known and available to those skilled in the art and may be used in the practice of the present invention. Positive transformants were re-designated NS3119 (containing pCL8) and NS3120 (containing pCLlOOO). NS3120 (containing plasmid pCLlOOO) was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on April 27, 1993 and given ATCC Accession No. 69291. In Example 2, positive transformants were tested to confirm that the glyoxylate cycle was operating.
  • Example 1 In order to confirm that host cells described in Example 1 were transformed with functional glyoxylate cycle genes, assays were conducted to determine the activity of glyoxylate cycle enzymes.
  • cell extracts were prepared by growing cultures of transformed host cells overnight in M63 medium [Chung, et ah, J. Bacteriol., 770:386-391 (1988), incorporated by reference herein] in a rotary /incubator shaker at 37° C and 300 rpm. A 20 ml aliquot of the culture was transferred into a 50 ml polypropylene centrifuge tube (Corning, Corning NY) and centrifuged for 10 minutes at 10,000 g and 4° C in a Beckman S2-21 centrifuge (Beckman). The pellet was resuspended in 4 ml of 0.1 M potassium phosphate buffer, pH 7.0.
  • the cells were then disrupted using a French Pressure Cell (SLM Instruments, Urbana, IL) at 1000 psi according to the manufacturer's instructions.
  • SLM Instruments, Urbana, IL French Pressure Cell
  • the resulting cell debris was removed by centrifuging at 1300 g for 8 minutes in an MSE microcentrifuge (MSE, Micro Centaur, U.K.).
  • MSE Micro Centaur, U.K.
  • the cell lysates described above were then tested for isocitrate lyase and malate synthase activity.
  • the isocitrate lyase assay was based on the protocol described in Dixon et al. , Biochem. J., 72:3V (1959) incorporated by reference herein, with 0.1 M potassium phosphate buffer substituted for the tris buffer described in the assay reported in Dixon.
  • the assay is based upon the rate of increase in optical density with increased isocitrate lyase activity as measured by the accumulation of glyoxylic acid phenylhydrazine. To conduct the assay, 0.10 ml of 100 mM phenylhydrazine,
  • 0.05 ml of 20 M cysteine, 0.05 ml of 100 mM MgCl 2 , and 0.05 ml of the cell lysate described above were placed in a 1 ml quartz cuvette with 0.10 ml 100 mM potassium phosphate buffer in 1.10 ml distilled water at pH7.2.
  • the optical density of the above mixture was adjusted to zero in a Hewlett-Packard model 8452A diode array spectrophotometer equipped with a kinetics software package in a Hewlett-Packard operating software package A.02.0.
  • the reaction whereby glyoxylate (and ultimately glyoxylic acid phenylhydrazine) is formed from isocitrate was begun by addition to the cuvette of 0.05 ml 20 mM potassium isocitrate. Absorbance was read at 324 nm and the amount of isocitrate lyase was determined by an increase in optical density.
  • isocitrate lyase activity increases significantly in cell lysates from transformants containing the glyoxylate cycle genes as compared to the same measurements in cell lysates from untransformed controls.
  • the malate synthase assay measures the decrease in optical density with an increase in breakdown of the thio-ester bond in acetyl COA which reacts with malate synthase to form malate in the presence of glyoxylate.
  • the malate synthase assay was conducted by first making a stock solution comprising 4.0 ml 0.1 M tris, 0.50 ml 0.002 M acetyl COA and 0.1 M MgCl 2 .
  • Example 3 provides fermentation and growth conditions which were used to prepare cultures for carbon conversion efficiency measurements. Table 1
  • transformed cells were fermented as follows.
  • Fermentations were conducted using 20 L fermentors (LSL Biolafitte, France). The temperature, pH, agitation, and air flow were kept constant at 32° C, 7.2, 500 rpm, and 11 L/minutes, respectively. Glucose feed, dissolved oxygen, and other parameters were varied and may be set at values determined by the skilled artisan.
  • the fermentor contained 10 L of K12 medium [Konstantinov et al. , J. Fermentation and Bio Engineering, 70(4):253-260 (1990), incorporated by reference herein] supplemented with 1 g/L yeast extract and was sterilized at 121° C for 30 minutes. The fermentor was then inoculated with 1 L of the cell culture containing either the untransformed strains or the glyoxylate cycle transformants. The back pressure of the fermentor was regulated at 10 psi and the dissolved oxygen concentration was initially 100% and was allowed to reach a value of 15% where it was maintained throughout the fermentation. A 70% (weight per volume) glucose solution was initially added until the fermentor contained 3.0 g/L glucose.
  • the glucose was then continuously added into the fermentor to maintain glucose in excess during exponential growth.
  • the final biomass in the fermentor was limited by tyrosine (E. coli strain ATCC 13281 is a tyrosine auxotroph).
  • the concentration of glucose was then allowed to decrease to approximately 0.2 g/L and was maintained at that concentration for the rest of the fermentation.
  • the total fermentation time was 48 hrs.
  • the concentrations of glucose, L-phenylalanine, prephenic acid, phenylpyruvic acid, and acetate were then measured to determine carbon conversion efficiency as taught in Example 4.
  • Carbon conversion efficiency relates to the ratio of carbon (glucose or another carbon source) metabolized to the amount of carbon ultimately incorporated in biosynthetic precursors and end products. It is apparent to any skilled artisan that numerous biosynthetic precursors and end products may be used as a basis for the calculation of carbon conversion efficiency. Examples of such compounds are amino acids, especially the aromatic amino acids, nucleotides, various enzymes, and structural proteins.
  • the methods of calculating carbon conversion efficiency are exemplified herein using L-phenylalanine and its two immediate precursors as a measure of the incorporation of carbon from the precursor, glucose. The amount of L-phenylalanine, prephenic acid, and phenylpyruvic acid and acetate which accumulated in the fermentor and the amount of glucose consumed were measured in order to calculate carbon conversion efficiency as provided below.
  • the accumulation of L-phenylalanine was measured using flow- switching High Performance Liquid Chromatography.
  • a flow-switching column allows faster clearance of phases not containing L-phenylalanine through the column.
  • High performance liquid chromatography is well-known in the art and the skilled artisan is aware of the application of numerous such techniques to determine the concentration of any biosynthetic product, such as L-phenylalanine.
  • the chromatographic apparatus comprised two Waters (Waters, Milford, MA) Model 600 A solvent delivery systems, an Applied Biosystems (Foster City, CA) 983 programmable detector, a Waters Model 710B auto sampler and model 680 automatic gradient controller, all used according to the manufacturer's instructions.
  • a Vici (Valco Instruments, Houston, TX) 10-part column-switching valve was used according to the manufacturer's instruction to achieve flow reversal and a Fisions Multichrome Data Collection System (Danvers, MA) was used to analyze the data.
  • Phenylpyruvic acid is the immediate precursor of phenylalanine in the phenylalanine biosynthetic pathway. Phenylpyruvic acid concentration provides, in part, a measure of carbon conversion because, at any point in time, not all the carbon from glucose which has been transferred to the biosynthesis of cellular components will be in the form of L-phenylalanine. Some of the carbon will be in the form of the immediate precursors of phenylalanine. Thus, measurement of those precursors in addition to phenylalanine provides a more accurate measure of carbon conversion than would phenylalanine measurements alone.
  • Phenylpyruvic acid concentration was measured in samples taken from fermentation broths containing either transformed or untransformed E. coli cells as described above by reverse-phase High Performance Liquid Chromatography at a detection wavelength of 214 nm, 0.2 AUFS (Absorbance Units Full Scale) using a Whatman RAC II Partisil 5 ODS-3 10 cm column (Whatman, Hillsboro, OR) according to the manufacturer's instructions.
  • An isocratic method was used, with a mobile phase comprising 10 mis of 1 % 0.5 M pentane sulfonic acid solution, 950 ml water, and 40 ml acetonitrile, pH.2.5.
  • a volume of 10 ⁇ l was injected onto the column at a flow rate of 2 ml/minutes and the run time was 10 minutes
  • Phenylpyruvic acid standards were also run at concentrations of 0.02, 0.08, and 0.4 mg/ml.
  • Prephenic acid is the immediate precursor of phenylpyruvic acid in the pathway leading to synthesis of phenylalanine.
  • concentration of prephenic acid was determined by High Performance Liquid Chromatography at a detection wavelength of 220 nm, 0.2 AUFS, using a Supelco LC10 column (Supelco). An isocratic method was used, with the mobile phase comprising 10% acetonitrile in
  • the amount of glucose concentration of the feed stock was measured using High Performance Liquid Chromatograph equipped with a refractive index detector and a column heater. A 400 mg sample was obtained from the glucose being fed into the fermentor and diluted to 100 ml.
  • a Biorad A Biorad
  • HPX-878 column equilibrated to 85° C was used with a mobile phase comprising Milli-Q water (Millipore, Bedford, MA). The flow rate was 0.6 ml/minutes and the run time was 20 minutes. A 20 ⁇ l volume of sample was injected on the column at time zero. The column was equilibrated for 30 minutes until a stable base line was reached. External standards were also run using Glucose obtained from Sigma Chemical (G-5500, Sigma St. Louis, MO).
  • glucose may be broken down to form acetate. While such glucose will be detected as being consumed, it should not properly be incorporated into calculations of carbon efficiency according to the presently-claimed methods. The reason for this is that any glucose used to produce acetate is not available to directly produce biosynthetic products. Therefore, acetate production should be calculated in order to form a basis for subtracting glucose which is diverted from pathways generating biosynthetic products and intermediates as noted above.
  • Ion chromatography was used to detect acetate in fermentation broths according to the procedure set forth in the Dionex (Sunnvale, CA) Ion Chromatography Cookbook, Issue I, p. 11-16.
  • a 50 ⁇ l aliquot of sample was injected on an HPIcE-ASl column (Dionex) with a flow rate of 0.8 ml/min.
  • the eluant was 1.0 mM octane-sulfonic acid in 2% 2-propanol.
  • the regenerant was 5 mM tetrabutyl ammonium hydroxide. Standards were also run at concentrations of 0.02, 0.1, 0.2 mg/ml.
  • Carbon conversion efficiency of transformed and untransformed cells was calculated as the total combined grams of L-phenylalanine, phenylpyruvic acid, and prephenic acid produced at 48 hrs. of fermentation divided by the total grams of glucose consumed during that fermentation. If acetate accumulates during the fermentation, carbon conversion efficiency may be calculated by subtracting 180.16 g (1 mole) of glucose for every 121 g (2 moles) of acetate produced and then calculating as indicated above.
  • the carbon conversion efficiencies for various batches of transformed and untransformed host cells are shown in Table 2. As is evident in that Table, carbon conversion efficiency significantly increased in cells which had been transformed with glyoxylate cycle genes.
  • results obtained in one fermentation run are used herein to demonstrate the calculation of carbon conversion efficiency according to the invention.
  • the results obtained in fermentation run 4 in Table 2 wherein E. coli cells had been transformed with plasmid pCLlOOO as described above.
  • the total phenylalanine (6.11 g) prephenic acid (15.55 g), and phenylpyruvic acid (1.81) was 23.47 g/L in a fermentation volume of 13.11 L. Additionally, 2.86 g/L acetate were produced and 2,844.18 grams of glucose were consumed during fermentation.
  • CE _ f [g/L (p e) * g/L(PPA) * g/UPA) ] x fermentation volume ( ) * j ⁇ 10Q [ g (glucose consumed) - g( lucose used to prod, acetate) j
  • Host cells which had been transformed with genes encoding enzymes of the glyoxylate cycle utilized carbon in a significantly more efficient manner as compared to untransformed controls. This effect is likely due to the fact that transformed cells utilized the glyoxylate cycle to bypass the CO 2 -evolving steps of the TCA cycle, thus making available greater amounts of carbon for incorporation into biosynthetic products. The greater efficiency of carbon utilization further results in increases in the synthesis of those products.
  • Mutations in the glyoxylate genes may also be used to obtain increases in carbon conversion efficiency.
  • a mutated aceK gene which possesses primarily kinase activity, may be used in methods according to the invention. The foregoing parameters are known in the art and may be varied according to the use to which the presently-claimed methods are put.

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Abstract

L'invention concerne des procédés permettant d'augmenter l'efficacité de la conversion du carbone d'un micro-organisme qui consistent à introduire des enzymes codant l'ADN du cycle de glyoxylate dans ledit micro-organisme.
EP94918169A 1993-05-27 1994-05-27 Procede d'augmentation de l'efficacite de la conversion du carbone dans des micro-organismes. Withdrawn EP0660875A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US68433 1987-06-30
US6843393A 1993-05-27 1993-05-27
PCT/US1994/006084 WO1994028154A1 (fr) 1993-05-27 1994-05-27 Procede d'augmentation de l'efficacite de la conversion du carbone dans des micro-organismes

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EP0660875A1 true EP0660875A1 (fr) 1995-07-05
EP0660875A4 EP0660875A4 (fr) 1998-08-05

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EP (1) EP0660875A4 (fr)
JP (1) JPH07509375A (fr)
KR (1) KR950702637A (fr)
AU (1) AU6960894A (fr)
CA (1) CA2140527A1 (fr)
WO (1) WO1994028154A1 (fr)

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US20050124047A1 (en) * 2001-07-18 2005-06-09 Degussa Ag Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an attenuated acek gene
JP5602982B2 (ja) * 2006-07-03 2014-10-08 三菱化学株式会社 コハク酸の製造方法
CN105296411B (zh) * 2015-11-24 2019-03-08 南京工业大学 一株利用单糖发酵产l-天冬氨酸的基因工程菌及其构建方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0066994A2 (fr) * 1981-06-04 1982-12-15 Imperial Chemical Industries Plc Production et utilisation de microorganismes génétiquement modifiés
US5169768A (en) * 1983-10-07 1992-12-08 Biotechnica International, Inc. Method of biosynthesis of phenylalanine
EP0530765A2 (fr) * 1991-09-02 1993-03-10 Kyowa Hakko Kogyo Co., Ltd. ADN-régulateur pour l'expression de gènes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0066994A2 (fr) * 1981-06-04 1982-12-15 Imperial Chemical Industries Plc Production et utilisation de microorganismes génétiquement modifiés
US5169768A (en) * 1983-10-07 1992-12-08 Biotechnica International, Inc. Method of biosynthesis of phenylalanine
EP0530765A2 (fr) * 1991-09-02 1993-03-10 Kyowa Hakko Kogyo Co., Ltd. ADN-régulateur pour l'expression de gènes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
S. R. MALOY ET AL.: "Elevated Levels of Glyoxylate Shunt Enzymes in Escherichia coli Strains Constitutive for Fatty Acid Degradation." J. BACTERIOL., vol. 143, no. 2, 1980, pages 720-725, XP002066359 *
See also references of WO9428154A1 *

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KR950702637A (ko) 1995-07-29
AU6960894A (en) 1994-12-20
JPH07509375A (ja) 1995-10-19
CA2140527A1 (fr) 1994-12-08
EP0660875A4 (fr) 1998-08-05
WO1994028154A1 (fr) 1994-12-08

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