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WO2013019052A2 - Micro-organisme du genre escherichia contenant ou ne contenant pas un gène ybbo amplifié et procédé de production de rétinoïde faisant appel à ce micro-organisme - Google Patents

Micro-organisme du genre escherichia contenant ou ne contenant pas un gène ybbo amplifié et procédé de production de rétinoïde faisant appel à ce micro-organisme Download PDF

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WO2013019052A2
WO2013019052A2 PCT/KR2012/006074 KR2012006074W WO2013019052A2 WO 2013019052 A2 WO2013019052 A2 WO 2013019052A2 KR 2012006074 W KR2012006074 W KR 2012006074W WO 2013019052 A2 WO2013019052 A2 WO 2013019052A2
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seq
gene
gene encoding
protein
microorganism
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WO2013019052A9 (fr
WO2013019052A3 (fr
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김선원
장희정
윤상활
하보경
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Gyeongsang National University GNU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

Definitions

  • the present invention relates to a microorganism of the genus Escherichia and a method for producing a retinoid using the microorganism.
  • Retinoids are a class of lipophilic isoprenoid molecules chemically associated with vitamin A.
  • the retinoid may be combined with an alcohol (eg retinol), aldehyde (eg retinal), carboxylic acid (eg retinoic acid), or ester (eg retinyl acetate) functional group to form ⁇ - It consists of inon rings and polyunsaturated side chains. They are known to play an essential role in human health, such as protecting eyesight, developing and regenerating bone, and antioxidant effects, and to reduce the risk of certain cancers.
  • alcohol eg retinol
  • aldehyde eg retinal
  • carboxylic acid eg retinoic acid
  • ester eg retinyl acetate
  • Retinoids have received great attention in recent years as effective cosmetic and pharmaceutical ingredients for wrinkle improvement and skin disease treatment.
  • the retinoid market is estimated to be around $ 16 billion worldwide.
  • Chemically synthesized retinoids are representative commercial raw materials.
  • Retinol is produced from acidification or hydrolysis of retinal chemically synthesized by reduction of pentadiene derivatives.
  • this chemical process has disadvantages such as complicated purification steps and formation of unwanted byproducts.
  • Animals produce retinoids from carotenoids obtained from fruits and vegetables, while plants cannot synthesize retinoids.
  • the entire route of retinoid synthesis is only possible in microorganisms comprising bacteriododocin or proteorodosin with retinal as a prosthetic group.
  • microorganisms produce a protein-binding form of retinal and are therefore not suitable for mass production of free retinoids.
  • enzymes for biological production, but no successful results.
  • biotechnological methods for retinoid production using metabolically transformed microorganisms there is a need for the development of biotechnological methods for retinoid production using metabolically transformed microorganisms.
  • Retinoids are chemically very unstable due to their reactive conjugated double bonds and are easily oxidized and isomerized by heat, oxygen and light. Retinoids are also readily degraded biologically through retinoic acid. Therefore, there is a need for a method of producing retinoids more efficiently.
  • a gene encoding YbbO protein is attenuated or deleted, or amplified, or a foreign gene having a homologous or greater than 80% homology with the nucleotide sequence of the gene is introduced to provide amplified Escherichia microorganism.
  • Another aspect provides a method of producing retinoids using this microorganism.
  • YbbO protein Another aspect is YbbO protein; Or an enzyme composition comprising a protein encoded by a gene having at least 80% homology with a nucleotide sequence of a gene encoding a YbbO protein.
  • a gene encoding the YbbO protein of a parent strain of Escherichia genus having retinoid production ability is attenuated or deleted, or amplified, or a foreign gene having at least 80% homology with the nucleotide sequence of the gene is introduced.
  • the parent strain may be a wild type Escherichia spp microorganism having a retinoid producing ability or a transformed Escherichia spp microorganism.
  • Wild-type Escherichia microorganisms are known to have a MEP pathway as an intrinsic retinoid synthesis pathway.
  • the transformed Escherichia genus microorganism may be introduced with a gene associated with the intrinsic MEP pathway of retinoid synthesis, a gene associated with the foreign MVA pathway, or a combination thereof.
  • the MVA pathway gene may be a gene encoding an enzyme of the foreign mevalonate pathway involved in producing IPP from acetyl-CoA.
  • Fig. 1 is a diagram schematically showing the MEP pathway of retinal biosynthesis and the foreign MVA pathway.
  • the wild type Escherichia microorganism can be, for example, Escherichia coli.
  • the E. coli may be DH5 ⁇ , MG1655, BL21 (DE), S17-1, XL1-Blue, BW25113, or a combination thereof.
  • the parent strain is, for example, a gene encoding acetyl-CoA acetyltransferase / hydroxymethylglutaryl (HMG) -CoA reductase from Enterococcus faecalis of SEQ ID NO: 1, SEQ ID NO: 2 Gene encoding HMG-CoA synthase derived from Enterococcus faecalis, gene encoding mevalonate kinase derived from Streptococcus pneumoniae of SEQ ID NO: 3, Streptococcus pneumoniae of SEQ ID NO: 4 Gene encoding the phosphomevalonate kinase derived from, Gene encoding the mevalonate diphosphate decarboxylase derived from Streptococcus pneumoniae of SEQ ID NO: 5, Isopentenyl diphosphate from E.
  • HMG hydroxymethylglutaryl
  • the parent strain was transformed with the genes of SEQ ID NOs: 1 to 10, and also encoded the ⁇ -carotene monooxygenase from uncultured marine bacterium 66A03 of SEQ ID NO: 13, the mouse of SEQ ID NO: 14 Br-like protein 2 derived from Natronomonas pharaonis ATCC35678, a gene encoding ⁇ -carotene 15,15'-monooxygenase from Mus musculus : one or more genes selected from the group consisting of a gene encoding brp2) and a gene encoding ⁇ -carotene monooxygenase from Halobacterium salinarum ATCC700922 of SEQ ID NO: 16 or 17 It may have been switched.
  • the parent strain may be to produce a retinoid, which is further transformed with a gene encoding IPP isomerase from Haematococcus pluvialis of SEQ ID NO: 12.
  • the parent strain may be transformed with a gene encoding E. coli-derived 1-deoxyxylulose-5-phosphate (DXP) synthase (dxs) of SEQ ID NO: 11. Since DXP is an enzyme that corresponds to the rate determining step in the intrinsic MEP pathway, the additional gene encoding the DXP synthase allows microorganisms to produce high concentrations of ⁇ -carotene.
  • DXP E. coli-derived 1-deoxyxylulose-5-phosphate synthase
  • the parent strain is, for example, Escherichia coli DH5 ⁇ / pTDHB / pSNA (deposited on Jan. 2, 2008) or Escherichia coli DH5 ⁇ / pTDHBSR / pSNA on accession no. KCTC 11254BP (KOREAN COLLECTION FOR TYPE CULTURE) 1.
  • KCTC 11254BP KOREAN COLLECTION FOR TYPE CULTURE
  • E. coli DH5 ⁇ / pTDHBSR / pSNA can produce high productivity of retinoids from carbon sources in the medium.
  • the microorganism is Enterococcus faecalis of SEQ ID NO: 1 ( Enterococcus faecalis Gene encoding the acetyl-CoA acetyltransferase / hydroxymethylglutaryl (HMG) -CoA reductase derived from c), HMG-CoA synthase from Enterococcus faecalis of SEQ ID NO: 2, sequence Streptococcus pneumoniae (number 3 Streptococcus pneumoniae Gene encoding the mevalonate kinase derived from), phosphomevalonate kinase derived from Streptococcus pneumoniae of SEQ ID NO: 4, mevalonate diphosphate dekar from Streptococcus pneumoniae of SEQ ID NO: 5 Gene encoding a carboxylase, isopentenyl diphosphate (IPP) isomerase derived from Escherichia coli of SEQ ID NO: 6, panto
  • Uncultured marine bacterium 66A03 of SEQ ID NO: 13 Gene encoding the ⁇ -carotene monooxygenase derived may be one having a nucleotide sequence of SEQ ID NO: 18 optimized for codon use in E. coli.
  • retinoids refers to a class of chemicals chemically related to vitamin A.
  • the structure of the retinoid consists of cyclic end groups, polyene side chains and polar end groups.
  • Many retinoids are chromophores.
  • Various retinoids can be produced by changing side chains and end groups.
  • the retinoid may be retinal, retinol, retinoic acid, retinyl acetate, or a combination thereof.
  • the retinoid may be an in vivo degradation product of retinal, retinol, retinoic acid, retinyl acetate, or a combination thereof.
  • the retinoid is a substance having a basic carbon number of 20, and the final carbon number may vary depending on the fatty acid auxiliary group to be bonded.
  • the final carbon number may be 22 for acetate bonding and 38 for oleic acid bonding.
  • YbbO protein may be a protein having an amino acid sequence of SEQ ID NO: 23, which may be encoded by a gene having a nucleotide sequence of SEQ ID NO: 24.
  • the foreign gene having at least 80% homology with the nucleotide sequence of the gene encoding the YbbO protein may have a nucleotide sequence of SEQ ID NOs: 49 to 56.
  • the foreign gene has at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology with the gene encoding the YbbO protein.
  • SEQ ID NO: 49 is the nucleotide sequence of the GENE ID: 3666309 ybbO gene of Shigella sonnei Ss046, which is 100% homologous to the gene encoding YbbO protein herein.
  • SEQ ID NO: 50 is the nucleotide sequence of a gene having GENE ID: 8710313 ROD_05481 of Citrobacter rodentium ICC168 which is 92% homology with the gene encoding YbbO protein herein.
  • SEQ ID NO: 51 shows Salmonella enterica subsp. 91% homology with the gene encoding YbbO protein herein. enterica serovar Typhimurium str.
  • GENE ID: 1247022 of LT2 is the nucleotide sequence of the ybbO gene.
  • SEQ ID NO: 52 is the nucleotide sequence of the GENE ID: 11485505 ybbO gene of Enterobacter cloacae EcWSU1 which is 87% homologous to the gene encoding YbbO protein herein.
  • SEQ ID NO: 53 is the nucleotide sequence of the GENE ID: 10792827 EAE_13085 gene of Enterobacter aerogenes KCTC 2190 with 84% homology with the gene encoding YbbO protein herein.
  • SEQ ID NO: 54 is the nucleotide sequence of the GENE ID: 6935503 KPK_4209 gene of Klebsiella pneumoniae 342, which is 83% homologous to the gene encoding YbbO protein herein.
  • SEQ ID NO: 55 is the nucleotide sequence of the GENE ID: 11664596 KOX_13160 gene of Klebsiella oxytoca KCTC 1686 with 83% homology with the gene encoding YbbO protein herein.
  • SEQ ID NO: 56 is the nucleotide sequence of the GENE ID: 5548810 ESA_02773 gene of Cronobacter sakazakii ATCC BAA-894, which is 83% homologous to the gene encoding YbbO protein herein.
  • genes having the nucleotide sequences of SEQ ID NOs: 49 to 56 may encode proteins having the amino acid sequences of SEQ ID NOs: 41 to 48, respectively, in the order described.
  • the retinal production capacity or the ratio of retinal in the retinoid may be increased.
  • the amplification of the YbbO gene or a foreign gene having a homology of 80% or more may increase the retinol production capacity or the ratio of retinol in the retinoid.
  • the amplification may be performed by introduction of a gene encoding a foreign retinol dehydrogenase.
  • the amplification may be performed by introduction of a foreign YbbO gene in one aspect.
  • the term “attenuation” refers to a decrease in the expression of the gene of interest compared to the parent strain.
  • deletion refers to the loss of expression of a gene of interest.
  • Such attenuation or deletion may be caused by mutations in the gene sequence, eg, substitutions, deletions, insertions or combinations thereof.
  • the attenuation or deletion may be caused by a change in the sequence of the gene regulatory moiety, eg, substitution, deletion, insertion or combination thereof.
  • amplification refers to an increase in gene copy and / or an increase in the expression level of a gene.
  • the amplification may be in one aspect by introducing a foreign gene or by increasing the number of copies of an endogenous gene.
  • a promoter or a ribosomal binding site (RBS) may be substituted to increase the expression level of the gene for the amplification.
  • Escherichia microorganism amplified by attenuation or deletion, or amplification of a gene encoding the YbbO protein of a parent strain of Escherichia sp., Or by introduction of a foreign gene having at least 80% homology with the nucleotide sequence of the gene.
  • retinoids ie, retinal, retinol, retinyl ester, retinoic acid, or a combination thereof may have increased production capacity.
  • the microorganism may be a production capacity of at least one of retinoids, that is, retinal, retinol, retinyl ester, retinoic acid, or a combination thereof compared to the parent strain.
  • the parent strain was a gene encoding ethanolamine utilization protein E (EutE); A gene encoding Putrescine utilization pathway protein C (PuuC); Or a combination thereof may be a microorganism of the genus Escherichia that is further attenuated or deleted.
  • Ethanolamine-using protein E may have an amino acid sequence of SEQ ID NO: 19, and the gene encoding the coding may have a nucleotide sequence of SEQ ID NO: 21.
  • Putrescine pathway protein C may have an amino acid sequence of SEQ ID NO: 20, the gene encoding it may have a nucleotide sequence of SEQ ID NO: 22.
  • Another aspect includes culturing the microorganism; And separating the retinoid from the culture.
  • the method includes culturing the microorganism described above.
  • the microorganism may be a microorganism of the genus Escherichia attenuated or deleted or amplified by the gene encoding the YbbO protein as described above, and the above-described information regarding the microorganism of the genus Escherichia may be applied to the microorganism of the present method.
  • the culturing may include culturing in a medium containing a lipophilic substance.
  • the lipophilic substance may be one having lipophilic as an organic compound having 8 to 50 carbon atoms.
  • the lipophilic material may be an alkane compound having 8 to 50 carbon atoms, a compound of Formula 1; A compound of Formula 2; Or combinations thereof:
  • R 2 Each independently represent alkyl having 8 to 50 carbon atoms, and CO represents a carbonyl group
  • R 3 , R 4 and R 5 each independently represent alkyl having 8 to 50 carbon atoms, and CO represents a carbonyl group).
  • Alkanes having 8 to 50 carbon atoms may be linear alkanes, branched alkanes, cyclic alkanes, or combinations thereof.
  • the alkane compound is, for example, 8 to 46, 8 to 40, 8 to 36, 8 to 30, 8 to 26, 8 to 20, 8 to 16, 8 to 12, 8 to 10, 10 to 50, 10 to 46, 10 to 40, 10 to 36, 10 to 30, 10 to 26, 10 to 20, 10 to 16, 10 to 12, 10 to 50, 10 to 46, 12 to 50, 12 to 46, 12 to 36, It may be 12 to 30, 12 to 26, 12 to 20, or 12 to 16 alkane compound.
  • Straight alkanes contain 8 (octane), 10 (decane), 12 (dodecane), 14 (tetradecane), 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 alkanes, or a combination thereof.
  • Branched alkanes have 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 alkanes, Or combinations thereof.
  • Branched alkanes may be saturated analogs of terpene compounds. For example, it may be phytoscualan.
  • the combination of straight alkanes, branched alkanes, and cyclic alkanes may be mineral oil.
  • the mineral oil may be a mixture of alkanes having 15 to 40 carbon atoms from non-vegetable raw materials (minerals).
  • Alkanes having 15 to 40 carbon atoms include 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, It may be a mixture of two or more of alkanes of 36, 37, 38, 39, 40.
  • the mineral oil may be light mineral oil or heavy mineral oil.
  • Light mineral oils generally have a density of 880-920kg / m 3 and a specific gravity of 820-860 kg / m 3 at 20 ° C and a fluid viscosity of 14-18cst at 40 ° C.
  • By weight mineral oil (heavy mineral oil) is of generally a density of 920kg / m 3 and a specific gravity of 860 ⁇ 900 kg / m 3, liquid viscosity of 85cst at 65 ⁇ 40 °C at 20 °C material.
  • One And R 2 are each independently straight, branched or cyclic alkyl having 8 to 50 carbon atoms.
  • R One And R 2 are each independently alkyl having 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 Can be.
  • R 1 and R 2 each have 8 to 50 carbon atoms, for example, 8 to 46, 8 to 40, 8 to 36, 8 to 30, 8 to 26, 8 to 20, 8 to 16, 8 to 12, 8 carbon atoms.
  • R 1 may be straight alkyl having 13 carbon atoms and R 2 may be isopropyl.
  • R 1 may be an ethylpentyl group and R 2 may be cetyl.
  • R 3 , R 4 and R 5 are each independently straight, branched or cyclic alkyl having 8 to 50 carbon atoms.
  • R 3 , R 4 , and R 5 are each having 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 alkyl.
  • the compound may have R 3 , R 4 , and R 5 , each having 8 to 50 carbon atoms, for example, 8 to 46, 8 to 40, 8 to 36, 8 to 30, 8 to 26, 8 to 8 carbon atoms.
  • the lipophilic material may be octane, decane, dodecane, tetradecane, phytoscualan, mineral oil, isopropyl myristate, cetyl ethylhexanoate, dioctanoyl decanoyl glycerol, squalane, or a combination thereof.
  • lipophilic substances can increase the productivity of retinoids by microorganisms.
  • the lipophilic substance may be one that does not affect or grows little in the growth of microorganisms.
  • Cultivation may be in synthetic, semisynthetic, or complex culture media.
  • a culture medium a medium consisting of a carbon source, a nitrogen source, vitamins and minerals can be used.
  • a Man-Rogosa-Sharp (MRS) liquid medium or a liquid medium added with milk can be used.
  • Starch glucose, sucrose, galactose, fructose, glycerol, glucose or mixtures thereof may be used as the carbon source of the medium.
  • glycerol can be used as the carbon source.
  • nitrogen source ammonium sulfate, ammonium nitrate, sodium nitrate, glutamic acid, casamino acid, yeast extract, peptone, tryptone, soybean meal or mixtures thereof may be used.
  • the mineral may be sodium chloride, dipotassium phosphate, magnesium sulfate or mixtures thereof.
  • the culture is in a fermentor, it is preferable to use glucose as the carbon source of the medium.
  • glucose is preferable to use in the case of in vitro culture.
  • glycerol is preferably used as the carbon source of the medium.
  • Each of the carbon source, nitrogen source, and mineral in the microbial culture medium may use, for example, 10 to 100 g, 5 to 40 g, and 0.5 to 4 g per liter.
  • the vitamin added to the conventional culture medium may be vitamin A, vitamin B, vitamin C, vitamin D, vitamin E or mixtures thereof.
  • the vitamin may be added to the conventional culture medium with the above-mentioned carbon source, nitrogen source, minerals, etc., or separately added to the medium prepared by sterilization.
  • Cultivation may be carried out under conventional E. coli culture conditions. Incubation is for example about 15-45 °C, for example 15-44 °C, 15-43 °C, 15-42 °C, 15-41 °C, 15-40 °C, 15-39 °C, 15-38 °C, 15-37 ° C, 15-36 ° C, 15-35 ° C, 15-34 ° C, 15-33 ° C, 15-32 ° C, 15-31 ° C, 15-30 ° C, 20-45 ° C, 20-44 ° C, 20-43 ° C, 20-42 ° C, 20-41 ° C, 20-40 ° C, 20-39 ° C, 20-38 ° C, 20-37 ° C, 20-36 ° C, 20-35 ° C, 20-34 ° C, 20-33 ° C, 20-32 ° C, 20-31 ° C, 20-30 ° C, 25-45 ° C, 20
  • Centrifugation or filtration may be performed to remove the culture medium in the culture and recover or remove only the concentrated cells, and this step may be performed according to the needs of those skilled in the art.
  • the concentrated cells can be preserved so as not to lose their activity by freezing or lyophilizing according to a conventional method.
  • the culture may be in a medium containing glycerol as a carbon source.
  • Glycerol may be the only carbon source in the medium.
  • 0.5-5.0% (w / v), e.g. 0.5-4.5% (w / v), 0.5-4.0% (w / v), 0.5-3.5% (w / v), 0.5-3.0% (w / v), 0.5-2.5% (w / v), 0.5-2.0% (w / v), 1-5.0% (w / v), 1-4.5% (w / v), 1-4.0% (w / v), 1-3.5% (w / v), 1-3.0% (w / v) or 1-2.5% (w / v) may be made in a medium containing glycerol.
  • the medium may be YT medium added with glycerol and arabinose.
  • YT medium may comprise 1.6 wt% tryptone, 1 wt% yeast
  • Cultivation may be performed in a culture medium in the presence of a lipophilic substance, for example, with a dodecane phase of a lipophilic substance placed on the surface of the medium. Cultivation can be carried out while being agitated.
  • 100 to 300 rpm for example, 100 to 280 rpm, 100 to 260 rpm, 100 to 240 rpm, 100 to 220 rpm, 100 to 200 rpm, 100 to 180 rpm, 100 to 160 rpm, 100 to 140 rpm, 100 to 120 rpm, 120 to 120 300 rpm, 120 to 280 rpm, 120 to 260 rpm, 120 to 240 rpm, 120 to 220 rpm, 120 to 200 rpm, 120 to 180 rpm, 120 to 160 rpm, 120 to 140 rpm, 150 to 300 rpm, 150 to 280 rpm, 150 to 260 rpm, 150 to 240 rpm, It may be stirred at 150 to 220 rpm, 150 to 200 rpm, 150 to 180 rpm, 140 to 160 rpm, 200 to 300 rpm, 200 to 280 rpm, 200 to 260 rpm, 200 to 240 rpm, 200 to 220 rpm, or
  • the lipophilic material such as dodecane
  • the lipophilic substance may be dispersed in a medium to increase the area in contact with the microorganisms, thereby allowing the retinoid to be efficiently separated from the cells during the cultivation, thereby stabilizing and / or lysing.
  • the retinoid production may peak at some point and then decrease. This may be because additional retinoid synthesis is stopped during the stagnant state of microbial growth, while its oxidative degradation occurs in the cell.
  • the produced retinoid may be absorbed onto the lipophilic substance such as dodecane before being degraded in the cell, thereby improving retinoid production.
  • the lipophilic material such as the dodecane phase, may be one that is hydrophobic and has low volatility for extraction of hydrophobic retinoids without affecting the cell growth of Escherichia microorganisms.
  • 2 shows the conversion of ⁇ -carotene to retinoids including retinal, retinol, retinoic acid, and retinyl esters.
  • the volume ratio of the medium to the lipophilic material is not limited to a specific range of ratios, for example, the volume ratio of the medium to the lipophilic material is 1: 0.1-3.0, 1: 0.2-3.0, 1: 0.5-3.0, 1: 1.0-3.0, 1: 1.5-3.0, 1: 2.0-3.0, 1: 2.5-3.0, 1: 0.2-2.5, 1: 0.2-2.0, 1: 0.2-1.5, 1: 0.2-1.0, 1: 0.2-0.5, 1: 0.5-2.5, 1: 0.5-2.0, 1: 0.5-1.5, 1: 0.5-1.0, 1: 0.8-2.5, 1: 0.8-2.0, 1: 0.8-1.5, 1: 0.8-1.2, 1: 0.8- 1.0 and the like are possible.
  • the medium comprises a concentration of about 2.0% glycerol, the genus Escherichia microorganism is E. coli DH5 ⁇ or MG1655, the culturing step is a culture medium of about 7 ml, about 29 °C It may be to.
  • the method also includes the step of separating the retinoid from the lipophilic phase.
  • separating the retinoids such as retinal, retinol, retinyl esters, or combinations thereof are well known in the art. For example, it can be separated by ion exchange chromatography, HPLC, or the like. Specifically, in order to obtain a high-purity product after extraction with a solvent such as acetone after recovering the cells may be separated and purified through HPLC or crystallization operation.
  • Retinoids are widely used as ingredients in cosmetics, food or medicine.
  • YbbO protein In another aspect, YbbO protein; Alternatively, the conversion of retinal to retinol may be promoted by adding an enzyme composition including a protein encoded by a gene having a homology of 80% or more with the amino acid sequence of the gene encoding the YbbO protein.
  • the YbbO protein may have an amino acid sequence of SEQ ID NO: 23, and a protein encoded by a gene having at least 80% homology with the amino acid sequence of the gene encoding the YbbO protein may be any one of SEQ ID NOs: 41 to 48. It may have any one amino acid sequence.
  • a gene having at least 80% homology with the amino acid sequence of the gene encoding YbbO protein is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the amino acid sequence of the gene encoding YbbO protein.
  • the YbbO protein having the amino acid sequence of SEQ ID NO: 23 may be encoded by a gene having the nucleotide sequence of SEQ ID NO: 24, and the protein having the amino acid sequence of any one of SEQ ID NOs: 41 to 48 may be any one of SEQ ID NOs: 49 to 56 It can be encoded by a gene having a nucleotide sequence of.
  • the enzyme composition including the YbbO protein may further include components included in conventional enzyme compositions such as solvents, stabilizers, and various additives.
  • a component that promotes the conversion of retinal to retinol other than the YbbO protein may be further included.
  • the genus Escherichia microorganism has increased retinoid production capacity.
  • certain products of the retinoids can be produced in increased proportions.
  • the method for producing a retinoid According to the method for producing a retinoid according to another aspect, it is possible to efficiently produce a retinoid. In addition, certain products of the retinoids can be produced in increased proportions.
  • the gene encoding the YbbO protein of the present invention is attenuated or deleted, or amplified, or a lipophilic microorganism of the genus Escherichia amplified by introducing a gene having at least 80% homology with the amino acid sequence of the gene encoding the YbbO protein.
  • the retinoid can be produced more efficiently by culturing in a medium containing the substance to separate the retinoid from the lipophilic substance.
  • Retinoids obtained through the retinoid production method using the microorganism of the present invention can be widely used as a material for cosmetics, food, medicines, etc., if the effective production of specific retinoids for the manufacture of cosmetics, foods, medicines, etc. It is suitable to utilize the retinoid production method.
  • Fig. 1 is a diagram schematically showing the MEP pathway of retinal biosynthesis and the foreign MVA pathway.
  • FIG. 2 shows the conversion of ⁇ -carotene to retinoids including retinal, retinol, retinoic acid, and retinyl esters.
  • 3 is a diagram showing the effect of YbbO gene introduction on retinoid production.
  • 5 is a diagram showing the effect of eutE gene or puuC gene deletion on retinoid production.
  • FIG. 6 is a diagram showing retinoid production and cell growth according to dodecane volume of Escherichia coli (pT-DHBSR / pS-NA) in culture using a medium containing dodecane.
  • FIG. 7 is a diagram showing the distribution of retinoids according to incubation time and dodecane volume of Escherichia coli (pT-DHBSR / pS-NA) in culture using a medium containing dodecane as a percentage of each component relative to total retinoids. .
  • FIGS 10, 11 and 12 are diagrams showing the results of retinoid production, cell growth, and cell specific retinoids productivity of E. coli (pT-DHBSR / pS-NA) in the presence of various volumes of lightweight mineral oil.
  • FIG. 13 and 14 show the retinoid production and cell growth results of Escherichia coli (pT-DHBSR / pS-NA) in the presence of heavy mineral oil.
  • FIG. 15 and FIG. 16 show the retinoid production and cell growth results of Escherichia coli (pT-DHBSR / pS-NA) when cultured by tilting the test tube in the presence of heavy mineral oil.
  • FIG. 17 shows cell growth and pH of Escherichia coli (pT-DHBSR / pS-NA) in the presence of a skin-friendly lipophilic substance.
  • FIGS 18 and 19 are diagrams showing the results of retinoid production of Escherichia coli (pT-DHBSR / pS-NA) according to various kinds and amounts of skin-friendly lipophilic substances.
  • the YbbO gene was amplified or deleted in Escherichia coli DH5 ⁇ (pTDHBSR / pSNA) (Accession No. KCTC 11255BP), which has a retinoid producing ability, and the effect of the amplification or deletion on retinoid production was confirmed.
  • the pBBR1MCS2 (YbbO) plasmid was prepared by inserting the E. coli YbbO gene (SEQ ID NO: 24) into the KpnI and XhoI restriction sites of the pBBR1MCS2 plasmid.
  • pBBR1MCS2 (YbbO) plasmid was introduced into E. coli DH5 ⁇ (pTDHBSR / pSNA) by transformation to obtain E. coli DH5 ⁇ (pTDHBSR / pSNA / pBBR1MCS2-YbbO). This process is described in detail as follows.
  • Genomic DNA preparation, restriction enzyme cleavage, transformation and standard molecular biology techniques were performed as described in the literature (Sambrook and Russell 2001). PCR was performed using pfu DNA polymerase (Solgent Co., Korea) by its standard protocol.
  • the resulting strain was mixed with 5 mL of 2YT medium (16 g trypton, 10 g yeast extract, and 5 g NaCl) containing 2% (w / v) glycerol and 0.2% (w / v) arabinose and 5 mL of dodecane Incubated for 72 hours.
  • 2YT medium (16 g trypton, 10 g yeast extract, and 5 g NaCl) containing 2% (w / v) glycerol and 0.2% (w / v) arabinose and 5 mL of dodecane Incubated for 72 hours.
  • the culture was inoculated with each strain into a test tube of 15 cm in length and 25 mm in diameter, and then inoculated with each strain, followed by incubation at about 250 ° C. with shaking at a shaking incubator at about 250 rpm.
  • DH5 ⁇ pTDHBSR / pSNA / pBBR1MCS2 into which a vector without the YbbO gene was introduced was used.
  • FIG. 3 is a diagram showing the effect of YbbO gene introduction on retinoid production.
  • the ratio of retinol and retinyl acetate in the total retinoids was increased in the YbbO transgenic strain (pBBR2-ybbO) compared to the control strain (pBBR1MCS-2).
  • Total retinoid production was not significantly different.
  • the retinal: retinol: retinyl acetate was 70 mg / L: 45 mg / L: 20 mg / L in the control group, but 21 mg / L: 67 mg / L: 39 mg / L in the YbbO gene strain.
  • retinyl acetate ratio in retinoid production by the YbbO transgenic strain is believed to be caused by an increase in the retinol ratio.
  • a small amount of retinal was produced during retinoid production by the YbbO transgenic strain.
  • E. coli DH5 ⁇ ⁇ ybbO strain was prepared according to the method of Datsenko and Wanner (Datsenko KA and Wanner BL., 2000 Proc Natl Acad Sci U.S.A., 97 (12): 6640-6645).
  • kanamycin gene of pKD13 plasmid (Datsenko KA and Wanner BL., 2000 Proc Natl Acad Sci USA, 97 (12): 6640-6645) PCR primers having nucleotide sequences of the upstream and downstream ends of the ybbO gene were prepared while binding.
  • the forward primer consists of 50bp nucleotide sequence upstream of the ybbO gene followed by kanamycin gene binding nucleotide sequence 20bp
  • the reverse primer consists of 50bp nucleotide sequence downstream of the ybbO gene followed by kanamycin gene binding nucleotide sequence 20bp.
  • the primers used are shown in Table 3. PCR reactions were carried out using oligonucleotides of SEQ ID NOs: 28 and 29 as primers and pKD13 as a template.
  • the purified PCR reaction product was transformed into DH5 ⁇ competent cells including pKD46 (Datsenko KA and Wanner BL., 2000 Proc Natl Acad Sci U.S.A., 97 (12): 6640-6645). Colony PCR of colonies of kanamycin-resistant plate medium confirmed that the kanamycin gene replaced the ybbO gene by homologous recombination. Primers used for colony PCR were prepared to bind to the region immediately adjacent to the ybbO gene on the E. coli DH5 ⁇ chromosome.
  • the resulting strain was mixed with 5 mL of 2YT medium (16 g trypton, 10 g yeast extract, and 5 g NaCl) containing 2% (w / v) glycerol and 0.2% (w / v) arabinose and 5 mL of dodecane Incubated for 60 hours.
  • the culture was inoculated with each strain into a test tube of 15 cm in length and 25 mm in diameter, and then inoculated with each strain, followed by incubation at about 250 ° C. with shaking at a shaking incubator at about 250 rpm.
  • DH5 ⁇ pTDHBSR / pSNA
  • FIG. 4 shows the effect of YbbO gene deletion on retinoid production. As shown in FIG. 4, the total retinoid production was higher in the YbbO gene deletion strain (DH5 ⁇ ybbO) compared to the control strain (DH5 ⁇ ).
  • the retinal: retinol: retinyl acetate was 43 mg / L: 22 mg / L: 9.7 mg / L, but in the YbbO gene deletion strain, 65 mg / L: 25 mg / L: 9.5 mg / L It was.
  • the strains obtained can be used to produce retinoids with high retinal ratios.
  • retinoids having a high retinal ratio can be produced by deleting EutE, PuuC, or a combination thereof.
  • Example 2 Escherichia coli deleted from EutE gene and PuuC gene and retinoid production using the same
  • the EutE gene and PuuC gene were deleted from DH5 ⁇ (pTDHBSR / pSNA) (Accession No. KCTC 11255BP), a microorganism of the genus Escherichia, having a retinoid producing ability, and the effect of the deletion on retinoid production was confirmed.
  • PCR primers were prepared having the nucleotide sequences of the upstream and downstream ends of the EutE gene while binding.
  • the forward primer consists of 50bp nucleotide sequence upstream of the EutE gene followed by kanamycin gene binding nucleotide sequence 20bp
  • the reverse primer consists of 50bp nucleotide sequence downstream of the EutE gene followed by kanamycin gene binding nucleotide sequence 20bp.
  • the primers used are shown in Table 4 below. PCR reaction was performed using oligonucleotides of SEQ ID NO: 32 and SEQ ID NO: 33 as primers and pKD13 as a template.
  • the purified PCR reaction product was transformed into DH5 ⁇ competent cells including pKD46 (Datsenko KA and Wanner BL., 2000 Proc Natl Acad Sci U.S.A., 97 (12): 6640-6645). Colonies of kanamycin resistant plate medium were confirmed by colony PCR to determine whether the kanamycin gene replaced the EutE gene by homologous recombination. Primers used for colony PCR were prepared to bind to the region immediately adjacent to the EutE gene on the E. coli DH5 ⁇ chromosome.
  • PCR primers were prepared having the sequence of the upstream and downstream ends of the PuuC gene while binding.
  • the forward primer consists of 20bp of the upstream end of the PuuC gene followed by 20bp of chloramphenicol gene binding nucleotide sequence
  • the reverse primer consists of 20bp of the chloramphenicol gene binding base sequence of the downstream end of the PuuC gene.
  • the primers used are shown in Table 4 below. PCR reaction was carried out using oligonucleotides of SEQ ID NO: 36 and SEQ ID NO: 37 as primers and pKD3 as a template.
  • the purified PCR reaction product was transformed into DH5 ⁇ competent cells including pKD46 (Datsenko KA and Wanner BL., 2000 Proc Natl Acad Sci U.S.A., 97 (12): 6640-6645). Colonies of chloramphenicol resistant plate media were confirmed by colony PCR to determine whether the chloramphenicol gene replaced the PuuC gene by homologous recombination. Primers used for colony PCR were prepared to bind to the region immediately adjacent to the PuuC gene on the E. coli DH5 ⁇ chromosome.
  • EutE was the primers of SEQ ID NO: 34 and SEQ ID NO: 35 in Table 4 below
  • PuuC was the primers of SEQ ID NO: 38 and SEQ ID NO: 39 of Table 4 below. Confirmed.
  • the strain obtained in (1) was treated with 5 mL of 2YT medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl) and dodecane containing 2% (w / v) glycerol and 0.2% (w / v) arabinose. Incubated for 60 hours in 5 mL of mixed medium.
  • 2YT medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl
  • the culture was inoculated with each strain into a 15 cm long, 25 mm diameter test tube, and inoculated with each strain, followed by incubation at about 250 ° C. with a shaking incubator at about 250 rpm.
  • DH5 ⁇ pTDHBSR / pSNA
  • FIG. 5 is a diagram showing the effect of EutE gene, or PuuC gene deletion on retinoid production.
  • the amount of total retinoid total amount of retinal, retinol and retinyl acetate
  • the ratio of retinal in the deletion strain increased significantly at 60 hours of culture.
  • the total 60-hour total retinoid production was 75 mg / L, 110.3 mg / L when the EutE gene was deleted, and 102.2 mg / L when the PuuC gene was deleted.
  • the EutE gene and the PuuC gene encode oxidase activity that oxidizes retinal to retinoic acid. However, it is not limited to a specific mechanism.
  • Example 3 E. coli culture and retinoid production in the presence of dodecane
  • Escherichia coli (pT-DHBSR / pS-NA) was cultured in a dodecane-containing medium to confirm the effect of dodecane on retinoid production and cell growth.
  • FIG. 6 shows retinoid production and cell growth of Escherichia coli (pT-DHBSR / pS-NA) in a dodecane-containing medium. Retinal, retinol and retinyl acetate are shown in light gray, dark gray and black in retinoid production, respectively.
  • Dodecane volumes 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL overlayed during cell culture are indicated by ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • FIG. 7 shows the distribution of retinoids according to incubation time and dodecane volume as a percentage of each component relative to total retinoids. Retinal, retinol and retinyl acetate are shown in light gray, dark gray and black, respectively.
  • Retinoid content in the figure is a value measured by HPLC separating and recovering only the dodecane layer in the culture medium by centrifugation.
  • the retinoid was present in the cell or cell lysate only in a negligible or negligible amount, and was present in the culture medium in which the cells were removed, ie, the dodecane layer.
  • retinoid production increased by the addition of dodecane as compared to the absence of dodecane.
  • retinoid production was proportional to dodecane volume.
  • the highest retinoid production of 136 mg / L was obtained at 72 hours of incubation with 5 mL dodecane, which was about 2 times higher than with 1 mL dodecane (65 mg / L).
  • Extended cultures for more than 72 hours with 5 mL dodecane resulted in no further increase in retinoid production and maintained its peak without degradation (data not shown).
  • Dodecane addition volume was increased to 6 mL by adding 2 mL of dodecane to the culture at 0, 24, and 48 hours. There was no increase in total retinoid production compared to the culture with 5 mL dodecane in the 6 mL dodecane addition culture. There was no increase in retinoid production even in the initial addition of 6 mL dodecane (data not shown). Cell growth in all cultures with dodecane was slightly higher than without dodecane (FIG. 6).
  • FIG. 7 is a view showing the distribution of retinoids obtained according to the dodecane addition volume.
  • retinoids obtained according to the dodecane addition volume.
  • the ratio of retinal in retinoids is about 51% (w / w) in dodecane addition cultures and 23% in cultures without dodecane addition, while the retinol ratio is 30% to 39% in dodecane addition cultures. 59% in culture without dodecane addition.
  • dodecane addition increases the ratio of retinal but decreases the ratio of retinol.
  • the retinal Given the reaction sequence of retinol formation from the retinal in the cell, the retinal is thought to be extracted from the cell before its conversion to retinol by dodecane.
  • the retinyl acetate ratio at 48 hours is less than 20% in both with and without dodecane addition, which is relatively low compared to retinal and retinol.
  • the retinyl acetate ratio decreases with longer incubation time, indicating that cell activity for retinyl acetate formation decreases during the culture.
  • dodecane addition prevented the reduction of retinoid production and increased retinoid production in the stagnant state of cell growth.
  • In-drill extraction of the retinoids of the present invention does not require lysozyme for cell wall degradation.
  • Retinoids C20, isoprenoid molecules
  • ⁇ -carotene In a two-phase culture of retinoid production, ⁇ -carotene must be maintained in cells because it is a direct precursor of retinoids.
  • BCD (M) O located in the cytosol.
  • ⁇ -carotene cannot be released from cells because of its molecular size and is not extracted by dodecane, it can be maintained in cells in a two-phase culture of ⁇ -carotene.
  • Escherichia coli (pT-DHBSR / pS-NA) was cultured in various lipophilic medium-containing media, and the effect of the lipophilic substance on retinoid production and cell growth was confirmed.
  • Lightweight mineral oils have the advantage of being cheaper than alkanes. 5 ml of 2YT medium (16 g trypton per liter, 10 g yeast extract, and 5 g NaCl) containing 2% (w / v) glycerol and 0.2% (w / v) arabinose and a different volume of light mineral oil (Sigma) , Catalog No. M8410) was incubated for 72 hours in a mixed medium.
  • 2YT medium 16 g trypton per liter, 10 g yeast extract, and 5 g NaCl
  • the culture was inoculated with each strain into a test tube of 15 cm in length and 25 mm in diameter, and then inoculated with each strain, followed by incubation at about 250 ° C. with shaking at a shaking incubator at about 250 rpm.
  • 10 is a view showing the results of retinoid production in the presence of light mineral oil.
  • 11 is a view showing the results of growth of strains in the presence of light mineral oil.
  • FIG. 12 is a diagram showing cell specific retinoids productivity per cell. As shown in FIG. 12, irrespective of the amount of mineral oil, it showed a specific productivity of about 5 mg / L / OD 600 nm.
  • Heavy mineral oils are cheaper than lightweight mineral oils.
  • 5 ml of 2YT medium (16 g trypton, 10 g yeast extract, and 5 g NaCl) containing 2% (w / v) glycerol and 0.2% (w / v) arabinose and heavy mineral oil (Daejung, Catalog No. 5658-4400) were incubated for 96 hours in 2 ml of mixed medium.
  • the culture was inoculated with each strain into a test tube of 15 cm in length and 25 mm in diameter, and then inoculated with each strain, followed by incubation at about 250 ° C. with shaking at a shaking incubator at about 250 rpm.
  • FIG. 13 shows the results of retinoid production in the presence of heavy mineral oil.
  • 14 is a diagram showing the results of growth of strains in the presence of heavy mineral oil. As shown in FIGS. 13 and 14, the light mineral oil and the heavy mineral oil had less cell growth than dodecane. In addition, 96.5 mg / L of retinoids were produced. This is expected to be due to the poor mixing of the medium and the mineral oil due to the viscosity of the heavy mineral oil.
  • the cells were cultured in the same manner as above except that the test tubes used were tilted and placed in the incubator. By tilting the test tube, the effect of agitation was increased, allowing the medium and mineral oil to mix better.
  • Figure 15 is a view showing the results of retinoid production when incubated with test tubes.
  • 16 is a view showing the results of strain growth when cultured by tilting the test tube. As shown in FIG. 15 and FIG. 16, the cell growth and the retinoid production increased when the culture was inclined. Specifically, when the test tube was upright, 88.2 mg / L retinoids were produced at 96 hours, but when the test tube was tilted, 173.9 mg / L was produced.
  • Retinoids were produced in media containing skin friendly lipophilic substances.
  • Skin-friendly lipophilic materials used isopropyl myristate (IPM), dioctanoyl-decanoyl glycerol (ODO), cetyl ethylhexanoate (CEH) and phytosqualane.
  • strain DH5 ⁇ (pT-DHBSR / pSNA) transformed with pT-DHBSR / pSNA to DH5 ⁇
  • 2 ml of heavy mineral oil were added to 5 ml medium, respectively, 2% (w / v) glycerol and 0.2% (w / v) 5 ml of 2YT medium (16 g trypton, 10 g yeast extract, and 5 g NaCl) with arabinose and isopropyl myristate (IPM, Sigma, Catalog No.172472), dioctanoyl-decanoyl glycerol (ODO), cetyl ethylhexanoate (CEH), or phytosqualane (PHYTOSQUALAN®, Sophim; molecular formula C 30 H 62 ; hydrogenated form of squalane; extracted from Olive) incubated for 72 hours in 2 mL or 5 mL of mixed medium. .
  • the culture was inoculated with each strain into a test tube of 15 cm in length and 25 mm in diameter, and then inoculated with each strain, followed by incubation at about 250 ° C. with shaking at a shaking incubator at about 250 rpm.
  • 17 shows cell growth and pH in the presence of skin friendly lipophilic substances.
  • 18 and 19 show the results of retinoid production according to the amount of skin-friendly lipophilic substances.
  • the amount of retinoid production was higher at 2 mL compared to 5 mL in the lipophilic substance except dodecane. That is, when about 2 mL was used with respect to 5 mL of the medium of light mineral oil, IPM, ODO, CEH, and phytoscualan, the retinoid production amount was large. Among the IPM, ODO, CEH and phytosqualane, the most retinoids were produced in IPM. In particular, when 2 mL of IPM was added, 180 mg / L retinoids were produced. In the case of IPM, considering the similar cell growth, the specific productivity per cell is expected to be high.

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Abstract

La présente invention concerne un micro-organisme du genre escherichia contenant ou ne contenant pas un gène YbbO amplifié et un procédé de production de rétinoïde faisant appel à ce micro-organisme. Le procédé de production de rétinoïde comprend plus spécifiquement : l'utilisation d'un micro-organisme du genre Escherichia contenant ou ne contenant pas un gène diminué ou amplifié codant pour une protéine YbbO d'une souche parent du genre Escherichia qui est capable de produire un rétinoïde ; et la séparation du rétinoïde d'un produit cultivé qui est obtenu par la culture du micro-organisme. La présente invention est basée sur la confirmation de l'activité enzymatique de la protéine YbbO sur le rétinal et un rétinoïde.
PCT/KR2012/006074 2011-07-29 2012-07-30 Micro-organisme du genre escherichia contenant ou ne contenant pas un gène ybbo amplifié et procédé de production de rétinoïde faisant appel à ce micro-organisme Ceased WO2013019052A2 (fr)

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