WO2013019051A9 - Method for producing retinoid from microorganism - Google Patents

Abstract

The present invention relates to a method for producing retinoid from a microorganism, and more specifically, to a method for effectively obtaining retinoid, which lacks stability, from a microorganism by cultivating the microorganism capable of producing retinoid in a medium containing a lipophilic substance, and separating retinoid from the lipophilic substance.

Description

How to produce retinoids from microorganisms

The present invention relates to a method for producing a retinoid from a microorganism having a retinoid producing ability.

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 β- It consists of inon rings and polyunsaturated side chains. They are known to play an essential role in human health, such as vision protection, bone development and regeneration, antioxidant effects, and skin aging prevention and reduce the risk of certain cancers.

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. However, 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. However, microorganisms produce a protein-binding form of retinal and are therefore not suitable for mass production of free retinoids. To date, there have been some limited attempts to use enzymes for biological production, but no successful results. Thus, 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.

One aspect provides a method for efficiently producing retinoids from microorganisms.

One aspect includes the steps of culturing a microorganism having a retinoid production capacity in a medium containing a lipophilic substance; And separating the retinoid from the lipophilic substance. It provides a method for producing a retinoid from a microorganism.

The method includes culturing the microorganism having the retinoid production capacity in a medium containing a lipophilic substance.

The term “microorganism” can be a cell that can be cultured in a liquid medium. The microorganism may be one that can be cultured in a liquid medium as prokaryotic cells, eukaryotic cells, or isolated animal cells. The microorganism may be, for example, a bacterium, fungus, or a combination thereof. The bacteria may be Gram positive bacteria, Gram negative bacteria, or a combination thereof. Gram-negative bacteria can be of the genus Escherichia . Gram-positive bacteria can be of the genus Bacillus, Corynebacterium, lactic acid bacteria or combinations thereof. The fungus may be yeast, cloberomyces, or a combination thereof. The microorganism may be a natural or foreign gene is introduced. The foreign gene can be a gene involved in retinoid production, such as one or more genes of the MEP or MVA pathway. Animal cells may be those used for recombinant protein production. For example, it can be CHO cells, BHK cells, or a combination thereof.

The Escherichia genus microorganism having the retinoid producing ability may be a native Escherichia microorganism or a transformed Escherichia genus microorganism. Natural microorganisms of the genus Escherichia 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. In addition, it may be a strain introduced with a gene encoding an enzyme involved in synthesizing β-carotene from the IPP. Two or more copies of IPP isomerase may have been introduced to facilitate the conversion from IPP to DMAPP. Thus, the microorganism can produce a high concentration of retinoids. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram schematically showing the MEP pathway of retinal biosynthesis and foreign MVA pathway.

The natural Escherichia genus microorganism can be, for example, Escherichia coli. The E. coli may be DH5α, MG1655, BL21 (DE), S17-1, XL1-Blue, BW25113, or a combination thereof.

Transformed Escherichia microorganisms encode, for example, acetyl-CoA acetyltransferase / hydroxymethylglutaryl (HMG) -CoA reductase from Enterococcus faecalis of SEQ ID NO: 1 A gene encoding the HMG-CoA synthase derived from Enterococcus faecalis of SEQ ID NO: 2, a gene encoding the mevalonate kinase derived from Streptococcus pneumoniae of SEQ ID NO: 3, SEQ ID NO: 4 Gene encoding a phosphomevalonate kinase derived from Streptococcus pneumoniae, gene encoding the mevalonate diphosphate decarboxylase derived from Streptococcus pneumoniae of SEQ ID NO: 5, iso derived from E. coli Fenthenyl diphosphate (IPP) isomerase gene, Pantoea agglomerans gene of SEQ ID NO: 7 Gene encoding the geranyl geranyl pyrophosphate (GGPP) synthase, gene encoding the phytoene synthase derived from Pantoea agglomerans of SEQ ID NO: 8, Pantoea agglomer of SEQ ID NO: 9 And a gene encoding a phytoene dehydrogenase derived from Lance, and a gene encoding a lycopene-β-cyclase derived from Pantoea ananatis of SEQ ID NO: 10.

The transformed Escherichia genus microorganism is transformed with the genes of SEQ ID NOs: 1 to 10, and also encodes the β-carotene monooxygenase from uncultured marine bacterium 66A03 of SEQ ID NO: 13 , A gene encoding β-carotene 15,15'-monooxygenase from the musculus mouse of SEQ ID NO: 14, brp-like protein 2 from Natronomonas pharaonis ATCC35678 of SEQ ID NO: 15 (brp-like protein 2: brp2) and a gene encoding β-carotene monooxygenase from Halobacterium salinarum ATCC700922 of SEQ ID NO: 16 or 17 It may be further transformed with one or more genes.

The microorganism 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 microorganism having a retinoid production ability may be transformed with a gene encoding E. coli derived 1-deoxyxylulose-5-phosphate (DXP) synthase (dxs). 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 β-carotene.

When the microorganism having the retinoid production capacity of the present invention belongs to the genus Escherichia, for example, E. coli DH5α / pTDHB / pSNA of Accession No. KCTC 11254BP (deposited KOREAN COLLECTION FOR TYPE CULTURE, January 2. 2008) or Accession No. KCTC 11255BP E. coli DH5α / pTDHBSR / pSNA (KOREAN COLLECTION FOR TYPE CULTURE, deposited on Jan. 2, 2008). In particular, E. coli DH5α / pTDHBSR / pSNA can produce high productivity of retinoids from carbon sources in the medium.

In one embodiment, the microorganism is Enterococcus faecalis of SEQ ID NO: 1 (Enterococcus faecalisGene 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 3Streptococcus pneumoniaeGene 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, pantoea agglomerans of SEQ ID NO: 7 (Pantoea agglomeransGene encoding geranyl geranyl pyrophosphate (GGPP) synthase, gene encoding phytoene synthase derived from Pantoea agglomerans of SEQ ID NO: 8, pantoea aglu of SEQ ID NO: 9 Pantoea ananatis (SEQ ID NO: 10), a gene encoding a phytoene dehydrogenase derived from Merans (Pantoea ananatisGene encoding lycopene-β-cyclase, gene encoding 1-deoxyxylulose-5-phosphate (DXP) synthase derived from E. coli of SEQ ID NO: 11 and hematococcus fluvi of SEQ ID NO: 12 Alis (Haematococcus pluvialis), An uncultured marine bacterium of SEQ ID NO: 13, which is transformed with a gene encoding IPP isomerase from A gene encoding β-carotene monooxygenase derived from mouse of SEQ ID NO: 14 (MusculusGene encoding β-carotene 15,15'-monooxygenase from Natronomonas pharaonics (SEQ ID NO: 15)Natronomonas pharaonis) A gene encoding brp-like protein 2 derived from ATCC35678, and Halobacterium salinarum of SEQ ID NO: 16 or 17 (Halobacterium salinarumA) a microorganism of the genus Escherichia that is further transformed with one or more genes selected from the group consisting of genes encoding β-carotene monooxygenase from ATCC700922. Uncultured marine bacterium 66A03 of SEQ ID NO: 13 Gene encoding the derived β-carotene monooxygenase may be one having a nucleotide sequence of SEQ ID NO: 32 optimized codon use in E. coli.

As used herein, the term "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. A conjugated system formed by alternating C = C double bonds of the polyene side chains gives the color of the retinoid (usually yellow, orange or red). 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. In addition, 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. For example, the final carbon number may be 22 for acetate bonding and 38 for oleic acid bonding.

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:

[Formula 1]

R ₁ (CO) OR ₂

(Wherein_OneAnd R₂Each independently represent alkyl having 8 to 50 carbon atoms, and CO represents a carbonyl group),

[Formula 2]

(Wherein 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 ℃ at 20 ℃ material.

R in the compound of Formula 1_OneAnd R₂Are each independently straight, branched or cyclic alkyl having 8 to 50 carbon atoms. R_OneAnd R₂Are 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. 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, 12 to 30, 12 to 26, 12 to 20, or 12 to 16 alkyl. R ₁ may be straight alkyl having 13 carbon atoms and R ₂ may be isopropyl. In addition, R ₁ may be an ethylpentyl group and R ₂ may be cetyl.

In the compound of formula 2, 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. For example, 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. 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, 12 to 30, 12 to 26, 12 to 20, or 12 to 16 alkyl.

The lipophilic material may be octane, decane, dodecane, tetradecane, phytoscualan, mineral oil, isopropyl myristate, cetyl ethylhexanoate, dioctanoyl decanoyl glycerol, squalane, or a combination thereof.

In addition to stabilizing the retinoids produced, 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. As the culture medium, a medium consisting of a carbon source, a nitrogen source, vitamins and minerals can be used. For example, 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. For example, glycerol can be used as the carbon source. As the 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.

If the culture is in a fermentor, it is preferable to use glucose as the carbon source of the medium. 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 ℃, for example 15-44 ℃, 15-43 ℃, 15-42 ℃, 15-41 ℃, 15-40 ℃, 15-39 ℃, 15-38 ℃, 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, 25-44 ° C, 25-43 ° C, 25-42 ° C, 25-41 ° C, 25-40 ° C, 25-39 ° C, 25-38 ° C, 25-37 ° C, 25-36 ° C, 25-35 ° C, 25-34 ° C, 25-33 ° C, 25-32 ° C, 25-31 ° C, 25-30 ° C, 27-45 ° C, 27-44 ° C, 27-43 ° C, 27-42 ° C, 27-41 ° C, 27-40 ° C, 27-39 ° C, 27-38 ° C, 27-37 ° C, 27-36 ° C, It may be carried out at 27-35 ℃, 27-34 ℃, 27-33 ℃, 27-32 ℃, 27-31 ℃ or 27-30 ℃.

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.

In one example of the culture, 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 extract, and 0.5 wt% NaCl.

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.

When stirred, 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 150 rpm.

When agitated, the lipophilic material, such as dodecane, is dispersed in medium and contacted with the cells. 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.

When culturing microorganisms producing the retinoids described above without a lipophilic substance such as the dodecane phase, 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.

When the microorganism is cultured in the culture medium in the presence of a lipophilic substance such as dodecane, 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 β-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.

According to one embodiment the culturing step the medium comprises a concentration of about 2.0% glycerol, the genus Escherichia microorganism is E. coli DH5α or MG1655, the culturing step is a culture medium of about 7 ml, about 29 ℃ It may be to.

The method also includes the step of separating the retinoid from the lipophilic phase. It is well known in the art to separate such retinoids such as retinal, retinol, retinoic acid, retinyl esters, or combinations thereof. 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.

One embodiment comprises the steps of culturing the Escherichia genus microorganism having a retinoid production capacity in a medium containing a lipophilic substance; And separating the retinoid from the lipophilic substance, wherein the lipophilic substance is an alkane compound having 8 to 50 carbon atoms, a compound of Formula 1, a compound of Formula 2, or a combination thereof. It may be a method for producing a retinoid from.

According to the method for producing the retinoid of the present invention, retinol can be produced with high efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram schematically showing the MEP pathway of retinal biosynthesis and the foreign MVA pathway.

2 shows the conversion of β-carotene to retinoids including retinal, retinol, retinoic acid, and retinyl esters.

Figure 3 shows retinal production, β-carotene production and cell growth of E. coli, including pT-HB, pT-HBblh, pT-HBbrp, pT-HBbrp2, pT-HBBCMO1, and pT-HBSR.

4 shows retinal production of E. coli, including pT-HB, pT-HBSR, pT-DHB, and pT-DHBSR, and pT-DHB, or E. coli, including pT-DHBSR, with pS-NA, the MVA pathway plasmid. Carotene production and cell growth.

5 shows retinoid production and cell growth of various E. coli strains with pT-DHBSR and pS-NA.

6 shows retinoid production and cell growth of Escherichia coli with pT-DHBSR and pS-NA according to the culture test volume.

7 shows the retinoid production and cell growth of E. coli with pT-DHBSR and pS-NA at different culture temperatures.

8 shows the retinoid production and cell growth of Escherichia coli with pT-DHBSR and pS-NA according to the carbon source.

9 and 10 show retinoid production and cell growth according to the concentration of glycerol, the carbon source of E. coli, including pT-DHBSR and pS-NA.

11 and 12 show the results of retinoid production and cell growth of various E. coli strains in the presence of dodecane.

FIG. 13 shows the retinoid production and cell growth of Escherichia coli (pT-DHBSR / pS-NA) according to the concentration of glycerol as a carbon source in a two-phase culture comprising 1 mL dodecane on 5 ml culture medium.

FIG. 14 shows retinoid production and cell growth according to dodecane volume of Escherichia coli (pT-DHBSR / pS-NA) in two-phase culture.

FIG. 15 shows the distribution of retinoids according to the incubation time and dodecane volume of Escherichia coli (pT-DHBSR / pS-NA) in two-phase culture as a percentage of each component relative to the total retinoid.

FIG. 16 shows the effect of dodecane addition on beta-carotene production and cell growth of Escherichia coli with pT-DHB and pS-NA.

17 and 18 are diagrams showing the results of retinoid production and cell growth of Escherichia coli (pT-DHBSR / pS-NA) in the presence of various alkanes.

19, 20 and 21 is a diagram showing the results of the retinoid production, cell growth, cell specific retinoids (cell specific retinoids productivity) of E. coli (pT-DHBSR / pS-NA) in the presence of various volumes of light mineral oil.

22 and 23 are diagrams showing the results of retinoid production and cell growth of Escherichia coli (pT-DHBSR / pS-NA) in the presence of heavy mineral oil.

24 and 25 are diagrams showing the results of retinoid production and cell growth of Escherichia coli (pT-DHBSR / pS-NA) when cultured by tilting the test tube in the presence of heavy mineral oil.

FIG. 26 shows the cell growth and pH of Escherichia coli (pT-DHBSR / pS-NA) in the presence of a skin-friendly lipophilic substance.

FIG. 27 and FIG. 28 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. The following experimental materials and methods were used in the examples below.

Bacteria Strains and Culture Conditions

E. coli DH5α was used for gene cloning and retinoid production. E. coli MG1655, BL21 (DE3), XL1-Blue, S17-1 and BW25113 were used to find the optimal strain for retinol production. Cultivation for retinoid production was performed using a stirred incubator at 29 ° C. and 250 rpm in 2YT medium (16 g tryptone per liter, 10 g yeast extract, and 5 g NaCl). Glycerol and arabinose as primary and secondary carbon feedstocks, respectively, were added at concentrations of 0.5% to 2% (w / v) and 0.2% (w / v), respectively. Glucose, galactose, xylose and maltose were compared with glycerol as the carbon feedstock for retinoid production. Empicillin (100 μg / mL) and chlorampeticol (50 μg / mL) were added to the required cultures. Cultivation was performed in test tubes containing 7 mL medium. Cell growth was determined by measuring optical density at ₆₀₀ nm (OD ₆₀₀ ). In a two-phase culture of retinoid production, 1 mL of dodecane (Cat. No. 297879, Sigma, USA) was placed over 5 mL of culture medium.

Analytical Conditions for β-carotene and Retinoids

β-carotene and retinoids were extracted from bacterial cell pellets with acetone. In a two-phase culture with dodecane capping, the dodecane phase with retinoids was collected and centrifuged at 14,000 rpm for 10 minutes to remove all cell debris. Acetone extract and dodecane phase were analyzed by HPLC (LC-20A, Shimadzu, Kyoto, Japan) at detection wavelengths of 370 nm (retinal), 340 nm (retinol and retinyl acetate) and 454 nm (β-carotene). Analysis was performed using an HPLC column of Symmetry C18 (250 mm × 4.6 mm, 5 m) with Sentry Guard C18 (15 mm × 4.6 mm, 5 m). Mobile phases were 95: 5 and 70:30 methanol and acetonitrile, respectively, for retinoid and β-carotene analysis. HPLC analysis was applied with a flow rate of 1.5 ml / min and a column temperature of 40 ° C. Retinal (Cat. No. R2500), Retinol (Cat. No. R7632), Retinyl Acetate (Cat. No. R4632) and β-carotene (Cat. No. C4582) were purchased from Sigma (USA) and in acetone It was dissolved and used as a standard compound. Results are expressed as mean ± SD from three independent experiments.

Example 1 Preparation of Vectors for the Preparation of β-carotene and Retinal High Productivity E. Coli

Conventional procedures involving 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 blh gene of uncultured marine bacteria 66A03 (Genbank Permit No. AAY68319) was synthesized with Genofocus (Daejeon, Korea) following codon-optimization by Gene Designer software (DNA 2.0, Menlo Park, USA) for expression in E. coli. It was.

In this embodiment, E. coli, which has an MEP pathway, additionally introduces a gene encoding DXP synthase, which is an enzyme corresponding to the rate determining step, into E. coli, and simultaneously encodes a gene encoding an enzyme that participates in the mevalonate pathway. Selected from the source and introduced, β-carotene high productivity E. coli was prepared.

(1) Preparation of pSNA vector containing gene encoding enzyme of mevalonate pathway involved in synthesizing IPP from carbon source

The gene encoding the enzyme of the mevalonate pathway involved in synthesizing IPP from the carbon source used in this section is shown in Table 1 below.

Table 1

Enzyme Name	gene	Gene sequence (Genbank authorization number)
Acetyl-CoA Acetyltransferase / Hydroxymethylglutaryl (HMG) -CoA Reductase from Enterococcus faecalis	mvaE	SEQ ID NO: 18 (AF290092)
HMG-CoA Synthase from Enterococcus faecalis	mvaS	SEQ ID NO: 19 (AF290092)
Mevalonate kinase from Streptococcus pneumoniae	mvaK1	SEQ ID NO: 20 (AF290099)
Phosphomevalonate Kinase from Streptococcus pneumoniae	mvaK2	SEQ ID NO: 21 (AF290099)
Mevalonate diphosphate decarboxylase from Streptococcus pneumoniae	mvaD	SEQ ID NO: 22 (AF290099)
Isopentenyl diphosphate (IPP) isomerase from E. coli	idi	SEQ ID NO: 23 (U00096)

Primers and Restriction Enzymes for Amplifying the Genes of Table 1

TABLE 2

gene	Primer sequence		Restriction enzymes
mvaE	F	SEQ ID NO: 37	SacI
	R	SEQ ID NO: 38	SmaI
mvaS	F	SEQ ID NO: 39	SmaI
	R	SEQ ID NO: 40	BamHI
mvaK1, mvaK2, mvaD	F	SEQ ID NO: 41	KpnI
	R	SEQ ID NO: 42	XbaI
idi	F	SEQ ID NO: 43	SmaI
	R	SEQ ID NO: 44	Sph

Table 2 shows the primer sequences and restriction enzymes used for cloning the genes of Table 1. mvaK1, mvaK2, and mvaD existed on the chromosome as one operon, and PCR cloning of the operon was performed at once without the PCR cloning of each gene.

The genes of Table 1 were amplified by PCR using the primers listed in Table 3 and using the chromosomal DNA of the strain containing the gene as a template. The amplified product was introduced into the pSTV28 vector (Takara Korea, Korea) (SEQ ID NO: 45) using the restriction enzymes listed in Table 2 to prepare a vector pSNA. The vector pSNA contains all of the genes encoding enzymes of the mevalonate pathway that can produce IPP from acetyl-CoA.

(2) Preparation of vectors pT-HB and pT-DHB comprising genes encoding enzymes involved in synthesizing β-carotene from IPP

The gene encoding the enzyme involved in synthesizing β-carotene from the IPP used in this section and the DXP synthase gene, which is an enzyme in the rate determining step of the MEP pathway, are shown in Table 3 below.

TABLE 3

Enzyme Name	gene	Gene sequence (Genbank authorization number)
IPP Isoformerase from Haematococcus pluvialis	ipiHp1	SEQ ID NO: 24 (AF082325)
Escherichia coli-derived 1-deoxyxylulose-5-phosphate (DXP) synthase	dxs	SEQ ID NO: 25 (U00096)
Geranylgeranyl pyrophosphate (GGPP) synthase from pantoea agglomerans	crtE	SEQ ID NO: 26 (M87280)
Phytoene synthase from Pantoea agglomerans	crtB	SEQ ID NO: 27 (M87280)
Phytoen dehydrogenase from Pantoea agglomerans	crtI	SEQ ID NO: 28M87280
Lycopene-β-cyclase from Pantoea ananatis	crtY	SEQ ID NO: 29 (D90087)

Table 4

gene		primer	Restriction enzymes
ipiHp1	F	SEQ ID NO: 46	SmaI SphI
	R	SEQ ID NO: 47
dxs	F	SEQ ID NO: 48	Eco RI Sna BI
	R	SEQ ID NO: 49
crtE	F	SEQ ID NO: 50	Bsp HI Eco RI
	R	SEQ ID NO: 51
crtB, crtI	F	SEQ ID NO: 52	EcoRI SacI
	R	SEQ ID NO: 53
crtY	F	SEQ ID NO: 54	SalI PstI
	R	SEQ ID NO: 55

Primer sequences and restriction enzymes used for gene cloning in Table 3 are shown in Table 4. crtB and crtI exist on the chromosome as one operon, and PCR cloning of the operon was performed at once without PCR cloning of each gene.

The genes of Table 3 were amplified by PCR using the primers listed in Table 4 and using the chromosomal DNA of the strain containing the gene as a template. The amplified product was introduced into the pTrc99A vector (Genbank License No. M22744) (SEQ ID NO: 30) using the restriction enzymes listed in Table 4 to prepare a vector pT-DHB. The vector pTDHB contains both the gene encoding the enzyme involved in synthesizing β-carotene from IPP and the DXP synthase (dxs) gene, which is an enzyme in the rate determining step of the MEP pathway. In addition, all genes except dxs among the genes of Table 3 were introduced into the pTrc99A vector using the restriction enzymes listed in Table 4 to prepare a vector pT-HB.

(3) Preparation of a vector comprising a gene encoding an enzyme involved in synthesizing retinal from β-carotene

Genes encoding enzymes involved in synthesizing retinal from β-carotene used in the present invention are shown in Table 5 below. In Table 5, the gene encoding β-carotene monooxygenase from uncultured marine bacterium 66A03 was used as SR gene, the coli codon optimization sequence of blh .

Table 5

Enzyme Name	gene	Gene sequence (Genbank authorization number)
Uncultured marine bacterium 66A03 Β-carotene monooxygenase derived from	blh	SEQ ID NO: 31 (DQ065755)
Uncultured marine bacterium 66A03 Β-carotene monooxygenase derived from	SR(blhE. coli codon optimization sequence)	SEQ ID NO: 32
mouse (MusculusΒ-carotene 15,15'-monooxygenase derived from	bcmo1	SEQ ID NO: 33 (NM_021486)
Natronomonas Pharaohs (Natronomonas pharaonis) Brp-like protein 2 derived from ATCC35678	brp2	SEQ ID NO: 34 (CR936257)
Halobacterium Salinarum (Halobacterium salinarumΒ-carotene monooxygenase from ATCC700922	blh	SEQ ID NO: 35 (AE004437)
Halobacterium Salinarum (Halobacterium salinarumΒ-carotene monooxygenase from ATCC700922	brp	SEQ ID NO: 36 (AE004437)

            

Table 6

gene		Primer sequence	Restriction enzymes
SR	F	SEQ ID NO: 56	EcoR1 SpeI
	R	SEQ ID NO: 57
bcmo1	F	SEQ ID NO: 58	EcoR1 SpeI
	R	SEQ ID NO: 59
brp2	F	SEQ ID NO: 60	EcoR1 SpeI
	R	SEQ ID NO: 61
blh	F	SEQ ID NO: 62	EcoR1 SpeI
	R	SEQ ID NO: 63
brp	F	SEQ ID NO: 64	EcoR1 SpeI
	R	SEQ ID NO: 65

Primer sequences and restriction enzymes used for gene cloning in Table 5 are shown in Table 6. The genes of Table 5 were amplified by PCR using the primers listed in Table 6 as a template of the chromosomal DNA of the strain containing the gene and the cDNA library of the mouse. The amplified products were introduced into the vectors pT-HB using the restriction enzymes listed in Table 6, respectively, to prepare vectors pT-HBSR, pT-HBBcmo1, pT-HBbrp2, pT-HBblh, pT-HBbrp. The vectors pT-HBSR, pT-HBBcmo1, pT-HBbrp2, pT-HBblh and pT-HBbrp are vectors in which SR, Bcmo1, brp2, blh and brp genes are introduced into the pT-HB vector, respectively. It contains all the genes that encode enzymes involved in synthesizing retinal. The SR gene from pT-HBSR was cut with Spe I and inserted into the corresponding portion of pT-DHB to obtain pT-DHBSR.

Example 2: Comparison of Various BCM (D) O Genes for Retinal Production

Retinal is a recombinant that produces β-carotene E. coli can be produced by introducing a BCM (D) O gene encoding β-carotene mono (di) oxygenase. We found two bacteria, Halobacterium spp. (Halobacteriumsp) NRC-1 (blh AndbrpGenes) and Natronomymonas pharaonics (Natronomonas pharaonis) (brp2 Genes) and Vertebrate Moose MusculusMusculus) (Bcmo1BCM (D) O gene from the gene) was cloned. The inventors also note that uncultured marine bacteria 66A03blhCodon-optimized BCDO gene (SR) was synthesized based on the amino acid sequence of the gene. The BCM (D) O gene was used to prepare retinal synthetic plasmids pT-HBblh, pT-HBbrp, pT-HBbrp2, pT-HBBcmo1 and pT-HBSR, respectively. Recombinant E. coli cells containing each retinal plasmid were incubated at 29 ° C. for 48 hours in 2YT medium containing 0.5% (w / v) glycerol and 0.2% (w / v) arabinose as the carbon source.

Figure 3 shows retinal production, β-carotene production and cell growth of E. coli, including pT-HB, pT-HBblh, pT-HBbrp, pT-HBbrp2, pT-HBBCMO1, and pT-HBSR. White bars and gray bars represent the values at 24 hours and 48 hours, respectively.

As shown in FIG. 3, recombinant E. coli comprising pT-HBblh, pT-HBbrp, or pT-HBSR produced 2.2, 0.8, or 1.4 mg / L of retinal at 24 hours, respectively. However, retinal production of Escherichia coli containing pT-HBblh or pT-HBbrp decreased to 0.7 or 0.4 mg / L, respectively, at 48 hours, whereas the retinal production of Escherichia coli (pT-HBSR) was slightly increased. The decrease in retinal production after 24 hours may be due to intracellular oxidative degradation of the retinal. The amount of retinal obtained from the culture depends on both intracellular synthesis and degradation of the retinal.

For E. coli containing pT-HBblh or pT-HBbrp, the retinal production rate after 24 hours of incubation time will be lower than their degradation rate. Trace amounts of retinal were detected in E. coli strain cultures containing pT-HBbrp2 or pT-HBBcmo1. E. coli (pT-HB) without the BCM (D) O gene produced 35 mg / L of β-carotene and no retinal. Because β-carotene is just the precursor of retinal, the β-carotene consumption by BCM (D) O would be exactly proportional to the retinal production if there was retinal degradation. Since β-carotene remained in the culture of E. coli containing BCM (D) O except SR, the β-carotene cleavage activity of SR was estimated to be the highest among the tested BCM (D) O. Thus, SR enzyme was selected for retinal production in further experiments. Cell growth was not affected by overexpression of the BCM (D) O gene, except for the N. pharaonis brp gene, which showed growth retardation.

Example 3: Genetic Engineering with MEP and MVA Pathways for Building Block Supply

Retinal building blocks, IPP and DMAPP, can be synthesized in E. coli via the inherent MEP pathway and the foreign MVA pathway (FIG. 1). It is reported that 1-deoxy-d-xylose-5-phosphate (DXP) synthesis is an important rate-limiting step in the MEP pathway. Thus, overexpression of D XP synthase (encoded by dxs) increased lycopene and β-carotene production in our previous invention. The pT-DHBSR was prepared by introducing the dxs gene before the MEP pathway in pT-HBSR.

4 shows the retinal production of pT-DHB, or E. coli, including pT-DHBSR, with pS-NA, which is E. coli, including pT-HB, pT-HBSR, pT-DHBSR, and pT-DHBSR; β-carotene production and cell growth. White bars and gray bars represent the values at 24 hours and 48 hours, respectively.

As shown in Figure 4, retinal production of Escherichia coli (pT-DHBSR) was slightly higher than that of Escherichia coli (pT-HBSR) at 24 hours, but there was no difference at 48 hours. Β-carotene production from Escherichia coli (pT-DHB) increased 1.5 fold compared to Escherichia coli (pT-HB) by dxs overexpression. Exogenous MVA pathways in E. coli are known to significantly increase isoprenoid production by providing sufficient amounts of IPP and DMAPP building blocks. Escherichia coli (pT-DHBSR / pS-NA) containing an additional foreign MVA pathway produced 8.7 mg / L retinal at 48 hours, which is four times higher than the production of Escherichia coli (pT-DHBSR). In E. coli strains containing the SR gene, there was no or a small amount of β-carotene remaining in the cells, presumably due to an effective β-carotene cleavage reaction by SR. There was a big difference between the amount of β-carotene (substrate) consumed and the amount of retinal (product) produced. This may be due to the presence of a cellular response that metabolizes the retinal in E. coli in addition to the biological degradation of the retinal. Thus, the formation of other retinoids derived from retinal by any enzyme in E. coli is contemplated. Because retinal can be converted to retinol, retinoic acid and retinyl esters by cellular enzymatic reactions (FIG. 2), retinal derivatives in E. coli cultures were analyzed. The formation of derivatives except retinoic acid was confirmed and the production of retinal, retinol and retinyl acetate was measured in further experiments.

Example 4 Effect of Escherichia Coli Strains, Culture Conditions and Carbon Raw Materials on Retinoid Production

(1) strain

The effect of E. coli strains on the production of retinoids including retinal, retinol and retinyl acetate was investigated. Retinoid production was performed using five E. coli strains, MG1655, DH5α, XL1-Blue, S17-1, and BL21 (DE3), including pT-DHBSR and pS-NA. Table 5 shows the characteristics of the six E. coli strains, including the five strains.

TABLE 7

Escherichia coli strain	Explanation
MG1655	K12, wild type
DH5α	F -, φ f 80d lac Z △ M15, △ (lacZYA - argF) U169, deoR, recA1endA1, hsdR 17 (r K - m K +), phoA, supE 44, λ-, thi -1, gyrA 96, relA One
XL1-Blue	hsdR 17, supE 44, recA 1, endA 1, gyrA 46, thi , relA 1, lac / F '[ proAB + , lacI q , lacZ ΔM15 :: Tn10 (tet r )]
S17-1	recA pro hsdR RP4-2-Tc :: Mu-Km :: Tn7
BL21 (DE3)	F -, ompT, hsdS B ( r B - m B -), gal (lc I 857, ind 1, Sam 7, nin 5, lac UV5-T7 gene 1), dcm (DE3)
BW25113	△ (araD - araB) 567, △ lacZ4787 (:: rrnB-3), lambda -, rph-1, △ (rhaD-rhaB) 568, hsdR 514

5 shows retinoid production and cell growth of five E. coli strains with pT-DHBSR and pS-NA. Cultivation was performed at 29 ° C. for 48 hours in 2YT medium containing 0.5% (w / v) glycerol and 0.2% (w / v) arabinose. Retinal, retinol, and retinyl acetate are shown in light gray, dark gray, and black, respectively, and in cell growth, MG1655, DH5α, XL1-Blue, S17-1, and BL21 (DE3), respectively, ■, ●, ▲, ▼ and ◆.

As shown in FIG. 5, E. coli DH5α showed the highest amount of retinoid as 40 mg / L at 36 hours, and E. coli S17-1 and XL1-Blue then produced about 22 mg / L retinoids. However, very small amounts of retinoids were obtained with E. coli MG1655 and BL21 (DE3). Therefore, E. coli DH5α was selected as the retinoid producing strain.

(2) culture conditions

The effect of dissolved oxygen on retinoid production was tested in different test volumes in a 30 mm diameter test tube.

6 shows retinoid production and cell growth of Escherichia coli with pT-DHBSR and pS-NA according to test volume. In FIG. 6, retinal, retinol, and retinyl acetate are shown in light gray, dark gray and black, respectively, and test volumes 3, 5, 7 mL, and 10 mL, respectively, in cell growth. , ▼. Cultivation was performed at 29 ° C. for 48 hours in 2YT medium containing 0.5% (w / v) glycerol and 0.2% (w / v) arabinose.

As shown in FIG. 6, when having a low test volume, retinoid production peaked faster (corresponding to higher dissolved oxygen), and probably decreased faster due to its oxidative degradation. Both cell growth and retinoid production were delayed at a test volume of 10 mL, but small product degradation was observed. The optimal test volume for retinoid production was found to be 7 mL.

In addition, retinoid production with temperature was investigated. 7 shows the retinoid production and cell growth of E. coli with pT-DHBSR and pS-NA at different culture temperatures. In Figure 7, retinal, retinol, and retinyl acetate are shown in light gray, dark gray and black, respectively, and in cell growth the incubation temperatures of 29 ° C, 34 ° C and 37 ° C are indicated by ■, ●, ▲, respectively. Cultivation was performed for 48 hours in 2YT medium containing 0.5% (w / v) glycerol and 0.2% (w / v) arabinose.

As shown in FIG. 7, retinoid production was affected by incubation temperature and the highest production was obtained at 29 ° C.

(3) carbon source

The effect of different carbon feedstocks on retinoid production was compared.

8 shows the retinoid production and cell growth of Escherichia coli with pT-DHBSR and pS-NA according to the carbon source. In Figure 8, retinal, retinol, and retinyl acetate are shown in light gray, dark gray and black, respectively, and have no carbon source, carbon source glycerol, glucose, xylose, maltose, and galactose, respectively, in cell growth. , ▲, ▼, ◆, and ◀. Cultivation was performed at 29 ° C. for 48 hours in 2YT medium containing 0.2% (w / v) arabinose and 0.5% (w / v) glycerol, glucose, xylose, maltose, or galactose.

As shown in Figure 8, glycerol was the best carbon source for retinoid production. When glucose or galactose was used as the carbon source, the production of retinoids was lower than without the carbon source.

The effect of glycerol concentration on retinoid production and cell growth was then investigated. E. coli DH5α (pT-DHBSR / pSNA) was grown at 29 ° C. in 2YT medium containing 0.0% to 2.0% (w / v) glycerol.

9 shows the production of retinoids (retinal, retinol and retinyl acetate) of E. coli, including pT-DHBSR and pS-NA. Retinal, retinol and retinyl acetate are shown in light gray, dark gray and black, respectively.

10 shows cell growth of Escherichia coli including pT-DHBSR and pS-NA. The provided glycerol concentrations of 0%, 0.5%, 1%, and 2% are indicated by ■, ●, ▲, and ▼, respectively.

As shown in Figures 9 and 10, cell growth increased in proportion to glycerol concentrations. Stabilization reached 36, 48, and 72 hours at glycerol concentrations of 0.5, 1.0 and 2.0 (w / v), respectively. At this time its maximum retinoid production was obtained and thereafter the yield decreased significantly during stagnation. Retinoid production appeared to increase mainly after 24 hours. Retinoid production was highest among the various glycerol concentrations at 95 mg / L at 2.0% (w / v) glycerol, which was 2.4 times higher than the maximum retinoid production of 0.5% (w / v) glycerol. Increasing glycerol concentrations delayed stagnation and prolonged the period of retinoid production.

A significant decrease in retinoid production was observed in all cultures in the stagnant state of cell growth, likely due to disruption of retinoid synthesis and its intracellular oxidative degradation during the stagnant state.

(4) culturing in the presence of dodecane

The strains transformed with pT-DHBSR / pSNA for the six strains of Table 7 were used, 1 ml of dodecane was added to 5 ml of medium and cultured according to the conditions described in "Bacteria strains and culture conditions". As a medium, 2YT medium containing 0.2% (w / v) arabinose and 0.5% (w / v) glycerol was used.

11 is a view showing the retinoid production results according to different retinoid production strains in the presence of dodecane. As shown in FIG. 11, the most retinoids were produced in DH5α and MG1655. In the case of MG1655, cell growth and retinoid production were increased compared to the case where dodecane was not added. Cell growth and retinoid production rate of MG1655 was faster than that of DH5α. The BL21 (DE3) strain had high cell growth and little retinyl acetate production. As a result, DH5α and MG1655 were more suitable than the other strains among the six strains.

12 shows the results of growth of retinoid producing strains in the presence of dodecane.

13 is a view showing the results of retinoid production and growth according to the concentration of glycerol as a carbon source in the presence of dodecane.

Example 5: Two-Phase Cultivation Using Dodecane for Phosphorus Extraction of Retinoids

To prevent intracellular retinoid degradation, a two-phase culture with hydrophobic solvent dodecane was performed for in-situ extraction of retinoids from the cells. Dodecane was selected with low toxicity to E. coli, high hydrophobicity (log P _{O / W} , 6.6) for the extraction of hydrophobic retinoids _and no evaporation loss due to low volatility.

In this example, 1 mL of dodecane was added onto 5 mL of culture. FIG. 13 shows retinoid production and cell growth of Escherichia coli (pT-DHBSR / pS-NA) in a two-phase culture comprising 1 mL dodecane on 5 mL culture medium. Retinal, retinol and retinyl acetate are shown in light gray, dark gray and black in retinoid production, respectively. Glycerol concentrations 0.5%, 1%, and 2% provided in cell growth are indicated by ■, ●, and ▲, respectively.

Retinoids were extracted onto dodecane and negligible amounts of retinoids were detected in cell mass and culture (data not shown). As a result, retinoid production was measured from the dodecane phase. As shown in FIG. 13, in-drill extraction by dodecane could minimize intracellular degradation of retinoids. Retinoids in the dodecane phase appeared to be relatively stable and remained without significant oxidative degradation. Compared to the results of FIGS. 9 and 10 (without dodecane addition), the addition of 1 mL of dodecane significantly increased retinoid production even at 24 hours, and retinoid production without cell growth being affected by dodecane addition. The decrease of did not appear in the stationary state. However, retinoid production in the culture of 2% (w / v) glycerol ranged from 1% (w / v) to 2% (w / v), even though cell growth increased significantly with increasing glycerol concentrations. / v) not higher than that obtained with glycerol. 1 mL of dodecane addition volume may be insufficient for effective retinoid in-drill extraction in culture of 2% (w / v) glycerol.

To investigate the effect of dodecane addition volume on retinoid production and cell growth, 1 mL to 5 mL of dodecane was initially added onto a culture comprising 2% (w / v) glycerol (FIG. 14). .

FIG. 14 shows retinoid production and cell growth according to dodecane volume of Escherichia coli (pT-DHBSR / pS-NA) in two-phase culture. 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 ■, ●, ▲, □, ○, Δ, and ☆, respectively.

FIG. 15 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.

As shown in Figures 14 and 15, the total retinoid production also improved as the dodecane addition volume increased. 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. 14).

Figure 15 shows the distribution of retinoids obtained according to the volume of dodecane addition. There is a significant difference in retinoid distribution in the retinal and retinol ratios obtained with and without dodecane addition. At 48 hours, 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. Thus, dodecane addition increases the ratio of retinal but decreases the ratio of retinol. 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. In dodecane-added cultures, the retinyl acetate ratio decreases with longer incubation time, indicating that cell activity for retinyl acetate formation decreases during the culture. In conclusion, 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) can be effectively released from cells without loss of cell walls. In a two-phase culture of retinoid production, β-carotene must be maintained in cells because it is a direct precursor of retinoids. When β-carotene is extracted on dodecane, it can be cleaved by BCD (M) O located in the cytosol.

Since β-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 β-carotene (FIG. 16).

FIG. 16 shows the effect of dodecane addition on beta-carotene production and cell growth of Escherichia coli with pT-DHB and pS-NA. Cultivation was performed at 29 ° C. for 48 hours with addition of 1 mL dodecane on 5 mL of 2YT medium containing 0.5% (w / v) glycerol and 0.2% (w / v) arabinose. Gray bars and black bars represent 24 and 48 hours, respectively.

As shown in FIG. 16, a negligible amount of β-carotene was detected on dodecane and almost all β-carotene remained in the cell. There was no significant difference in β-carotene production and cell growth between cultures with and without dodecane addition.

Cultures with 5 mL dodecane addition resulted in 122 mg / L total retinoid production at 48 hours, whereas cultures without dodecane addition produced half of that amount (60 mg / L) at the same time. Thus, the dodecane-added two-phase culture system may be applied to other transformation systems that produce lipophilic small molecules.

Example 6: Production of Retinoids in Medium Containing Lipophilic Substances

In this example, it was confirmed whether there is an effect of increasing retinoid production for various lipophilic substances.

(1) Production of Retinoids in Medium Containing Alkanes

Using strain DH5α (pT-DHBSR / pSNA) transformed with pT-DHBSR / pSNA to DH5α, 5 ml of octane, decane, dodecane and tetradecane were respectively added to 5 ml medium and subjected to "bacterial strain and culture conditions". The culture was carried out according to the conditions described. As a medium, 2YT medium containing 0.2% (w / v) arabinose and 2.0% (w / v) glycerol was used.

17 shows the results of retinoid production in the presence of alkanes. 18 is a diagram showing the results of growth of a retinoid producing strain in the presence of alkanes.

As shown in FIG. 17, a total of 108 mg / L retinoids were produced when using decane. In addition, cell growth, pH and the amount of β-carotene in the cells did not show a significant difference according to the alkanes. As a result, decane seems to be more advantageous for retinoid production than dodecane. When octane was used, the production of retinal and retinol was associated with other alkanes, but little retinyl acetate. Tetradecane had lower total retinoid production than other alkanes.

(2) the production of retinoids in the medium containing mineral oil

(2.1) lightweight mineral oil

Lightweight mineral oils have the advantage of being cheaper than alkanes. Using strain DH5α (pT-DHBSR / pSNA) transformed with pT-DHBSR / pSNA to DH5α, adding different volumes of lightweight mineral oil to 5 ml medium, respectively, and cultivating according to the conditions described in "Bacterial strains and culture conditions" It was. As a medium, 2YT medium containing 0.2% (w / v) arabinose and 2.0% (w / v) glycerol was used.

19 shows the results of retinoid production in the presence of light mineral oil. 20 is a view showing the growth results of strains in the presence of light mineral oil.

As shown in FIG. 19, when 136.1 mg / L was produced in 5 ml of dodecane, 158 mg / L was produced in 2 ml light mineral oil. As shown in FIG. 20, the pH did not show a large difference except for dodecane. Cell growth, on the other hand, decreased as the amount of light mineral oil increased. This is expected to be due to insufficient mixing of the medium and mineral oil due to the high viscosity and specific gravity of the lightweight mineral oil. Retinoid production also decreased due to reduced cell growth.

21 is a diagram showing cell specific retinoids productivity per cell. As shown in FIG. 21, irrespective of the amount of mineral oil, it showed a specific productivity of about 5 mg / L / OD ₆₀₀ nm.

(2.2) weight mineral oil

Heavy mineral oils are cheaper than lightweight mineral oils. Using strain DH5α (pT-DHBSR / pSNA) transformed with pT-DHBSR / pSNA to DH5α, 2 ml of heavy mineral oil was added to 5 ml medium, respectively, and cultured according to the conditions described in "bacterial strains and culture conditions". . As a medium, 2YT medium containing 0.2% (w / v) arabinose and 2.0% (w / v) glycerol was used.

22 shows the results of retinoid production in the presence of heavy mineral oil. Fig. 23 shows the growth results of strains in the presence of heavy mineral oil. As shown in FIGS. 22 and 23, the light mineral oil and the heavy mineral oil had less cell growth than dodecane. In addition, 104.6 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 24 is a view showing the results of retinoid production when incubated with test tubes. 25 is a view showing the results of strain growth when cultured by tilting the test tube. As shown in FIG. 24 and FIG. 25, 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.

This indicates that lightweight and heavy mineral oils are important factors for retinoid production due to their high viscosity. Thus, it can be used for retinoids by providing adequate agitation during incubation.

(3) production of retinoids in media containing skin-friendly lipophilic substances

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.

 Using strain DH5α (pT-DHBSR / pSNA) transformed with pT-DHBSR / pSNA to DH5α, 2 ml of heavy mineral oil was added to 5 ml medium, respectively, and cultured according to the conditions described in "bacterial strains and culture conditions". . As a medium, 2YT medium containing 0.2% (w / v) arabinose and 2.0% (w / v) glycerol was used. The control group was added 5 ml of dodecane.

FIG. 26 shows cell growth and pH in the presence of skin friendly lipophilic material. FIG. 27 and 28 are diagrams showing the results of retinoid production according to the amount of skin-friendly lipophilic material. As shown in FIG. 27 and FIG. 28, 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.

[Revision under Rule 91 02.10.2012]

[Revision under Rule 91 02.10.2012]

[Revision under Rule 91 02.10.2012]

[Revision under Rule 91 02.10.2012]

[Revision under Rule 91 02.10.2012]

[Revision under Rule 91 02.10.2012]

[Revision under Rule 91 02.10.2012]

Claims (19)

1. Culturing the microorganism having the retinoid production capacity in a medium containing a lipophilic substance; And

   Separating the retinoid from the lipophilic phase;

   Including, the method for producing a retinoid from a microorganism.
2. The method of claim 1, wherein the microorganism is a bacterium, fungus, isolated animal cell, or a combination thereof.
3. The method of claim 1, wherein the microorganism is genus Escherichia, Bacillus, Corynebacterium, Yeast, Cluberomyces or a combination thereof.
4. The method according to claim 1, wherein the lipophilic material is an alkanes compound having 8 to 50 carbon atoms; A compound of Formula 1; A compound of Formula 2; Or a combination thereof:

   [Formula 1]

   R ₁ (CO) OR ₂

   (Wherein_OneAnd R₂Each independently represent alkyl having 8 to 50 carbon atoms, and CO represents a carbonyl group),

   [Formula 2]

   

   (Wherein R ₃ , R ₄ and R ₅ each independently represent alkyl having 8 to 50 carbon atoms, and CO represents a carbonyl group).
5. The method of claim 1, wherein the lipophilic material is octane, decane, dodecane, tetradecane, phytoscualan, mineral oil, isopropyl myristate, cetyl ethylhexanoate, dioctanoyl decanoyl glycerol, squalane, or a combination thereof That's how.
6. The method of claim 1, wherein the volume ratio of the medium to lipophilic material is 1: 0.2-3.0.
7. The method of claim 1, wherein the culturing is with stirring.
8. The method of claim 1, wherein the medium comprises glycerol.
9. The method of claim 1, wherein the medium comprises glucose.
10. The method of claim 1, wherein the separating comprises separating the retinoid from dodecane after removing the cells from the culture.
11. The method of claim 1, wherein the retinoid is at least one selected from the group consisting of retinal, retinol, retinyl ester and retinoic acid.
12. The method of claim 1, wherein the microorganism is Escherichia coli.
13. The method of claim 12, wherein the E. coli is DH5α, MG1655, BL21 (DE), S17-1, XL1-Blue, BW25113, or a combination thereof.
14. The method of claim 1, wherein the microorganism comprises: a gene encoding acetyl-CoA acetyltransferase / hydroxymethylglutaryl (HMG) -CoA reductase from Enterococcus faecalis of SEQ ID NO: 1; A gene encoding HMG-CoA synthase from Enterococcus faecalis of SEQ ID NO: 2; A gene encoding a mevalonate kinase from Streptococcus pneumoniae of SEQ ID NO: 3; A gene encoding a phosphomevalonate kinase from Streptococcus pneumoniae of SEQ ID NO: 4; A gene encoding mevalonate diphosphate decarboxylase from Streptococcus pneumoniae of SEQ ID NO: 5; A gene encoding isopentenyl diphosphate (IPP) isomerase from E. coli of SEQ ID NO: 6; A gene encoding geranylgeranyl pyrophosphate (GGPP) synthase from Pantoea agglomerans of SEQ ID NO: 7; A gene encoding a phytoene synthase derived from Pantoea agglomerans of SEQ ID NO: 8; A gene encoding a phytoene dehydrogenase from Pantoea agglomerans of SEQ ID NO: 9; And transformed with a gene encoding lycopene-β-cyclase from Pantoea ananatis of SEQ ID NO: 10.
15. The method of claim 14, wherein the microorganism comprises: a gene encoding β-carotene monooxygenase from uncultured marine bacterium 66A03 of SEQ ID NO: 13; A gene encoding β-carotene 15,15'-monooxygenase from a mouse of SEQ ID NO: 14 (Mus musculus); A gene encoding brp-like protein 2 derived from Natronomonas pharaonis ATCC35678 of SEQ ID NO: 15; And genes encoding β-carotene monooxygenase from Halobacterium salinarum ATCC700922 of SEQ ID NO: 16 or 17.
16. The method of claim 14, wherein the microorganism is further transformed with a gene having a nucleotide sequence of SEQ ID NO: 32 optimized for codon use in E. coli.
17. The method of claim 1, wherein the microorganism is transformed with a gene encoding E. coli derived 1-deoxyxylulose-5-phosphate (DXP) synthase.
18. The method of claim 1, wherein the microorganism is transformed with a gene encoding IPP isomerase from Haematococcus pluvialis of SEQ ID NO. 12.
19. The method according to claim 1, wherein the Escherichia microorganism is Escherichia coli DH5α / pTDHB / pSNA of Accession No. KCTC 11254BP or Escherichia coli DH5α / pTDHBSR / pSNA of Accession No. KCTC 11255BP.

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