WO2004009828A1 - Procede de fabrication biotechnologique d'acide citrique au moyen d'une levure genetiquement modifiee yarrowia lipolytica - Google Patents

Procede de fabrication biotechnologique d'acide citrique au moyen d'une levure genetiquement modifiee yarrowia lipolytica Download PDF

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WO2004009828A1
WO2004009828A1 PCT/DE2003/002458 DE0302458W WO2004009828A1 WO 2004009828 A1 WO2004009828 A1 WO 2004009828A1 DE 0302458 W DE0302458 W DE 0302458W WO 2004009828 A1 WO2004009828 A1 WO 2004009828A1
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seq
gene sequence
vector
isocitrate lyase
citric acid
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PCT/DE2003/002458
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Kordula Kruse
Andre Förster
Thomas Juretzek
Stephan Mauersberger
Gerold Barth
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Technische Universität Dresden
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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
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/48—Tricarboxylic acids, e.g. citric acid

Definitions

  • the invention relates to a method for the biotechnological production of citric acid (CS) with a genetically modified yarrow Yarrowia. lipolytica (Y. lipolytica)
  • citric acid which is sold in crystalline form, is used on a large scale in various industries, but mainly in the food and pharmaceutical industries as flavoring agents, preservatives, acidulants and antioxidants (Roehr et al. 1996, Citric acid, Biotechnology Vol 6, Rehm and Reed, eds).
  • Y. lipolytica with which a number of hydrophobic substrates, such as vegetable and animal oils and fats or n-alkanes, can be used as a C source due to the properties of this yeast. It has been known since the mid-1960s that, under specific conditions, Y. lipolytica is able to secrete citric acid (CS) and isocitric acid (ICS), as well as other metabolites of intermediate metabolism such as pyruvate and ⁇ -ketoglutarate. The corresponding cultivation conditions have since been extensively investigated.
  • CS citric acid
  • ICS isocitric acid
  • the most important prerequisite for an overproduction and excretion of these metabolites is a growth limitation of the production culture with a simultaneous excess of usable C source (Stottmeister et al. 1982, Z Allg Mikrobiol 22: 399-424).
  • the growth limitation the culture is thereby limited by necessary nutrient medium components such. B. minerals, vitamins and amino acids.
  • the type of metabolite formed depends on the limiting nutrient component and other cultivation conditions, such as the C source used or the pH of the production medium.
  • citric and isocitric acid can be caused by a lack of nitrogen, phosphorus, sulfur or magnesium (Stottmeister et al. 1982, Z Allg Mikrobiol 22: 399-424).
  • the CS / ICS formation can also be induced by a lack of amino acids (Barth and Krebs 1984, DD 227 448 A1).
  • a growth limitation due to a lack of thiamine which is not produced by Y. lipolytica itself, initiates the formation of 2-ketoglutaric acid and pyruvate (Weissbrodt et al. 1988, DD 267999A1, Chernyavskaya et al. 2000, Appl Microbiol Biotechnol 53: 152- 158).
  • the product ratio CS / ICS and the product yield for wild-type strains of Y. lipolytica depend on the C sources used. Y. lipolytica forms about 90% CS and 10% ICS on glucose and glycerol (Treton et al. 1978, Appl Microbiol Biotechnol 6: 67-77). Similar product ratios were found when ethanol was used as the C source (Arzumanov et al. 2000, Appl Microbiol Biotechnol 53: 525-529). On the hydrophobic substrates of interest for industrial use, such as vegetable oils or n-alkanes, the product ratio shifts to an ICS content of 40 to 50% (reviews by Stottmeister et al.
  • citric acid and glyoxylate cycle enzymes influence the product range, they are Biochemical and molecular biological relationships have not been fully clarified to date. With the previously known methods, a targeted production of high-performance production strains for CS production with low ICS accumulation is not possible.
  • the object of the invention is to develop a process for the production of citric acid with the yeast Y lipolytica, in which significantly less isocitric acid is obtained and with which an economically efficient citric acid production with the yeast Y lipolytica is made possible.
  • the object is achieved by a process for the biotechnological production of citric acid in yarrow cells from Yarrowia lipolytica comprising the steps: a) transformation of yeast cells which have a defect in a gene which is important for their growth, with a vector which is suitable for multiple genomic integration, which contains a gene which acts as a selection marker and which can compensate for the defect mentioned above, b) cultivation of the yeast under deficiency conditions and a carbon source, c) purification of the citric acid, the vector containing an expression cassette with a gene sequence coding for isocitrate lyase activity and
  • Clones are selected in a suitable nutrient medium which have stably integrated several copies of a gene sequence coding for the isocitrate lyase activity and the selection marker into their genome.
  • the multiple integration of a gene coding for isocitrate lyase activity into the yeast genome is achieved by the transformation with special multicopy plasmid vectors and subsequent selection pressure.
  • a gene sequence coding for isocitrate lyase activity (ICL activity) is cloned into a vector which contains a multicopy selection marker and the vector is transformed into a Y lipolytica strain suitable for the selection marker.
  • a gene sequence is preferably cloned into the vector, which for a protein according to SEQ ID No. 1 coded.
  • the ICL protein according to the invention according to SEQ ID No. 1 differs significantly in its sequence from the sequence known from Barth and Scheuber 1993, Mol Gen Genet 241: 422-430.
  • an expression cassette according to SEQ ID No. 2 cloned into the vector.
  • the expression cassette according to SEQ ID No. 2 shows a gene sequence which for the protein according to the invention according to SEQ ID No. 1 coded.
  • Suitable vectors and Y lipolytica strains are known and accessible to the person skilled in the art.
  • these vectors contain, for example, allelic variants of the a homologous transformation platform, such as the sequence region of the ribosomal DNA (rDNA) or the zeta-called LTR sequence (long terminal repeat) of the retrotransposon Ylt1 from Y lipolytica.
  • allelic variants of the a homologous transformation platform such as the sequence region of the ribosomal DNA (rDNA) or the zeta-called LTR sequence (long terminal repeat) of the retrotransposon Ylt1 from Y lipolytica.
  • the integrative transformation takes place with the linearized vectors using the lithium acetate method (modified according to Barth and Gaillardin 1996, Yarrowia lipolytica. In: Nonconventional Yeasts in Biotechnology, Wolf K, ed, Springer-Verlag, Berlin Heidelberg New York, pp 313 -388). It is transformed into a Y lipolytica strain that matches the marker gene. In the case of an lf? ⁇ 3 allele as a selection marker, this is for example a Y lipolytica strain in which the is broken. Y. lipolytica strains with defective URA3 gene and methods for their preparation are described in the literature (Barth and Gaillardin 1996, Yarrowia lipolytica. In: Nonconventional Yeasts in Biotechnology, Wolf K, ed, Springer-Verlag, Berlin Heidelberg New York, pp 313-388).
  • the vector used preferably contains an expression cassette according to SEQ ID No. Second
  • the transformation of the vectors p64ICL1 and p67ICL1 into the Y lipolytica strain H222-S4 (ura3-302) with a defect in the URA3 gene is described as an example.
  • the plasmids p64ICL1 and p67ICL1 contain a shortened allele of the URA3 gene, ura3d4, as a selection marker in the promoter region. By shortening the promoter range in the ura3d4- ⁇ e ⁇ , multiple integration of ura3d4 into the yeast genome is necessary to compensate for the defect in the URA3 gene.
  • Plasmids with other multicopy selection markers, e.g. B. of corresponding allelic variants of the EL / 2 gene and corresponding Y. lipolytica strains can also be used.
  • the expression of the gene product isocitrate lyase is significantly increased in Y lipolytica strains that have integrated this several times into their genome. At the same time, a stable increase in the specific isocitrate lyase activity is achieved. Surprisingly, neither the increased ICL 7 expression nor the isocitrate lyase activity is abolished by transcriptional, post-translational and metabolic counter-regulation mechanisms (see exemplary embodiment 2).
  • the Y lipolytica strains according to the invention which have integrated the ICL 1 gene several times into their genome, are cultivated under deficiency conditions which lead to an overproduction of CS and ICS, such as, for example, under nitrogen, phosphorus. Sulfur, magnesium or when using auxotrophic strains due to lack of amino acids.
  • the strains produced according to the invention can be used for citric acid production can therefore be easily used in large-volume bioreactors (fermenters).
  • Y lipolytica strains used for citric acid production can use a large number of different C sources, such as glucose, glycerol, ethanol, n-alkanes, vegetable or animal oils or fats.
  • C sources such as glucose, glycerol, ethanol, n-alkanes, vegetable or animal oils or fats.
  • the method according to the invention results in a significantly reduced accumulation of the undesired isocitric acid in comparison to wild-type strains. Surprisingly, this results in the production of citric acid with Y lipolytica strains that have integrated several times into their genome, a significant shift in the product ratio CS / ICS in favor of citric acid (see Example 3).
  • Another object of the invention is the expression cassette according to SEQ ID no. 2.
  • the invention also relates to vectors which the expression cassette according to SEQ ID No. 2 contain, preferably the vectors p64ICL1 or p67ICL1 whose sequences of SEQ ID No. 3 or SEQ ID No. 4 correspond.
  • the invention furthermore relates to a strain of Y lipolytica which has stably integrated several copies of a gene sequence coding for isocitrate lyase activity into its genome, preferably the sequence according to SEQ ID No.2.
  • strains of Y lipolytica which are transformed with one of the vectors p64ICL1 or p67ICL1 are also preferred.
  • the strain of Y lipolytica H222-S4 (p64ICL1) T1 with the deposit number DSM 15105 is also preferably deposited with the German Collection for Microorganisms and Cell Cultures (DSMZ) in Braunschweig in accordance with the Budapest Treaty.
  • the invention also relates to the use of a strain of Y lipolytica according to the invention for the production of citric acid.
  • yeast strains and vectors are used in these examples:
  • H222-S4 Production by gene destruction of the URA3 gene in H222 as a result of insertion of the SUC2 gene from Saccharomyces cerevisiae by integrative transformation with a 4.3 kb Sal ⁇ from the plasmid plNA302 (Mauersberger et al. 2001 , J Bacteriol 183: 5102-5109). Like H222, this strain contains no free zeta elements or intact Ylt1 (Juretzek et al. 2001, Yeast 18: 97-113).
  • E129L1 (MATA lys11-23 ura3-302 xpr2-322): Production by complementing the leucine auxotrophy from the laboratory strain E129 (MATA lys 11-23 leu2-270 ura3-302 xpr2-322) with the plasmid plNA62 (Barth and Gaillardin 1996, Yarrowia lipolytica. In: Nonconventional Yeasts in Biotechnology, Wolf K, ed, Springer-Verlag, Berlin Heidelberg New York, pp 313-388).
  • the strains used in the exemplary embodiments come from the strain collection of the Institute of Microbiology at the TU Dresden.
  • Example 1 The strains used in the exemplary embodiments come from the strain collection of the Institute of Microbiology at the TU Dresden.
  • Integrative vectors for the multicopy transformation of Yarrowia lipolytica For the multiple integration of the ICL 1 gene into the yeast genome, the multicopy vectors p64ICL1 and p67ICL1 (FIG. 1, SEQ ID No. 3 and 4) based on the plasmids p64IP and p67IP for constructed the heterologous expression of proteins under the control of the ICL 7 promoter in Y lipolytica (Juretzek et al. 2001, Yeast 18: 97-113).
  • the output vectors p64IP and p67IP used contain an ICL 7 promoter fragment from Y lipolytica.
  • the ura3c / 4 allele of the URA3 gene from • Y lipolytica was used as the multicopy selection marker.
  • This marker sequence enables the multicopy transformation of Y lipolytica strains with a defect in the URA3 gene (Ura " ).
  • the vectors carry the rDNA sequence (p64IP) or the long terminal repeat (LTR) sequence of the retrotransposon Ylt1 from Y. lipolytica (p67IP)
  • zeta does not occur in all Y lipolytica strains, so that the vectors p67IP and p67ICL1 can be used for the transformation of zeta-free strains with significantly lower transformation efficiency.
  • the 2.3 kb 8amHI fragment of the ICL1 structural gene was inserted into the vectors p64IP and p67IP.
  • the vectors p64ICL1 and p67ICL1 thus obtained thus contain the complete ICL1 gene (according to Seq ID No. 2) as an expression cassette.
  • the E. coli strain DH5 ⁇ c was used for the amplification of the integration vectors.
  • the E. coli cells were cultivated in LB medium with 100 ⁇ g / ml ampicillin at 37 ° C.
  • the vector DNA was isolated by means of plasmid preparation according to Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York).
  • the vectors (FIG. 1) were linearized with SacII (p64ICL1) or Not ⁇ (p67ICL1) using the modified lithium acetate method according to Barth and Gaillardin (Barth and Gaillardin 1996, Yarrowia lipolytica.
  • Nonconventional Yeasts in Biotechnology Wolf K, ed, Springer-Verlag, Berlin Heidelberg New York, pp 313-388) were transformed into the recipient strains H222-S4 and E129L1. 2-5 ⁇ g plasmid DNA were used for the transformation.
  • the transformants with compensation of the uracil defect as a result of the multicopy integration of the vector were modified on agar plates with minimal medium M (according to Reader, modified: Mauersberger et al. 1996, Candida maltosa.
  • Nonconventional Yeasts in Biotechnology, Wolf K, ed, Springer- Verlag, Berlin Heidelberg New York, pp 411-580 with glucose as a C source by culturing at 28 ° C for 3 to 10 days.
  • Transformants (TF) with the rDNA vector p64ICL1 were obtained with the two starting strains H222-S4 and E129L1 with a frequency of 30 to 70 TF / ⁇ g DNA.
  • the zeta vector p67ICL1 With the zeta vector p67ICL1, however, the zeta-free strain H222-S4 could only be transformed with the significantly lower efficiency of about 0.5-1 TF / ⁇ g DNA.
  • the strain E129L1 carrying many zeta copies was transformable with the vector p67ICL1 with an efficiency of 30 TF / ⁇ g DNA.
  • the multiple integration of the ICL 7 gene in selected transformants was demonstrated by Southern hybridization.
  • genomic DNA of the recipient strains and selected transformants was prepared using the method of Hoffmann and Winston (1987, Gene 57: 267-272) after mechanical cell disruption. Probe preparation and detection was carried out using the "Gene Images random prime labeling and detection system" from Amersham Life Sciences.
  • the genomic DNA was cleaved completely with the restriction enzyme ⁇ / col. After separation of the preparations in gel electrophoresis and subsequent membrane blotting, the 2.3 kb SamHI fragment of the / C / _7 structural gene was used as a detection probe for the hybridization.
  • 3 specific bands (bands a, d, e in FIG. 2) of the genomic copy of the ICL1 gene were detected at approximately 4,500, approximately 1,300 and 1,233 bp.
  • the examined transformand T1 of the type H222-S4 (p64ICL1) had additional bands at 2.9 kb and 1.9 kb (bands b and c in FIG.
  • the number of copies of the integrated vectors was also determined by detecting the ura3d4 allele used as the marker gene in the transformation vectors.
  • a 1.7 kb fragment from the URA gene from Y lipolytica was used as a detection probe for the Southern blot.
  • the starting strain H222-S4 had a specific band at 4.3 kb (band a in FIG.
  • the specific activity of the isocitrate lyase from the parent strain H222-S4 and selected ICL 1 multicopy transformants was determined when culturing on different C sources.
  • the strains were first overnight in 100 ml shake flasks with 25 ml yeast minimal medium M with 2% glucose as a C source and necessary supplements (0.3 ⁇ g / ml thiamine hydrochloride for all cultures, 40 mg / l uracil for H222-S4 ) cultivated at 28 ° C on a horizontal shaker at 230 rpm (revolutions per minute) (1st preculture).
  • 20 ml of the 1st preculture were transferred to 500 ml Erlenmeyer flasks with 100 ml of the same nutrient medium (2nd preculture).
  • the second preculture was incubated under the conditions mentioned above for 15 to 24 h and after pH adjustment for the inoculation of the main cultures in 500 ml Erlenmeyer flasks with 100 ml yeast minimal medium M and necessary supplements (thiamine, uracil) and various C sources (2% glucose, 1% ethanol, 5% sunflower oil or 5% hexadecane).
  • the inoculation was carried out with an inoculation concentration of 2 to 3 * 10 7 cells / ml.
  • the cultivation was carried out as described for the precultures.
  • the cell-free extract was obtained after 5 min centrifugation at 3000 rpm and 4 ° C and used for the determination of the isocitrate lyase activity and the protein concentration.
  • the isocitrate lyase activity was measured photometrically according to Dixon and Kornberg (1959, Biochem J 72: 3) on the basis of the formation of Phenylhydrazone from phenylhydrazine and glyoxylate over a period of 3 to 5 minutes at 324 nm and 30 ° C.
  • the protein concentrations of the cell-free extracts according to Lowry et al. (1951, J Biol Chem 193: 265-275).
  • H222-S4 and the transformant H222-S4 (p64ICL1) T1 were cultivated using glucose and ethanol as the C source (FIG. 4), a 15- to 16-fold higher specific was found on both substrates after an incubation time of 9 h Isocitrate lyase activity of the transformed strain compared to the parent strain was found. Under the chosen test conditions, a maximum of the specific isocitrate lyase activity occurs after approx. 9 h in all strains examined. Measurements before and after this activity maximum also confirmed the high relative increase in the specific ICL activity of the transformants compared to the parent strain.
  • the increased isocitrate lyase activity of the transformants H222-S4 (p64ICL1) T1 compared to the parent strain was also confirmed when using the hydrophobic substrates sunflower oil and hexadecane for citric acid production (FIG. 5).
  • yeast minimal medium M with a reduced ammonium nitrogen content of 1 g / l (NH) 2 S0 4 was used, which resulted in a growth limitation of the cultures due to a lack of nitrogen and the production of citric and isocitric acid was induced after about 3 days of cultivation.
  • Precultures for the cultivation for citric acid production The cultures were incubated in 500 ml shaking flasks in 100 ml YPD for 24 h on a horizontal shaker at 28 ° C. and 230 rpm, then centrifuged under sterile conditions at 5000 rpm for 5 min and the pellet in yeast minimal medium M washed twice without the addition of nitrogen. The cell pellet was then taken up in 5 ml of minimal medium M without addition of nitrogen and used for the inoculation of the main cultures.
  • the citric acid production was cultivated in 500 ml Erlenmeyer flasks each with 100 ml yeast minimal medium M with a reduced ammonium sulfate content of 1 g / l.
  • 3 to 10% glucose, sunflower oil, waste fat (vegetable fat mixture) or hexadecane were added as the C source.
  • Sunflower oil was processed without substrate preparation, but used fat as an aqueous emulsion (Emulsification of 20% used fat with 1% Tween 80 in water using ultrasound).
  • 0.3 ⁇ g / ml thiamine hydrochloride and, in the case of H222-S4, an additional 40 mg / l uracil were added to all cultures.
  • the cultivation was carried out after inoculation of the cultures (inoculation titers 0.5 to 2 OD - optical density at 600 nm) on a horizontal shaker at 230 rpm and 28 ° C. with regular pH adjustment to pH 6.0 using 2.5 mol / l, 5 mol / l or 10 mol / l NaOH over a period of 5 to 12 days.
  • the ammonium nitrogen content in the culture medium was analyzed using an enzymatic test kit from Dr. Lange GmbH & Co KG (Dusseldorf).
  • the citric acid and isocitric acid concentration was analyzed using enzymatic test kits (kit no. 0139076 or 0414433) from R-Biopharm GmbH (Darmstadt), or using an DX-320 ion chromatography system from Dionex (Sunnyvale, USA) - separation column lonPac AS 15, 2 mm diameter, conductivity detector CD 25a, Autosampier AS 40).
  • the product formation rates achieved under the selected conditions of the shake cultures with H222 on sunflower oil were between 190 to 250 mg / l * h for citric acid (total acidity CS + ICS 340 to 390 mg / l * h) with a biomass concentration (dry yeast mass) of 13 to 14 g / l.
  • the product formation shown in FIG. 7 on CS / ICS could be carried out by prolonged culture (up to 20 days) and Substrate concentrations from 8-10% sunflower oil to 100 to 150 g / l (total acidity) can be increased.
  • the transformants H222-S4 (p64ICL1) T8, T9, T11, T12, T16, T17 and T18 investigated under the same conditions showed a significantly more favorable CS / ICS product spectrum from 94: 6 to 96: 4 with a total acid content of 80 to 100 g / l on (Fig. 8). Comparable results were achieved with selected transformants of the type H222-S4 (p67ICL1). This significant shift in the product ratio in favor of citric acid in the ICL 7-multicopy transformants was confirmed when cultivating for glucose, used fat (mixture of vegetable fats) and hexadecane (Table 1).
  • Tab. 1 Relationship of CS / ICS production with Y lipolytica H222, H222-S4 (ura3-302) and the / C 7-multicopy transformants H222-S4 (p64ICL1) and H222-S4 (p67ICL1) on different C sources ,
  • the yeast cells were in 500 ml shake flasks with 100 or 200 ml minimal medium M with 1 g / l (NH 4 ) 2 SO 4 ) and 5-6% C source for 5 to 12 days or up to 18 days for the experiments cultivated with sunflower oil. In the case of longer cultivation, the C source was replenished when the substrate was exhausted (total 8-10% substrate). The data marked with 1) were obtained after a shorter culture time of 5-7 days.
  • Fig. 1 Structure of the multicopy transformation vectors p64ICL1 and
  • the multicopy vectors with rDNA sequence (p64ICL1) or the zeta sequence (p67ICL1) used for increasing the copy number of the ICL gene by integrative transformation are shown as homologous transformation sequences.
  • the vectors contain the / C 7 gene from Y lipolytica with promoter (plCL1), intron (ICLi), open reading frame of ICL1 (ICL1 ORF) and terminator (ICLt) as well as the ura3d4 allele of the URA3 gene from Y as a functional expression cassette lipolytica as a multicopy selection marker. After linearization with Sacll (p64ICL1) or ⁇ / ofl (p67ICL1), the vectors are integratively transformed into the genome of Y lipolytica.
  • Fig. 2 Evidence of the increased copy number of the ICL1 gene.
  • DNA by the restriction enzyme ⁇ / col and detection of specific bands by means of the 2.3 kb SamHI fragment of the / C 7 gene from Y lipolytica.
  • Transformand H222-S4 (p64ICL1) T1; MW: molecular weight standard.
  • Fig. 3 Evidence of the increased number of copies of the ura3d4 alley.
  • a Southern blot is shown after complete digestion of the genomic DNA by the restriction enzyme Sa / I and detection of specific bands by means of a 1.7 kb Sa / I fragment of the ⁇ / ra3d4 allele from Y lipolytica.
  • H222-S4 parent strain H222-S4; 2-4: Multicopy transformants H222-S4 (p64ICL1) T1, T9 and T11; MW: molecular weight standard.
  • Detected bands a: Genomic fragment with the ura3-302 allele at 4.3 kb; b: The t; ra3d4-containing fragment from the vector p64ICL1 at 2.8 kb.
  • Fig. 4 Evidence of the increase in specific isocitrate lyase activity due to multicopy integration of the ICL1 gene when cultivated on the C sources glucose and ethanol.
  • the figure shows the course of the specific isocitrate lyase activity when cultivating the parent strain H222-S4 and the multicopy transformande H222-S4 (p64ICL1) T1 on glucose or ethanol as a C source over a cultivation time of 27 h. Due to the ICL repression by glucose, significantly lower ICL activities are found on this substrate than with ethanol. Irrespective of this transcription regulation and other metabolic regulation mechanisms, at least 10 to 20 times higher specific ICL activity of transformants H222-S4 (p64ICL1) T1 compared to the original strain can always be detected from 6 h cultivation under comparable conditions. 5: Evidence of the increase in the specific isocitrate lyase activity as a result of multicopy integration of the ICL1 gene when cultivated on the C sources sunflower oil and hexadecane.
  • the figure shows the specific isocitrate lyase activities of the parent strain H222-S4 and the multicopy transformande H222-S4 (p64ICL1) T1 after cultivation on sunflower oil (oil) or hexadecane for 16, 40 and 162 hours.
  • the production of citric and isocitric acid was induced after approx. 40 h to 50 h (production phase).
  • p64ICL1 T1 Over the entire culture period (growth and production phase), a 20-fold higher ICL activity of the Transformande H222-S4 (p64ICL1) T1 compared to the original strain was detected.
  • the electrophoretic separation of cell-free protein extracts is shown in the SDS-PAGE after 3, 6, 9 and 12 hours of cultivation on ethanol as a C source (cf. FIG. 4).
  • the proteins were separated (20 ⁇ g protein applied per sample) in 8% polyacrylamide gel at 20 to 50 mA.
  • significantly stronger protein bands at approx. 60 kDa molecular weight of the isocitrate lyase were detected for the investigated / CL7-multicopy transformants H222-S4 (p64ICL1) T1 over the entire cultivation time.
  • Fig. 7 Culture course for citric acid production with Y. lipolytica under the conditions of nitrogen limitation using the example of H222 on sunflower oil.
  • the figure shows the course of the cultivation of Y. lipolytica H222 for citric acid production in modified yeast minimal medium M with a reduced ammonium sulfate content of 1 g / l (production medium) and 5% sunflower oil as a C source in a shake flask test.
  • the production of citric and isocitric acid starts after about 60 h to 70 h of cultivation after the nitrogen source has been used up and the growth of the culture begins to be limited.
  • the product formation rates achieved with H222 on sunflower oil under these conditions were between 190 to 250 mg / h for citric acid (total acid CS and ICS: 340 to 390 mg / h) with a biomass concentration (dry yeast mass) of 13 to 14 g / l.
  • Fig. 8 Evidence of the shift in the product ratio CS / ICS due to multicopy integration of the ICL1 gene when cultivated on sunflower oil.
  • the figure shows the product ratio citric acid / isocitric acid (CS / ICS) of the parent strain H222-S4 and the transformants H222 : S4 (p64ICL1) T8, T9, T11, T12, T16, T17 and T18 after 18 days of cultivation with 8% to 10 % Sunflower oil as a C source.
  • the starting strain had a CS / ICS ratio of about 60:40 (57:43 to 65:35) with a total acidity (CS and ICS) of 80 g / l to 85 g / l.
  • the transformants investigated under the same conditions had a significantly more favorable product range from 94: 6 to 96: 4 with a total acid content of 80 to 100 g / l.
  • the following abbreviations are used in the description text and in the figures:
  • Zeta LTR of the retrotransposon Ylt1 from Y lipolytica The yeast strain Yarrowia lipolytica H22-S4 (p64ICL1) T1 was released on July 16, 2002 after the Budapest contract with
  • the lawyer ensures that the 4 sequences on the diskette enclosed with the application are identical to the sequences in the application.

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Abstract

L'invention concerne un procédé de fabrication biotechnologique d'acide citrique au moyen d'une levure génétiquement modifiée Yarrowia lipolytica. Ledit procédé consiste à intégrer plusieurs fois, de façon stable, une séquence génétique codant l'activité isocitrate lyase, dans le génome de la levure. De manière avantageuse, le procédé selon l'invention permet de réduire considérablement la formation d'acide isocitrique, celui-ci étant un produit secondaire indésirable.
PCT/DE2003/002458 2002-07-16 2003-07-16 Procede de fabrication biotechnologique d'acide citrique au moyen d'une levure genetiquement modifiee yarrowia lipolytica WO2004009828A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003258457A AU2003258457A1 (en) 2002-07-16 2003-07-16 Method for the biotechnological production of citric acid by means of a genetically-modified yeast yarrowia lipolytica

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10233600.8 2002-07-16
DE10233600 2002-07-16

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WO2004009828A1 true WO2004009828A1 (fr) 2004-01-29

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PCT/DE2003/002458 WO2004009828A1 (fr) 2002-07-16 2003-07-16 Procede de fabrication biotechnologique d'acide citrique au moyen d'une levure genetiquement modifiee yarrowia lipolytica

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CN (1) CN1723286A (fr)
AU (1) AU2003258457A1 (fr)
DE (1) DE10333144B4 (fr)
RU (1) RU2005102929A (fr)
WO (1) WO2004009828A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007063133A3 (fr) * 2005-12-01 2007-09-13 Dsm Ip Assets Bv Nouveaux gènes utiles pour la production industrielle d'acide citrique
EP3536784A1 (fr) * 2018-03-05 2019-09-11 ACIB GmbH Cellule hôte conçue pour améliorer la production de métabolites

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011056290A1 (de) 2011-12-12 2013-06-13 Thyssenkrupp Uhde Gmbh Pilzstämme mit genetischer modifikation betreffend einen carbonsäure-transporter

Citations (3)

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DD227448A1 (de) * 1984-10-18 1985-09-18 Akad Wissenschaften Ddr Verfahren zur herstellung von zitronensaeure auf mikrobiellem wege
DD259637A1 (de) * 1987-04-09 1988-08-31 Adw Ddr Verfahren zur herstellung von isozitratlyase in rekombinanten mikroorganismen
WO2000003008A2 (fr) * 1998-07-10 2000-01-20 Technische Universität Dresden Cellules yarrowia lipolytica haploïdes ou diploïdes recombinees pour l'expression heterologue fonctionnelle de systemes du cytochrome p450

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DE19525282A1 (de) * 1995-06-29 1997-01-02 Max Delbrueck Centrum Expressionskassetten zur heterologen Expression von Proteinen in der Hefe Yarrowia lipolytica unter Kontrolle des regulierbaren Promotors der Isocitratlyase

Patent Citations (3)

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DD227448A1 (de) * 1984-10-18 1985-09-18 Akad Wissenschaften Ddr Verfahren zur herstellung von zitronensaeure auf mikrobiellem wege
DD259637A1 (de) * 1987-04-09 1988-08-31 Adw Ddr Verfahren zur herstellung von isozitratlyase in rekombinanten mikroorganismen
WO2000003008A2 (fr) * 1998-07-10 2000-01-20 Technische Universität Dresden Cellules yarrowia lipolytica haploïdes ou diploïdes recombinees pour l'expression heterologue fonctionnelle de systemes du cytochrome p450

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Title
AURICH ANDREAS ET AL: "Citric acid production from renewable resources by Yarrowia lipolytica.", BIOTECHNOLOGY ADVANCES, vol. 21, no. 5, August 2003 (2003-08-01), Moo-Young Symposium on Biotechnology and Bioengineering;Waterloo, Ontario, Canada; September 06-07, 2002, pages 454 - 455, XP002265486, ISSN: 0734-9750 *
BARTH G ET AL: "CLONING OF THE ISOCITRATE LYASE GENE (ICL1) FROM YARROWIA LIPOLYTICA AND CHARACTERIZATION OF THE DEDUCED PROTEIN", MOLECULAR AND GENERAL GENETICS, SPRINGER VERLAG, BERLIN, DE, vol. 241, no. 3/4, 1 November 1993 (1993-11-01), pages 422 - 430, XP000611459, ISSN: 0026-8925 *
DATABASE EMBL [online] 16 November 1993 (1993-11-16), BARTH G: "Y.lipolytica ICL1 gene for isocitrate lyase", XP002265487, retrieved from EBI Database accession no. X72848 *
JURETZEK THOMAS ET AL: "Vectors for gene expression and amplification in the yeast Yarrowia lipolytica", YEAST, vol. 18, no. 2, 30 January 2001 (2001-01-30), pages 97 - 113, XP008025954, ISSN: 0749-503X *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007063133A3 (fr) * 2005-12-01 2007-09-13 Dsm Ip Assets Bv Nouveaux gènes utiles pour la production industrielle d'acide citrique
US8637280B2 (en) 2005-12-01 2014-01-28 Adcuram Nutrition Holding Gmbh Genes useful for the industrial production of citric acid
EP3536784A1 (fr) * 2018-03-05 2019-09-11 ACIB GmbH Cellule hôte conçue pour améliorer la production de métabolites

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DE10333144B4 (de) 2006-06-29
AU2003258457A1 (en) 2004-02-09
CN1723286A (zh) 2006-01-18
RU2005102929A (ru) 2005-08-10
DE10333144A1 (de) 2004-02-05

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