WO2007017710A1 - Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, homoserine, or valine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression - Google Patents
Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, homoserine, or valine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression Download PDFInfo
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- WO2007017710A1 WO2007017710A1 PCT/IB2005/003071 IB2005003071W WO2007017710A1 WO 2007017710 A1 WO2007017710 A1 WO 2007017710A1 IB 2005003071 W IB2005003071 W IB 2005003071W WO 2007017710 A1 WO2007017710 A1 WO 2007017710A1
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- 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
- C12P13/20—Aspartic acid; Asparagine
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- 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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- 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
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
Definitions
- the present invention relates to increasing the production of aspartate or aspartate- 5 derived metabolites by boosting the activity of the glyoxylate shunt. This is accomplished by increasing the activity of glyoxylate shunt specific enzymes and decreasing the activity of reactions consuming glyoxylate and its precursors.
- Amino acids and their derivatives are important precursors in the pharmaceutical industry and added to a wide variety of food and feed as supplements.
- amino acids such as glutamate, lysine and threonine are produced using their natural biosynthetic pathways, hi natural amino acid biosynthesis the amino acid aspartate serves as the precursor for the production of other amino acids, such as lysine, threonine, isoleucine and
- Aspartate is produced from oxaloacetate, which is a central metabolite of the citric acid cycle.
- Oxaloacetate can be transformed into a molecule of citrate by accepting a molecule of acetyl-CoA, a reaction that is part of the citrate cycle or it can leave the citrate cycle via a transamination reaction that yields aspartate.
- oxaloacetate fulfils a pure acceptor role and is regenerated during a round of the cycle. Keeping the
- PEP phosphoenol pyruvate
- PEP carboxylase a reaction catalyzed by PEP carboxylase
- the carbon source is acetate
- oxaloacetate is produced via the glyoxylate cycle (Tricarboxylic acid cycle and glyoxylate bypass, reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E.
- acetate see fig. 1
- malate synthase a reaction catalyzed by the enzyme, malate synthase, encoded by aceB.
- Malate also enters the citrate cycle and is converted into oxaloacetate.
- another oxaloacetate molecule is generated and can leave the citric acid cycle to be transformed into aspartate.
- the entry of carbon into the glyoxylate shunt is regulated on the transcriptional and posttranscriptional level. Transcriptional regulation is exerted on the ⁇ ceBAK operon by the IcIR repressor.
- AceBAK encode malate synthase, isocitrate lyase and isocitrate kinase/phosphatase, respectively.
- the iclR. gene is negatively autoregulated and activated by the FadR protein.
- the activity of isocitrate dehydrogenase, encoded by the icd gene is regulated posttranscriptionally. Isocitrate dehydrogenase and isocitrate lyase compete for the common substrate isocitrate.
- the entry into the glyoxylate pathway depends in part on the regulation of the isocitrate dehydrogenase enzyme.
- Isocitrate dehydrogenase activity is modulated by its phosphorylation or dephosphorylation catalyzed by AceK. Phosphorylation reduces the activity of led and dephosphorylation reactivates the led enzyme. If AceK acts as kinase or phosphatase depends on the presence of several metabolites. Depletion of isocitrate and 3-phosphoglycerate stimulates kinase activity; the presence of pyruvate and AMP inhibits the kinase function thus favoring the phosphatase activity (see also Neidhard).
- a strong production of aspartate is a prerequisite for the industrial production of aspartate-derived amino acids, hi E. coli this has been accomplished by increasing PEP carboxylase activity by expressing feed-back desensitized PEP carboxylase enzymes (Patent EP0723011B1 by Ajinomoto) or heterologous pyruvate carboxylases (patent US6455284).
- PEP carboxylase activity by expressing feed-back desensitized PEP carboxylase enzymes (Patent EP0723011B1 by Ajinomoto) or heterologous pyruvate carboxylases (patent US6455284).
- the deletion of oxaloacetate or malate consuming enzymes such as pyruvate carboxykinase, encoded by the pck gene, or the malic enzymes encoded by sfcA and maeB, is important for the production of aspartate derived metabolites.
- the invention is based on the discovery that an increase in the activity of the glyoxylate shunt can boost the production of aspartate and aspartate-derived metabolites.
- the invention relates to optimizing a method for the production of aspartate or aspartate-derived metabolites by up-regulating the activity of the glyoxylate shunt and/or decreasing isocitrate dehydrogenase activity. This can be accomplished by directly overexpressing the activity of the enzymes isocitrate lyase and malate synthase, by decreasing their repression through the attenuation, preferentially deletion of the gene ic ⁇ R, or through the deletion of fadR.
- a further increase in the production of aspartate or its derivatives can be achieved by reducing the consumption of glyoxylate and its precursors.
- the invention concerns a method for the production of aspartate or its derivatives by culture of a microorganism in an appropriate culture medium and recovery of the aspartate derivative from the culture medium, wherein the microorganism is transformed to enhance the activity of the glyoxylate shunt.
- the invention further relates to microorganisms, preferentially enterobacteriaceae, coryneform bacteria or yeast, into which the aforementioned modifications were integrated.
- the invention describes a method for the fermentative production and purification of aspartate and its derivatives using microorganisms with the described properties, comprising a) Fermentation of the microorganism producing the desired metabolite b) Concentration of the desired product in the cells of the bacteria or in the medium and c) Isolation of the desired metabolite/ constituents of the fermentation broth and/or the biomass optionally remaining in portions or in the total amount (0-100%) in the end product.
- aspartate derived metabolites or “aspartate derivatives” means any compounds or products synthesized/produced by a microorganism using aspartate as substrate or co-substrate upstream in the biosynthesis pathway.
- amino acids lysine, threonine, isoleucine and methionine are selected among the amino acids lysine, threonine, isoleucine and methionine, preferably methionine.
- Aspartate-derived metabolites especially the amino acids lysine, threonine and methionine are produced in industrial scale and mainly used in the nutrition of animals and in pharmaceutical applications. Difficult to synthesize chemically as pure L-stereoisomers, some of these amino acids are produced more economically by fermentation. This requires strong production of aspartate and thus a strong efflux of the acceptor molecule oxaloacetate from the citric acid cycle. To counteract the efflux of this acceptor C4 dicarboxylic acid, the oxaloacetate pool needs to be replenished via anaplerotic reactions.
- the object of this invention is thus an innovative way to increase the production of oxaloacetate that in turn can leave the citric acid cycle and be transaminated to aspartate. This is accomplished by (i) increasing the activity of the glyoxylate pathway specific enzymes AceA and AceB, (ii) decreasing the activity of isocitrate dehydrogenase that competes with the first enzyme of the glyoxylate shunt, isocitrate lyase, for the substrate isocitrate and (iii) by reducing glyoxylate consuming reactions.
- the glyoxylate shunt is an anaplerotic pathway that diverges from the citric acid cycle, m the glyoxylate shunt the common substrate isocitrate is converted into glyoxylate and succinate, a reaction catalyzed by isocitrate lyase encoded by the aceA gene.
- glyoxylate is transformed into malate using acetyl- CoA, a reaction catalyzed by malate synthase, encoded by the aceB gene.
- Entry into the glyoxylate shunt is regulated on the transcriptional level by the transcription factor IcIR that represses the aceBAK operon and by regulating the enzymatic activity of isocitrate dehydrogenase, encoded by the icd gene, via phosphorylation.
- the corresponding kinase/ phosphatase is encoded by the aceK gene.
- Object of the invention is the increased expression, especially overexpression of the glyoxylate pathway specific enzymes, isocitrate lyase and malate synthase.
- the corresponding genes aceA and aceB may be encoded chromosomally or extrachromosomally. Chromosomally there may be one or several copies on the genome that can be introduced by methods of recombination known to the expert in the field. Extrachromosomally genes may be carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell.
- the genes aceA and aceB may be expressed using promoters with different strength that need or need not to be induced by inducer molecules. Examples are the promoters Ptrc, Ptac, Plac, the lambda promoter cl or other promoters known to the expert in the field. Expression of the two genes may be boosted or reduced by elements stabilizing or destabilizing the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or the protein (e.g. GST tags, Amersham Biosciences)
- the present invention also relates to microorganisms that contain one or several alleles encoding isocitrate dehydrogenase and/or malate synthase according to the invention.
- Such strains are characterized by the fact that they possess a carbon metabolism that permits the increased synthesis of oxaloacetate.
- an increase in isocitrate lyase and malate synthase activity is achieved by attenuating the expression of the gene iclR encoding a repressor of the ⁇ ceBAK operon. Attenuation is defined as the reduced expression obtained by decreasing the force of the proper promoter or introducing artificial promoters upstream of iclR, or possibly as the complete elimination of the iclR gene.
- the activity of isocitrate lyase and malate synthase are increased by attenuating, or preferentially eliminating the activation of zc/R transcription through FadR. This may be accomplished by attenuating, preferentially eliminating the expression of FadR or by deleting the binding sites of FadR in the zc/R promoter.
- the activity of isocitrate dehydrogenase that competes with isocitrate lyase for substrate is attenuated. This can be accomplished by introducing artificial promoters that drive the expression of the icd gene or possibly by introducing mutations into the led enzyme that reduce its activity. Since the activity of led is reduced by phosphorylation it may also be controlled by introducing mutant aceK genes that have increased kinase activity or reduced phosphatase activity compared to the wildtype AceK enzyme.
- the production of oxaloacetate and thus aspartate and its derived metabolites is further increased by attenuating the expression of genes involved in the consumption of glyoxylate, such as the gene gel, encoding glyoxylate carboligase or eda encoding 2-keto 3-deoxygluconate 6-phosphate aldolase (Vlahos & Dekker (1986) JBC 261, pp. 11049-11055).
- a further increase of the production of aspartate and aspartate-derived metabolites is obtained by increasing the expression, preferentially by overexpressing other genes that are involved hi the production of aspartate or its precursor oxaloacetate.
- PEP phosphoenol pyruvate
- pyc pyruvate carboxylase
- the expressed pyruvate carboxylase enzyme may be insensible to feed-back inhibition by aspartate and not require activation by acetyl-phosphate.
- the activity of the enzyme phosphoenolpyruvate carboxykinase, encoded by the pck gene, may be attenuated, preferentially deleted. Additional mutations that reduce the activity of acetate kinase and phosphotransacetylase encoded by the pta-ackA operon may allow the strain to regain vitality. Attenuating the activity of the malic enzyme encoded by sfcA may also reduce futile cycling.
- Another embodiment of the invention describes the fermentative production of aspartate or one of its derived metabolites, in particular methionine, based on a strain incorporating the mutations described above.
- the production strain is fermented under industrial conditions that are known to the expert in the field.
- the desired aspartate derived metabolite can be present inside the cells or in the fermentation broth.
- the invention comprises also a method for the isolation of the desired metabolite that is known to the expert in the field.
- constituents or biomass from 0 to 100 percent of the fermentation broth may be retained during the preparation of the desired product.
- a microorganism that is optimized for the production of aspartate-derived metabolites that harbors the modifications described above is also object of the invention.
- This microorganism is preferentially yeast, a Corynebacterium or Enterobacterium.
- yeast a Corynebacterium or Enterobacterium.
- Figure 1 shows the Citrate and Glyoxylate cycle. Shown are the major reactions of the two metabolic pathways including metabolites and genes encoding the relevant enzyme activities.
- DfadRF (SEQ ID NO l) ggcgcaaagcccggcgggtttcgcggaagagtacattattgaaagtatctggaataaccgcttccctccgggactattTGT
- the oligonucleotides DfadRF and DfadRR were used to amplify the chloramphenicol resistance cassette from the plasmid pKD3.
- the PCR product obtained was then introduced by electroporation into the strain MG1655 (pKD46), in which the Red recombinase enzyme expressed permitted the homologous recombination.
- Chloramphenicol resistant transformants were selected and the insertion of the resistance cassette verified by a PCR analysis with the oligonucleotides fadRF and fadRR defined below.
- the strain retained was designated MG1655 ⁇ / ⁇ JR::Cm
- fadRF (SEQ ID NO 3) : ccggggagcagcgggtagcatttcagggcc (homologous to the sequence from 12323741 to 1233770).
- fadRR SEQ ID NO 4 : cgccggttccgactggctggaaacgctgc (homologous to the sequence from 1235176 to 1235348).
- Control tubes 100 ⁇ l cells 100 ⁇ l phages Pl of strain MG1655 (AfadR.::Cm) - Test tube: 100 ⁇ l of cells + 100 ⁇ l of phages Pl of the strain MG1655 (AfadRy.Cm)
- Chloramphenicol resistant transformants were then selected and the deletion of the gene (AfadKwCm) was verified by PCR analysis with the oligonucleotides fadRF and fadRR. The strain retained was designated MG1655 metA*ll AmetJ ⁇ fadRy.Cm.
- the chloramphenicol resistance cassette can then be eliminated.
- the plasmid pCP20 carrying FLP recombinase acting at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strain by electroporation. After a series of cultures at 42°C, the loss of the chloramphenicol resistance cassette was verified by PCR analysis with the same oligonucleotides as used previously (fadRF and fadRR). The strain retained is designated MGl 655 metA*ll AmetJ AfadK.
- a thrA* allele with reduced feed-back resistance to threonine is expressed from the plasmid pCL1920 (Lerner & Inouye, 1990, NAR 18, 15 p 4631) using the promoter Ptrc.
- plasmid pME101-thrA*l thrA was PCR amplified from genomic DNA using the following oligonucleotides: ⁇ spHlthrA (SEQ ID NO 5): ttaTCATGAgagtgttgaagttcggcggtacatcagtggc
- the plasmid pCL1920 is PCR amplified using the oligonucleotides PMElOlF and PMElOlR and the BstZlll-Xm ⁇ l fragment from the vector pTRC99A harboring the lad gene and the Ptrc promoter is inserted into the amplified vector.
- the resulting vector and the vector harboring the thrA gene are restricted by Apal and Smal and the thrA containing fragment is cloned into the vector pMElOl.
- the mutation F318S is introduced by site-directed mutagenesis (Stratagene) using the oligonucleotides ThrAF F318S for and ThrAR F318S, resulting in the vector pMEl 01 -thrA*l.
- ThrAR F318S (Smal) (SEQ ID NO 10):
- the iclR. gene deletion was introduced in the MG1655 metA*ll AmetJ strain using the strategy described by Datsenko and Wanner (see above) with the following oligonucleotides :
- DiClF (SEQ ID NO 11): Cgcacccattcccgcgaaacgcggcagaaaacccgccgttgccaccgcaccagcgactggacaggttcagtctttaacgc gTGTAGGCTGGAGCTGCTTCG with
- DiClR (SEQ ID NO 12): gcgcattccaccgtacgccagcgtcacttccttcgccgctttaatcaccatcgcgccaaactcggtcacgcggtcatcg gCATATGAATATCCTCCTTAG
- the oligonucleotides DiclF and DiclR were used to amplify the kanamycin resistance cassette from the plasmid pKD4.
- the PCR product obtained was then introduced by electroporation into the strain MG1655 metA*ll Ame ⁇ (pKD46). Kanamycin resistant transformants were selected and the insertion of the resistance cassette verified by PCR using the oligonucleotides iclF and iclR defined below.
- strain retained is designated MG1655 metA* ⁇ l Ame ⁇ ⁇ zc/R: :Km IcIF (SEQ ID NO 13) : cctttgaggtcgcatggccagtcggc (homologous to the sequence from 4221558 to 4221533).
- iclR SEQ ID NO 14: gctttttaatagaggcgtcgccagctccttgcc (homologous to the sequence from 4219917 to 4219949).
- the pME101-r7zrA*l plasmid was then introduced in the strain MG1655 metA*ll Ame ⁇ ⁇ zc/R: :Km.
- the kanamycin resistance cassette was eliminated using the strategy described above.
- the loss of the kanamycin resistance cassette was verified by a PCR analysis with the same oligonucleotides as those used previously (iclF and iclR).
- the strain retained is designated MGl 655 met A*l 1 Ame ⁇ AicIR.
- the deletion AfadRy.Cm was transferred into the strain MGl 655 met A* W AmetJ ⁇ zc/R: :Km by Pl phage transduction as previously described using the phage lysate of the strain MG1655 AfadRy.Cm. Chloramphenicol and at the same time kanamycin resistant transformants were selected and the deletions ⁇ zc/R: :Km and AfadRv.Cm verified by PCR with the oligonucleotides fadRF, fadRR and iclF, iclR. The strain retained was designated MG1655 met A* 11 Ame ⁇ AfadR.: : Cm ⁇ zc/R: :Km.
- the plasmid pME101-thrA*l was then introduced in the strain MG1655 metA*l ⁇ Ame ⁇ AfadR:. : Cm ⁇ zc/R: :Km.
- kanamycin and chloramphenicol resistance cassettes were eliminated and their loss verified by PCR analysis with the same oligonucleotides as those used previously
- the MG 1655 ⁇ gc/::Rm was constructed using the method of Datsenko and Wanner as described with the following oligonucleotides : DgClF (SEQ ID NO 15): ggcaaaaatgagagccgttgacgcggcaatgtatgtgctggagaaagaaggtatcactaccgccttcggtgttccgggagcT GTAGGCTGGAGCTGCTTCG with
- DgipR (SEQ ID NO 16) gcgttacgttttaacggtacggatccatccagcgtaaaccggcttccgtggtggtttggggtttatattcacacccaacccCATA TGAATATCCTCCTTAG with
- the oligonucleotides DgclF and DgipR were used to amplify the kanamycin resistance cassette from the plasmid pKD4.
- the PCR product obtained is then introduced by electroporation into the strain MGl 655 (pKD46). Kanamycin resistant transformants were selected and the insertion of the resistance cassette was verified by PCR with the oligonucleotides gclF and gipR defined below.
- the strain retained was designated MG1655 ⁇ gc/::Km.
- gclF SEQ ID NO 17
- ggatatgcccaccttgctgaagg homologous to the sequence from
- gipR SEQ ID NO 18: cgcttagtttcaatcggggaaatgg (homologous to the sequence from
- Kanamycin resistant transformants were selected and the deletion of the gene
- the strain retained was designated MGl 655 metA*l 1 AmetJ AiclK AgclwKm.
- the plasmid pME101-t/jrA*l was then introduced in the strain MG1655 metA*l 1
- the kanamycin resistance cassette was eliminated as described and its loss verified by PCR with the oligonucleotides gclF and gipR (see above).
- the strain retained was designated MGl 655 met AHl AmetJ AiclR AgcL MG1655 metA*U AmetJ AiclR Aedd-eda (pME101 ⁇ tfwA*l)
- the MG 1655 Aedd-eda:: ⁇ Lm was constructed using the method of Datsenko & Wanner as described with the following oligonucleotides :
- DeddF (SEQ IDNO 19) CgcgcgagactcgctctgcttatctcgcccggatagaacaagcgaaaacttcgaccgttcatcgttcgcagttggcatgcggT
- DedaR (SEQ ID NO 20) gcttagcgccttctacagcttcacgcgccagcttagtaatgcggtcgtaatcgcccgcttccagcgcatctgccggaaccCAT ATGAATATCCTCCTTAG with
- the oligonucleotides DeddF and DedaR were used to amplify the kanamycin resistance cassette from the plasmid pKD4.
- the PCR product obtained was then introduced by electroporation into the strain MGl 655 met A* 11 (pKD46). Kanamycin resistant transformants were then selected and the insertion of the resistance cassette was verified by PCR analysis with the oligonucleotides eddF and edaR defined below.
- the strain retained was designated MGl 655 met A*ll Aedd-edawKm.
- eddF Gggtagactccattactgaggcgtgggcg (homologous to the sequence from 1932996 to 1932968).
- edaR SEQ ID NO 22: ccacatgataccgggatggtgacg (homologous to the sequence from 1929754 to1929777).
- the kanamycin resistance cassette was eliminated as described and its loss was verified by PCR analysis with the oligonucleotides eddF and edaR, described above. The strain retained was designated MGl 655 met A* 11 Ame ⁇ ⁇ zc/R Aedd-eda.
- the MG1655 Aedd ⁇ eda::Cm was constructed in the strain MG1655 metA*ll as described for the kanamycin resistant mutant, only that in this case the vector pKD3 carrying a chloramphenicol resistance was used.
- the deletion Aedd-edav.Cm was then transduced into strain MG1655 metA*l ⁇ Ame ⁇ ⁇ zc/R ⁇ ge/::Km by Pl phage transduction using a phage lysate of the strain MG 1655 Aedd-edav.Cm.
- the plasmid pME101-t/zrA*l was then introduced in the strain MG1655 metA*U Ame ⁇ AiclR. Agc ⁇ vXm Aedd-edav.Cm. The kanamycin and chloramphenicol resistance cassettes were eliminated as described and their loss was verified by PCR with the oligonucleotides eddF, edaR and gclF, gipR described above. The strain retained was designated MGl 655 met A ⁇ W Ame ⁇ ⁇ zc/R Agcl Aedd-eda.
- Production strains were analyzed in small Erlenmeyer flask cultures using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sd. USA 32:120-128) that was supplemented with 5 g/1 MOPS and 5 g/1 glucose. Spectinomycin was added if necessary at a concentration of 50 mg/1. An overnight culture was used to inoculate a 30 ml culture to an OD600 of 0.2 and then grown to an OD600 of 4.5 to 5. Extracellular metabolites were analyzed during the batch phase. Amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using GC-MS after silylation.
- AceA isocitrate lyase
- AceB malate synthase
- isocitrate lyase activity For the determination of isocitrate lyase activity (AceA) five ⁇ l extract were assayed in 50 mM imidazole, 100 mM KCl, 1 mM EDTA, 5 inM MgSO4, 20 mM phenylhydrazine, 5 mM isocitrate for 30 minutes at 25°C. Isocitrate lyase activity was determined according to the rate of glyoxylate phenylhydrazine complex formed that absorbs at 324 nm.
- Malate synthase activity was determined by assaying 10 ⁇ l extract in 50 mM potassium phosphate buffer (pH 6.5), 5 mM MgCl 2 , 0.1 mM Acetyl-CoA, 2 mM glyoxylate. Malate synthase activity was determined according to the reduction in absorbance at 232 nm that is caused by the consumption of acetyl-CoA used for the production of malate.
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Abstract
The present invention relates to increasing the production of aspartate or aspartate-derived metabolites by boosting the activity of the glyoxylate shunt. This is accomplished by increasing the activity of glyoxylate shunt specific enzymes and decreasing the activity of reactions consuming glyoxylate and its precursors.
Description
PROCESS FOR THE PREPARATION OF ASPARTATE AND DERIVED AMINO ACIDS LIKE LYSINE, THREONINE, ISOLEUCINE, METHIONINE, HOMOSERINE, OR VALINE EMPLOYING A MICROORGANISM WITH ENHANCED ISOCITRATE LYASE AND/OR MALATE SYNTHASE EXPRESSION
Field of the invention
The present invention relates to increasing the production of aspartate or aspartate- 5 derived metabolites by boosting the activity of the glyoxylate shunt. This is accomplished by increasing the activity of glyoxylate shunt specific enzymes and decreasing the activity of reactions consuming glyoxylate and its precursors.
Prior art
10 Amino acids and their derivatives are important precursors in the pharmaceutical industry and added to a wide variety of food and feed as supplements. Several amino acids, such as glutamate, lysine and threonine are produced using their natural biosynthetic pathways, hi natural amino acid biosynthesis the amino acid aspartate serves as the precursor for the production of other amino acids, such as lysine, threonine, isoleucine and
15 methionine. Aspartate is produced from oxaloacetate, which is a central metabolite of the citric acid cycle. Oxaloacetate can be transformed into a molecule of citrate by accepting a molecule of acetyl-CoA, a reaction that is part of the citrate cycle or it can leave the citrate cycle via a transamination reaction that yields aspartate. In the citrate cycle oxaloacetate fulfils a pure acceptor role and is regenerated during a round of the cycle. Keeping the
20 oxaloacetate pool at a constant level is thus a prerequisite for sufficient citrate cycle activity, which in turn is important for the cellular energy balance. Therefore oxaloacetate that is withdrawn from the cycle needs to be replenished. In E, coli this is accomplished by several different reactions known as anaplerotic pathways. If E. coli grows on glucose in a synthetic minimal medium, most of the oxaloacetate required is produced by carboxylation
25 of phosphoenol pyruvate (PEP), a reaction catalyzed by PEP carboxylase (Kay et al., PNAS 1999, 96, 823-28 and references therein). If the carbon source is acetate, oxaloacetate is produced via the glyoxylate cycle (Tricarboxylic acid cycle and glyoxylate bypass, reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E.
30 Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology). In this cycle isocitrate is cleaved into succinate and glyoxylate, a reaction that is catalyzed by isocitrate lyase, encoded by the aceA gene. Succinate directly enters the citric acid cycle and is converted into oxaloacetate. Glyoxylate is converted into malate by incorporating a molecule of acetyl-CoA derived
35 from acetate (see fig. 1), a reaction catalyzed by the enzyme, malate synthase, encoded by
aceB. Malate also enters the citrate cycle and is converted into oxaloacetate. Thus another oxaloacetate molecule is generated and can leave the citric acid cycle to be transformed into aspartate.
The entry of carbon into the glyoxylate shunt is regulated on the transcriptional and posttranscriptional level. Transcriptional regulation is exerted on the αceBAK operon by the IcIR repressor. AceBAK encode malate synthase, isocitrate lyase and isocitrate kinase/phosphatase, respectively. The iclR. gene is negatively autoregulated and activated by the FadR protein. The activity of isocitrate dehydrogenase, encoded by the icd gene, is regulated posttranscriptionally. Isocitrate dehydrogenase and isocitrate lyase compete for the common substrate isocitrate. Since the Km value for isocitrate is significantly higher for the isocitrate lyase reaction, the entry into the glyoxylate pathway depends in part on the regulation of the isocitrate dehydrogenase enzyme. Isocitrate dehydrogenase activity is modulated by its phosphorylation or dephosphorylation catalyzed by AceK. Phosphorylation reduces the activity of led and dephosphorylation reactivates the led enzyme. If AceK acts as kinase or phosphatase depends on the presence of several metabolites. Depletion of isocitrate and 3-phosphoglycerate stimulates kinase activity; the presence of pyruvate and AMP inhibits the kinase function thus favoring the phosphatase activity (see also Neidhard).
A strong production of aspartate is a prerequisite for the industrial production of aspartate-derived amino acids, hi E. coli this has been accomplished by increasing PEP carboxylase activity by expressing feed-back desensitized PEP carboxylase enzymes (Patent EP0723011B1 by Ajinomoto) or heterologous pyruvate carboxylases (patent US6455284). In addition, the deletion of oxaloacetate or malate consuming enzymes, such as pyruvate carboxykinase, encoded by the pck gene, or the malic enzymes encoded by sfcA and maeB, is important for the production of aspartate derived metabolites. An increase of glyoxylate shunt activity has been shown to be beneficial for the production of succinate (Lin, H., Bennett, G. N. San, K-Y. 2005, Biotechnol and Bioeng 89, 148-156). Here we demonstrate that an increase in the activity of the glyoxylate shunt specific enzymes together with a decrease in the consumption of glyoxylate and its precursors can boost the production of aspartate-derived metabolites.
Summary of the invention
The invention is based on the discovery that an increase in the activity of the glyoxylate shunt can boost the production of aspartate and aspartate-derived metabolites. Thus the invention relates to optimizing a method for the production of aspartate or
aspartate-derived metabolites by up-regulating the activity of the glyoxylate shunt and/or decreasing isocitrate dehydrogenase activity. This can be accomplished by directly overexpressing the activity of the enzymes isocitrate lyase and malate synthase, by decreasing their repression through the attenuation, preferentially deletion of the gene icϊR, or through the deletion of fadR. A further increase in the production of aspartate or its derivatives can be achieved by reducing the consumption of glyoxylate and its precursors.
Thus the invention concerns a method for the production of aspartate or its derivatives by culture of a microorganism in an appropriate culture medium and recovery of the aspartate derivative from the culture medium, wherein the microorganism is transformed to enhance the activity of the glyoxylate shunt.
The invention further relates to microorganisms, preferentially enterobacteriaceae, coryneform bacteria or yeast, into which the aforementioned modifications were integrated. Finally the invention describes a method for the fermentative production and purification of aspartate and its derivatives using microorganisms with the described properties, comprising a) Fermentation of the microorganism producing the desired metabolite b) Concentration of the desired product in the cells of the bacteria or in the medium and c) Isolation of the desired metabolite/ constituents of the fermentation broth and/or the biomass optionally remaining in portions or in the total amount (0-100%) in the end product.
Detailed description of the invention
According to the present invention, "aspartate derived metabolites" or "aspartate derivatives" means any compounds or products synthesized/produced by a microorganism using aspartate as substrate or co-substrate upstream in the biosynthesis pathway. Preferred
"aspartate derived metabolites" or "aspartate derivatives" are selected among the amino acids lysine, threonine, isoleucine and methionine, preferably methionine.
Aspartate-derived metabolites, especially the amino acids lysine, threonine and methionine are produced in industrial scale and mainly used in the nutrition of animals and in pharmaceutical applications. Difficult to synthesize chemically as pure L-stereoisomers, some of these amino acids are produced more economically by fermentation. This requires strong production of aspartate and thus a strong efflux of the acceptor molecule oxaloacetate from the citric acid cycle. To counteract the efflux of this acceptor C4 dicarboxylic acid, the oxaloacetate pool needs to be replenished via anaplerotic reactions.
The object of this invention is thus an innovative way to increase the production of oxaloacetate that in turn can leave the citric acid cycle and be transaminated to aspartate. This is accomplished by (i) increasing the activity of the glyoxylate pathway specific enzymes AceA and AceB, (ii) decreasing the activity of isocitrate dehydrogenase that competes with the first enzyme of the glyoxylate shunt, isocitrate lyase, for the substrate isocitrate and (iii) by reducing glyoxylate consuming reactions.
The glyoxylate shunt is an anaplerotic pathway that diverges from the citric acid cycle, m the glyoxylate shunt the common substrate isocitrate is converted into glyoxylate and succinate, a reaction catalyzed by isocitrate lyase encoded by the aceA gene. In the second reaction of the glyoxylate cycle glyoxylate is transformed into malate using acetyl- CoA, a reaction catalyzed by malate synthase, encoded by the aceB gene. Entry into the glyoxylate shunt is regulated on the transcriptional level by the transcription factor IcIR that represses the aceBAK operon and by regulating the enzymatic activity of isocitrate dehydrogenase, encoded by the icd gene, via phosphorylation. The corresponding kinase/ phosphatase is encoded by the aceK gene.
Object of the invention is the increased expression, especially overexpression of the glyoxylate pathway specific enzymes, isocitrate lyase and malate synthase. For this purpose the corresponding genes aceA and aceB may be encoded chromosomally or extrachromosomally. Chromosomally there may be one or several copies on the genome that can be introduced by methods of recombination known to the expert in the field. Extrachromosomally genes may be carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell. They may be present as 1-5 copies, ca 20 or up to 500 copies corresponding to low copy number plasmids with tight replication (pSClOl, RK2), low copy number plasmids (pACYC, pRSFlOlO) or high copy number plasmids (pSK bluescript II).
The genes aceA and aceB may be expressed using promoters with different strength that need or need not to be induced by inducer molecules. Examples are the promoters Ptrc, Ptac, Plac, the lambda promoter cl or other promoters known to the expert in the field. Expression of the two genes may be boosted or reduced by elements stabilizing or destabilizing the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or the protein (e.g. GST tags, Amersham Biosciences)
The present invention also relates to microorganisms that contain one or several alleles encoding isocitrate dehydrogenase and/or malate synthase according to the invention.
Such strains are characterized by the fact that they possess a carbon metabolism that permits the increased synthesis of oxaloacetate. hi another embodiment of the invention an increase in isocitrate lyase and malate synthase activity is achieved by attenuating the expression of the gene iclR encoding a repressor of the αceBAK operon. Attenuation is defined as the reduced expression obtained by decreasing the force of the proper promoter or introducing artificial promoters upstream of iclR, or possibly as the complete elimination of the iclR gene.
In another object of the invention the activity of isocitrate lyase and malate synthase are increased by attenuating, or preferentially eliminating the activation of zc/R transcription through FadR. This may be accomplished by attenuating, preferentially eliminating the expression of FadR or by deleting the binding sites of FadR in the zc/R promoter.
In another embodiment of the invention the activity of isocitrate dehydrogenase that competes with isocitrate lyase for substrate is attenuated. This can be accomplished by introducing artificial promoters that drive the expression of the icd gene or possibly by introducing mutations into the led enzyme that reduce its activity. Since the activity of led is reduced by phosphorylation it may also be controlled by introducing mutant aceK genes that have increased kinase activity or reduced phosphatase activity compared to the wildtype AceK enzyme. hi another preferred embodiment of the invention the production of oxaloacetate and thus aspartate and its derived metabolites is further increased by attenuating the expression of genes involved in the consumption of glyoxylate, such as the gene gel, encoding glyoxylate carboligase or eda encoding 2-keto 3-deoxygluconate 6-phosphate aldolase (Vlahos & Dekker (1986) JBC 261, pp. 11049-11055). A further increase of the production of aspartate and aspartate-derived metabolites is obtained by increasing the expression, preferentially by overexpressing other genes that are involved hi the production of aspartate or its precursor oxaloacetate. These are the genes by preference ppc, encoding phosphoenol pyruvate (PEP) carboxylase and /or pyruvate carboxylase (pyc). rn a preferential application of the invention the PEP carboxylase may not be feed-back inhibited by aspartate. The expressed pyruvate carboxylase enzyme may be insensible to feed-back inhibition by aspartate and not require activation by acetyl-phosphate.
To reduce futile cycling between C4 and C3 metabolites the activity of the enzyme phosphoenolpyruvate carboxykinase, encoded by the pck gene, may be attenuated, preferentially deleted. Additional mutations that reduce the activity of acetate kinase and
phosphotransacetylase encoded by the pta-ackA operon may allow the strain to regain vitality. Attenuating the activity of the malic enzyme encoded by sfcA may also reduce futile cycling.
Expressing higher levels of aspC encoding aspartate aminotransferase may also increase aspartate production.
To further increase the production of aspartate the following genes encoding enzymes involved in the transformation of aspartate may be attenuated:
aspA encoding aspartase purC encoding SAICAR synthase pur A encoding adenylosuccinate synthase pyrB,pyrL encoding aspartate carbamoyl transferase asnA encoding aspartate ammonia ligase asnB encoding asparagine synthetase B argG encoding argininosuccinate synthase panB encoding aspartate decarboxylase nadB aspartate oxidase aral encoding aromatic-amino-acid: 2-oxoglutarate aminotransferase
Another embodiment of the invention describes the fermentative production of aspartate or one of its derived metabolites, in particular methionine, based on a strain incorporating the mutations described above. In this method the production strain is fermented under industrial conditions that are known to the expert in the field. After the fermentation the desired aspartate derived metabolite can be present inside the cells or in the fermentation broth. The invention comprises also a method for the isolation of the desired metabolite that is known to the expert in the field. In a specific embodiment constituents or biomass from 0 to 100 percent of the fermentation broth may be retained during the preparation of the desired product.
A microorganism that is optimized for the production of aspartate-derived metabolites that harbors the modifications described above is also object of the invention. This microorganism is preferentially yeast, a Corynebacterium or Enterobacterium. Here we have described and exemplified the application for an Escherichia coli strain. This, however, does not exclude similar applications in other microorganisms.
Brief description of the figures
Figure 1 shows the Citrate and Glyoxylate cycle. Shown are the major reactions of the two metabolic pathways including metabolites and genes encoding the relevant enzyme activities.
Examples:
1 Construction of strains with increased activity in the glyoxylate cycle
MG1655 metA*ll ΔmetJ ΔfadR (pME101-thrA*l)
To delete the fadR. gene the homologous recombination strategy described by Datsenko & Wanner (2000) was used. This strategy allows the insertion of a chloramphenicol or a kanamycin resistance cassette, while deleting the gene completely or in part. For this purpose the following oligonucleotides were used:
DfadRF (SEQ ID NO l) ggcgcaaagcccggcgggtttcgcggaagagtacattattgaaagtatctggaataaccgcttccctcccgggactattTGT
AGGCTGGAGCTGCTTCG with
- a region (lower case) homologous to the sequence (1234172-1234251) of the gene fadR. (reference sequence on the website http://genolist.pasteur.fr/ColibriΛ, - a region (upper case) for the amplification of the chloramphenicol resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640- 6645),
DfadRR (SEQ ID NO 2)) cccctgaatggctaaatcacccggcagatttttctgcatccggtgccaaatctcgccactctcatgcccatagcgacgC
ATATGAATATCCTCCTTAG with
- a region (lower case) homologous to the sequence (1234874-1234795) of the gene fadR (reference sequence on the website http://genolist.pasteur.fr/Colibri/). - a region (upper case) for the amplification of the chloramphenicol resistance cassette,
The oligonucleotides DfadRF and DfadRR were used to amplify the chloramphenicol resistance cassette from the plasmid pKD3. The PCR product obtained was then introduced by electroporation into the strain MG1655 (pKD46), in which the Red recombinase enzyme expressed permitted the homologous recombination.
Chloramphenicol resistant transformants were selected and the insertion of the resistance cassette verified by a PCR analysis with the oligonucleotides fadRF and fadRR defined below. The strain retained was designated MG1655 Δ/αJR::Cm
fadRF (SEQ ID NO 3) : ccggggagcagcgggtagcatttcagggcc (homologous to the sequence from 12323741 to 1233770).
fadRR (SEQ ID NO 4) : cgccggttccgactggctggaaacgctgc (homologous to the sequence from 1235176 to 1235348).
To boost methionine biosynthesis the mefl gene encoding the methionine repressor was deleted and feedback resistant mutants of met A (metA* 11) were introduced into the Ameύ mutant. These constructions have been described in patent application PCT IB2004/001901. The strain retained was designated MGl 655 metA *11 AmetJ. To transfer the deletion AfadRy.Cm into the strain MGl 655 metA* 11 AmetJ, the method of phage Pl transduction was used. The protocol followed was implemented in 2 steps with the preparation of the phage lysate of the strain MG1655 Δ/α<iR::Cm and the subsequent transduction into strain MGl 655 metA* 11 ΔmetJ.
Preparation of phage lysate Pl: - Inoculation with 100 μl of an overnight culture of the strain MG1655
AfadR.::Cm of 10 ml of LB + Km 50 μg/ml + glucose 0.2% + CaCl2 5 mM.
- Incubation for 30 min at 37°C with shaking.
- Addition of 100 μl of phage lysate Pl prepared on the strain MG1655 (about l.l09 phage/ml) - Shaking at 37°C for 3 hours until all the cells were lysed.
- Addition of 200 μl chloroform and vortexing. Centrifugation for 10 min at 4500 g to eliminate cell debris.
- Transfer of supernatant to a sterile tube and addition of 200 μl chloroform.
- Storage of lysate at 4°C. Transduction
- Centrifuging for 10 min at 1500 g of 5 ml of an overnight culture of the strain MG 1655 met A* 11 AmetJ in LB medium.
- Suspension of the cell pellet in 2.5 ml of 10 mM MgSO4, 5 mM CaCl2
- Control tubes: 100 μl cells 100 μl phages Pl of strain MG1655 (AfadR.::Cm)
- Test tube: 100 μl of cells + 100 μl of phages Pl of the strain MG1655 (AfadRy.Cm)
- Incubation for 30 min at 30°C without shaking.
- Addition of 100 μl of 1 M sodium citrate in each tube and vortexing. - Addition of 1 ml of LB
- Incubation for 1 hour at 37°C with shaking
- Spreading on dishes LB + Km 50 μg/ml after centrifuging of tubes for 3 min at 7000 rpm.
- Incubation at 370C overnight. Verification of the strain
Chloramphenicol resistant transformants were then selected and the deletion of the gene (AfadKwCm) was verified by PCR analysis with the oligonucleotides fadRF and fadRR. The strain retained was designated MG1655 metA*ll AmetJ ΔfadRy.Cm.
The chloramphenicol resistance cassette can then be eliminated. The plasmid pCP20 carrying FLP recombinase acting at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strain by electroporation. After a series of cultures at 42°C, the loss of the chloramphenicol resistance cassette was verified by PCR analysis with the same oligonucleotides as used previously (fadRF and fadRR). The strain retained is designated MGl 655 metA*ll AmetJ AfadK. To further boost the production of homoserine the aspartokinase/homoserine a thrA* allele with reduced feed-back resistance to threonine is expressed from the plasmid pCL1920 (Lerner & Inouye, 1990, NAR 18, 15 p 4631) using the promoter Ptrc. For the construction of plasmid pME101-thrA*l thrA was PCR amplified from genomic DNA using the following oligonucleotides: βspHlthrA (SEQ ID NO 5): ttaTCATGAgagtgttgaagttcggcggtacatcagtggc
SmαlthrA (SEQ ID NO 6): ttaCCCGGGccgccgccccgagcacatcaaacccgacgc The PCR amplified fragment is cut with the restriction enzymes BspRl and Smal and cloned into the Ncol I Smal sites of the vector pTRC99A (Stratagene). For the expression from a low copy vector the plasmid pMElOl is constructed as follows. The plasmid pCL1920 is PCR amplified using the oligonucleotides PMElOlF and PMElOlR and the BstZlll-Xmήl fragment from the vector pTRC99A harboring the lad gene and the Ptrc promoter is inserted into the amplified vector. The resulting vector and the vector harboring the thrA gene are restricted by Apal and Smal and the thrA containing fragment is cloned into the vector pMElOl. To relieve ThrA from feed-back inhibition the mutation
F318S is introduced by site-directed mutagenesis (Stratagene) using the oligonucleotides ThrAF F318S for and ThrAR F318S, resulting in the vector pMEl 01 -thrA*l.
PMElOlF (SEQ ID NO 7): Ccgacagtaagacgggtaagcctg
PMElOlR (SEQ ID NO 8): Agcttagtaaagccctcgctag ThrAF F318S (Smal) (SEQ ID NO 9):
Ccaatctgaataacatggcaatgtccagcgtttctggcccggg
ThrAR F318S (Smal) (SEQ ID NO 10):
Cccgggccagaaacgctggacattgccatgttattcagattgg
The plasmid pME101-t/*rA*lwas then introduced into the strain MG1655 metA*ll
AmetJ AfadRy.Cm, yielding MG1655 metAVl AmetJ AfadR (pMElOUhrA*!).
MG1655 metA*U AmetJ AiclR (pME101-^rA*l).
The iclR. gene deletion was introduced in the MG1655 metA*ll AmetJ strain using the strategy described by Datsenko and Wanner (see above) with the following oligonucleotides :
DiClF (SEQ ID NO 11): Cgcacccattcccgcgaaacgcggcagaaaacccgccgttgccaccgcaccagcgactggacaggttcagtctttaacgc gTGTAGGCTGGAGCTGCTTCG with
- a region (lower case) homologous to the sequence (4221202-4221120) of the gene zcZR (reference sequence on the website http://genolist.pasteur.fr/Colibri/),
- a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645),
DiClR (SEQ ID NO 12): gcgcattccaccgtacgccagcgtcacttccttcgccgctttaatcaccatcgcgccaaactcggtcacgcggtcatcg gCATATGAATATCCTCCTTAG
with
- a region (lower case) homologous to the sequence (4220386-4220465) of the gene iclR. (reference sequence on the website http://genolist.pasteur.fr/Colibri/\
- a region (upper case) for the amplification of the kanamycin resistance cassette, The oligonucleotides DiclF and DiclR were used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained was then introduced
by electroporation into the strain MG1655 metA*ll Ameύ (pKD46). Kanamycin resistant transformants were selected and the insertion of the resistance cassette verified by PCR using the oligonucleotides iclF and iclR defined below. The strain retained is designated MG1655 metA*ϊ l Ameύ Δzc/R: :Km IcIF (SEQ ID NO 13) : cctttgaggtcgcatggccagtcggc (homologous to the sequence from 4221558 to 4221533). iclR (SEQ ID NO 14) : gctttttaatagaggcgtcgccagctccttgcc (homologous to the sequence from 4219917 to 4219949).
The pME101-r7zrA*l plasmid was then introduced in the strain MG1655 metA*ll Ameύ Δzc/R: :Km.
The kanamycin resistance cassette was eliminated using the strategy described above. The loss of the kanamycin resistance cassette was verified by a PCR analysis with the same oligonucleotides as those used previously (iclF and iclR).
The strain retained is designated MGl 655 met A*l 1 Ameύ AicIR.
MG1655 metA*U AmetJ AfadR AicIR (pME10l-thrA*l)
The deletion AfadRy.Cm was transferred into the strain MGl 655 met A* W AmetJ Δzc/R: :Km by Pl phage transduction as previously described using the phage lysate of the strain MG1655 AfadRy.Cm. Chloramphenicol and at the same time kanamycin resistant transformants were selected and the deletions Δzc/R: :Km and AfadRv.Cm verified by PCR with the oligonucleotides fadRF, fadRR and iclF, iclR. The strain retained was designated MG1655 met A* 11 Ameύ AfadR.: : Cm Δzc/R: :Km.
The plasmid pME101-thrA*l was then introduced in the strain MG1655 metA*l\ Ameύ AfadR:. : Cm Δzc/R: :Km.
The kanamycin and chloramphenicol resistance cassettes were eliminated and their loss verified by PCR analysis with the same oligonucleotides as those used previously
(fadF, fadR and iclF, iclR). The strain retained was designated MGl 655 met A* W Ameύ
AfadR. Δzc/R. Subsequently the plasmid pME101-z7zrA*l was introduced giving the strain MG1655 metA*l 1 Ameύ AfadR Δzc/R (pME101-r7zrA*l).
MG1655 metAHl AmetJ AicIR Agcl (pME101-tfwA*l)
The MG 1655 Δgc/::Rm was constructed using the method of Datsenko and Wanner as described with the following oligonucleotides : DgClF (SEQ ID NO 15):
ggcaaaaatgagagccgttgacgcggcaatgtatgtgctggagaaagaaggtatcactaccgccttcggtgttccgggagcT GTAGGCTGGAGCTGCTTCG with
- a region (lower case) homologous to the sequence (533142-533222) of the region of the gene gel (reference sequence on the website http://genolist.pasteur.fr/ColibriΛ.
- a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645),
DgipR (SEQ ID NO 16) gcgttacgttttaacggtacggatccatccagcgtaaaccggcttccgtggtggtttggggtttatattcacacccaacccCATA TGAATATCCTCCTTAG with
- a region (lower case) homologous to the sequence (535720-535640) of the region of the gene gel (reference sequence on the website http://genolist.pasteur.fr/Colibri/).
- a region (upper case) for the amplification of the kanamycin resistance cassette, The oligonucleotides DgclF and DgipR were used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained is then introduced by electroporation into the strain MGl 655 (pKD46). Kanamycin resistant transformants were selected and the insertion of the resistance cassette was verified by PCR with the oligonucleotides gclF and gipR defined below. The strain retained was designated MG1655 Δgc/::Km. gclF (SEQ ID NO 17) : ggatatgcccaccttgctgaagg (homologous to the sequence from
532795 to 532817). gipR (SEQ ID NO 18) : cgcttagtttcaatcggggaaatgg (homologous to the sequence from
536114 to 536090). The deletion of Δgc/::Km was transduced into strain MG1655 metA*\l Anieβ
AiclK using a phage Pl lysate of the strain MG1655 Δgc/::Km
Kanamycin resistant transformants were selected and the deletion of the gene
(Agcl: :Km) was verified by PCR with the oligonucleotides gclF and gipR (defined above).
The strain retained was designated MGl 655 metA*l 1 AmetJ AiclK AgclwKm. The plasmid pME101-t/jrA*l was then introduced in the strain MG1655 metA*l 1
Ameύ AiclK Δge/::Km.
The kanamycin resistance cassette was eliminated as described and its loss verified by PCR with the oligonucleotides gclF and gipR (see above). The strain retained was designated MGl 655 met AHl AmetJ AiclR AgcL
MG1655 metA*U AmetJ AiclR Aedd-eda (pME101~tfwA*l)
The MG 1655 Aedd-eda::¥Lm was constructed using the method of Datsenko & Wanner as described with the following oligonucleotides :
DeddF (SEQ IDNO 19) CgcgcgagactcgctctgcttatctcgcccggatagaacaagcgaaaacttcgaccgttcatcgttcgcagttggcatgcggT
GTAGGCTGGAGCTGCTTCG with
- a region (lower case) homologous to the sequence (1932582-1932499) of the region of the genes edd-eda (reference sequence on the website http://genolist.pasteur.fr/Colibri/),
- a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, KA. & Wanner, B.L., 2000, PNAS, 97: 6640-6645),
DedaR (SEQ ID NO 20) gcttagcgccttctacagcttcacgcgccagcttagtaatgcggtcgtaatcgcccgcttccagcgcatctgccggaaccCAT ATGAATATCCTCCTTAG with
- a region (lower case) homologous to the sequence (1930144-1930223) of the region of the genes edd-eda (reference sequence on the website http://genolist.pasteur.fr/Colibri/). - a region (upper case) for the amplification of the kanamycin resistance cassette,
The oligonucleotides DeddF and DedaR were used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained was then introduced by electroporation into the strain MGl 655 met A* 11 (pKD46). Kanamycin resistant transformants were then selected and the insertion of the resistance cassette was verified by PCR analysis with the oligonucleotides eddF and edaR defined below. The strain retained was designated MGl 655 met A*ll Aedd-edawKm. eddF (SEQ ID NO 21) : Gggtagactccattactgaggcgtgggcg (homologous to the sequence from 1932996 to 1932968). edaR (SEQ ID NO 22) : ccacatgataccgggatggtgacg (homologous to the sequence from 1929754 to1929777).
The deletion Aedd-eda::Km was transduced into strain MGl 655 met A*ll AmetJ
Δzc/R using a Pl phage lysate of the strain MG1655 Aedd-eda::Km. Kanamycin resistant transformants were selected and the deletion of the gene (Aedd-eda: :Km) was verified by
PCR analysis with the oligonucleotides eddF and edaR. The strain retained was designated MG1655 met A* 11 Ameβ AiclR. Aedd-eda::Kxn.
The plasmid pME101-t/«-A*l was then introduced in the strain MG1655 ??ιetA*\ l Ameύ Δzc/R Aedd-edav.Km.
The kanamycin resistance cassette was eliminated as described and its loss was verified by PCR analysis with the oligonucleotides eddF and edaR, described above. The strain retained was designated MGl 655 met A* 11 Ameύ Δzc/R Aedd-eda.
MG1655 metA*U AmetJ AiclR Aedd-eda Agcl (pME101-ώrA*l)
The MG1655 Aedd~eda::Cm was constructed in the strain MG1655 metA*ll as described for the kanamycin resistant mutant, only that in this case the vector pKD3 carrying a chloramphenicol resistance was used.
The deletion Aedd-edav.Cm, was then transduced into strain MG1655 metA*l\ Ameύ Δzc/R Δge/::Km by Pl phage transduction using a phage lysate of the strain MG 1655 Aedd-edav.Cm.
Chloramphenicol and at the same time kanamycin resistant transformants were selected and the deletions of the genes Aedd-edav.Cm and Δgc/::Km were verified by PCR with the oligonucleotides eddF, edaR and gclF, gipR (defined above). The strain retained was designated MG1655.metA*ll Ameύ Δzc/R Δgc/::Km Aedd-edav.Cm.
The plasmid pME101-t/zrA*l was then introduced in the strain MG1655 metA*U Ameύ AiclR. AgcϊvXm Aedd-edav.Cm. The kanamycin and chloramphenicol resistance cassettes were eliminated as described and their loss was verified by PCR with the oligonucleotides eddF, edaR and gclF, gipR described above. The strain retained was designated MGl 655 met A^W Ameύ Δzc/R Agcl Aedd-eda.
2. Fermentation of strains with increased production of aspartate-derived metabolites
Production strains were analyzed in small Erlenmeyer flask cultures using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sd. USA 32:120-128) that was supplemented with 5 g/1 MOPS and 5 g/1 glucose. Spectinomycin was added if necessary at a concentration of 50 mg/1. An overnight culture was used to inoculate a 30 ml culture to an OD600 of 0.2 and then grown to an OD600 of 4.5 to 5. Extracellular metabolites were analyzed during the batch phase. Amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using GC-MS after silylation.
As can be seen from table 1 the amount of aspartate-derived metabolites is significantly increased upon deletion of iclR. and/or fadR thus demonstrating that the
deletion of the glyoxylate shunt regulation is crucial for the production of aspartate-derived metabolites.
Table 1. Sum of major aspartate-derived metabolites (O-succinylhomoserme, homoserine, threonine, valine, lysine, methionine, isoleucine) in mmol/g DW produced in batch culture.
3. Determination of glyoxylate shunt specific enzyme activities
The activities of isocitrate lyase (AceA) and malate synthase (AceB) were determined in vitro. E. coli strains were cultured in minimal medium with 5 g/1 glucose and harvested at mid log phase. Cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70W). After centrifugation, proteins contained in the supernatants were quantified (Bradford, 1976).
For the determination of isocitrate lyase activity (AceA) five μl extract were assayed in 50 mM imidazole, 100 mM KCl, 1 mM EDTA, 5 inM MgSO4, 20 mM phenylhydrazine, 5 mM isocitrate for 30 minutes at 25°C. Isocitrate lyase activity was determined according to the rate of glyoxylate phenylhydrazine complex formed that absorbs at 324 nm.
Malate synthase activity was determined by assaying 10 μl extract in 50 mM potassium phosphate buffer (pH 6.5), 5 mM MgCl2, 0.1 mM Acetyl-CoA, 2 mM glyoxylate. Malate synthase activity was determined according to the reduction in absorbance at 232 nm that is caused by the consumption of acetyl-CoA used for the production of malate.
As can be seen from table 2 the deletion of icϊR. increases significantly the activity of AceA and AceB. This can explain the increased production of aspartate-derived metabolites shown in table 1 above. In contrast in the OfadK mutant the activity of these two enzymes is not increased indicating that another independent unidentified mechanism must be operative that increases the production of aspartate-derived metabolite.
Table 2. Isocitrate lyase activity (AceA) and malate synthase activity (AceB) in mUI/mg protein in strains in which the regulatory genes fαdR and iclR were deleted.
Claims
Claims
1) A method for the production of aspartate or its derivatives by culture of a microorganism in an appropriate culture medium and recovery of the aspartate derivative from the culture medium, wherein the microorganism is transformed to enhance the activity of the glyoxylate shunt.
2) A method as claimed in claim 1 in which the expression of at least one gene selected among the following involved in glyoxylate production is increased. • ace A encoding isocitrate lyase
• aceB encoding malate synthase
3) A method as claimed in claims 1 or 2 in which the expression of one of the following genes is attenuated:
• iclR encoding the repressor of the glyoxylate shunt • fadR an activator of icϊR.
• icd encoding isocitrate dehydrogenase
• gel encoding glyoxylate carboligase
• eda encoding 2-keto 3-deoxy gluconate 6-phosphate aldolase.
4) A method as claimed in one of claims 1 to 3 in which the expression of additional genes involved in the production of aspartate or its derived metabolites is increased.
5) A method as claimed in one of claims 1 to 4 in which the expression of genes decreasing the production of aspartate or its derived metabolites is attenuated. 6) A method for the fermentative preparation of aspartate or a derived metabolite, in particular methionine its precursors or derivatives as claimed in one of claims 1 to 5 comprising a) Fermentation of the microorganism producing the desired metabolite b) Concentration of the desired product in the cells of the bacteria or in the medium and c) Isolation of the desired metabolite/ constituents of the fermentation broth and/or the biomass optionally remaining in portions or in the total amount (0- 100%) in the end product.
7) A microorganism used for the fermentative production of aspartate or its derived metabolites, in particular methionine, in which the biosynthesis of aspartate or its derivatives is optimized as described in claims 1 to 6.
Priority Applications (5)
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PCT/IB2005/003071 WO2007017710A1 (en) | 2005-08-11 | 2005-08-11 | Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, homoserine, or valine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression |
US11/997,732 US20080233617A1 (en) | 2005-08-11 | 2006-08-11 | Process for the Preparation of a Aspartate and Derived Amino Acids Like Lysine, Threonine, Isoleucine, Methionine, or Homoserine Employing Microorganism with Enhanced Isocitrate Lyase and/or Malate Synthase Expression |
EP06792801A EP1913128A1 (en) | 2005-08-11 | 2006-08-11 | Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, or homoserine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression |
EP10183233A EP2275529A3 (en) | 2005-08-11 | 2006-08-11 | Process for the preparation of aspartate and derived amino acids employing a microorganism with enhanced glyoxylate shunt |
PCT/EP2006/065270 WO2007017526A1 (en) | 2005-08-11 | 2006-08-11 | Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, or homoserine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression |
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PCT/IB2005/003071 WO2007017710A1 (en) | 2005-08-11 | 2005-08-11 | Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, homoserine, or valine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression |
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PCT/EP2006/065270 WO2007017526A1 (en) | 2005-08-11 | 2006-08-11 | Process for the preparation of aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, or homoserine employing a microorganism with enhanced isocitrate lyase and/or malate synthase expression |
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EP2275529A2 (en) | 2011-01-19 |
EP1913128A1 (en) | 2008-04-23 |
EP2275529A3 (en) | 2012-09-12 |
WO2007017526A1 (en) | 2007-02-15 |
US20080233617A1 (en) | 2008-09-25 |
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