CN104312934A - Method for establishing recombinant yeast for biologically synthesizing glucuronic acid - Google Patents
Method for establishing recombinant yeast for biologically synthesizing glucuronic acid Download PDFInfo
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- CN104312934A CN104312934A CN201410568261.3A CN201410568261A CN104312934A CN 104312934 A CN104312934 A CN 104312934A CN 201410568261 A CN201410568261 A CN 201410568261A CN 104312934 A CN104312934 A CN 104312934A
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- recombination microzyme
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/99—Miscellaneous (1.13.99)
- C12Y113/99001—Inositol oxygenase (1.13.99.1), i.e. myo-inositol oxygenase
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Abstract
The invention discloses a method for establishing recombinant yeast for biologically synthesizing glucuronic acid, belonging to the field of metabolic engineering. According to the method, inositol oxygenase genes (MIOX) of different resources are expressed in different yeast hosts, and biological synthesis of glucuronic acid is achieved by taking glucose or inositol as a substrate. 20mg/L of glucuronic acid is generated from a recombinant pichia pastoris strain in a YPD fermentation culture medium, and 50mg/L of pichia pastoris is generated from a YPD-MI (60mM of inositol is added into the YPD culture medium) fermentation culture medium. The method for establishing recombinant yeast for synthesizing glucuronic acid has wide development prospect, and a novel idea is provided for synthesizing glucuronic acid by using a biological method.
Description
Technical field
The present invention relates to a kind of method building recombination yeast biosynthesizing glucuronic acid, belong to metabolic engineering field.
Background technology
Glucuronic acid, full name D-(+)-glucuronic acid (D-glucuronic acid), it is the compound that the primary hydroxyl group of glucose is oxidized to carboxyl and is formed, its aqueous solution is unstable, easy conversion formation 3,6-Glucuronic acid lactone, the two is in tautomeric equilibrium state in aqueous.Glucuronic acid is in heating and when having strong acid to exist, easily decarboxylation occur and generate the product such as CO2, furans.
Glucuronic acid is extensively present in animals and plants, has important biological function.It in conjunction with the toxic substance containing groups such as hydroxyl, amino, carboxyl, sulfydryls, can strengthen toxicant water-soluble, makes it to discharge fast kidney, play detoxification.Meanwhile, glucuronic acid can be combined by active group-phenolic hydroxyl group on combined thing molecule, reduces the biological action of associated hormone and medicine.Its derivative Glucuronic acid lactone is also a kind of health medicine, and it is conducive to the removing toxic substances of liver, can prophylactic treatment epidemic hepatitis, liver cirrhosis, food and drug intoxication, simultaneously or functional drinks, protective foods additive.Along with the raising day by day strengthening and require quality of life of health of people idea, the demand for glucuronic acid can be increasing.
At present, the production method of glucuronic acid mainly contains: chemical oxidization method, polysaccharide hydrolysis method and biological fermentation process.
Chemical oxidization method mainly adopts inorganic oxide or catalyzed oxidation, and the main method of current China industrial production glucuronic acid and lactone thereof is inorganic oxide method.Inorganic oxide method poor selectivity, degree of oxidation can not effectively control, product separation reclaims difficulty, and environmental pollution is serious.Catalyzed oxidation is mainly divided into homogeneous catalytic oxidation and heterogeneous catalytic oxidation.
Polysaccharide hydrolysis method is the polyuronic acid that existed by acid or basic hydrolysis occurring in nature or containing the polysaccharide of uronic acid, extraction and isolation obtains aldehydic acid.But because the glycosidic link between glucuronic acid is highly stable, need strong reaction condition, as added strong acid or highly basic, but under this kind of condition, can produce glucuronic acid and destroy, the rate of recovery is low.
Biological fermentation process utilizes specific bacterial strain or biological enzyme to carry out fermentation to prepare glucuronic acid to the predetermined substance that can form glucuronic acid or its precursor.Research at present for biological fermentation process production glucuronic acid is relatively less, but biological fermentation process has, and cost is low, environmental pollution is little, specificity advantages of higher, utilizes specific microorganism strains to carry out fermentative production glucuronic acid and there is larger development prospect.
The production of current glucuronic acid is mainly raw material with starch, and through nitric acid oxidation, pressurized hydrolysis is produced, and this method exists this many drawback, as serious in environmental pollution, reclaim the problems such as difficulty, poor selectivity.In order to solve the problem, by the metabolism of genetic engineering techniques microorganism cells, carry out fermentative production object product with the performance improving cell, there is vast potential for future development.
Yeast is unicellular lower eukaryotes, and existing prokaryote is easily cultivated, fast growth, simple to operate, has again when eukaryote is expressed functions such as the processing of protein and modifications.Because nutritional requirement when it is cultivated is low, growth fast, substratum is cheap, can carry out high density fermentation cultivation, be therefore a kind of excellent host that can be used for productive target biological product.
Not yet find that there is the report being carried out fermentative production glucuronic acid by recombination yeast at present, original yeast strain can not generate glucuronic acid, Given this, and the novel recombinant bacterial strain built in the present invention, produce in glucuronic acid at fermentable, provide new method and thinking
Summary of the invention
The present invention, by expressing the inositol oxygenase gene (MIOX) of different sources in yeast recombinant strain, with glucose or inositol for substrate, realizes the biosynthesizing of glucuronic acid.
The invention provides a kind of recombination microzyme, is inositol oxygenase gene M IOX is cloned into the recombination microzyme built in the yeast host bacterium that sets out.
Described inositol oxygenase gene M IOX derive from following any one: mouse (mouse), pichia spp (Pichia pastoris), Candida albicans (Candida albicans).
Described inositol oxygenase gene M IOX is derive from pichia spp in one embodiment of the invention.
Described inositol oxygenase gene M IOX, in one embodiment of the invention, its aminoacid sequence is the sequence shown in SEQ ID NO.2.
Described inositol oxygenase gene M IOX, in one embodiment of the invention, its nucleotide sequence is the sequence shown in SEQ ID NO.1.
The described yeast host bacterium that sets out be following any one: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida lipolytica (Candida lipolytica), pichia spp (Pichia pastoris).
The described yeast host bacterium that sets out is pichia spp in one embodiment of the present invention.
In described recombination microzyme, the promotor of regulating and expressing inositol oxygenase gene is GAP, Gal1, AOX1 or TEF1.
The present invention also provides a kind of construction process of described recombination microzyme, is to be cloned on expression vector by inositol oxygenase gene M IOX to form recombinant vectors, then proceeds to after recombinant vectors linearizing in yeast host bacterium, obtains recombination microzyme.
Described expression vector be following any one: pGAPZA, pGAPZB, pGAPZC, pPICZA, pPICZB, pPICZC and pPIC9K.
The present invention also provides a kind of method utilizing described recombination microzyme synthesis of glucose aldehydic acid, is be inoculated in fermention medium by recombination microzyme seed liquor with the inoculum size of 5%-10%, and with 25 DEG C-30 DEG C, 180rpm-220rpm, cultivates 24h-96h.
The carbon source of described fermention medium be following any one or multiple: glucose, inositol, glycerine, sucrose.
Described fermention medium is YPD substratum or YPD-MI substratum (adding 60mM inositol in YPD substratum) in one embodiment of the invention
The method of described synthesis of glucose acid, in one embodiment of the present invention, specifically: recombination microzyme is cultivated in YDP substratum preparation seed liquor, be inoculated into containing in fermention medium by 10% inoculum size, in temperature 30 DEG C, shaking speed 200rpm, cultivates 60 hours.
Utilize recombination microzyme synthesis of glucose aldehydic acid of the present invention, recombinant yeast pichia pastoris produces glucuronic acid 20mg/L in YPD substratum, in the substratum of YPD-MI (adding 60mM inositol in YPD substratum), produce glucuronic acid 50mg/L, in contrast pichia spp Pichia pastoris GS115, aldehydic acid do not detected.The inositol oxygenase gene that what the present invention was used derive from pichia spp and the MIOX amino acid deriving from mouse of existing report differ greatly, and not deriving from MIOX gene in pichia spp can be converted into the relevant report of aldehydic acid by inositol at present.The present invention adopts the strategy of metabolic engineering, and transformation microorganism strains synthesizes object product glucuronic acid, for biological process High-efficient Production glucuronic acid lays a solid foundation.Expression of recombinant yeast inositol oxygenase gene carrys out synthesis of glucose aldehydic acid, and when also having a cultivation, nutritional requirement is low, growth is fast, substratum is cheap, inheritance stability, expression level are high, can many advantages such as high density fermentation.
Accompanying drawing explanation
Fig. 1: recombinant yeast pichia pastoris shake flask fermentation malaga uronic acid situation; 1 is control strain Pichia pastoris GS115, and 2 is the cultivation results of recombinant yeast pichia pastoris in YPD substratum, and 3 is the cultivation results of recombinant yeast pichia pastoris in YPD-MI.
The result of glucuronic acid in Fig. 2: LC-MS detection fermented liquid; A is glucuronic acid standard specimen, and B is recombinant yeast pichia pastoris fermented liquid.
Embodiment
The detection of glucuronic acid: LC-MS (LC-MS), instrument: Shimadzu ion-trap time-of-flight mass spectrometer LCMS-IT-TOF analysis condition:
Moving phase: A:1mM ammonium formiate+0.01% formic acid+water; B:1mM ammonium formiate+acetonitrile
Elution program: 0-12min30%B; 12-30min30%-65%; 30-31min65%-95%; 31-35min95%35-36min95%-30%B; 36-40min30%
Chromatographic column: Shim-pack VP-ODS (150L × 2.0)
Flow velocity: 0.15ml/min sample size: 5ul
Ionization mode: Negative electrospray ionization pattern
Atomization gas flow velocity: 1.5L/min
CDL temperature: 200 DEG C of HB temperature: 200 DEG C
Sweep limit: MS1, m/z150-300
The preparation of glucuronic acid standard specimen: accurately take 10mg glucuronic acid powder and be dissolved in deionized water, the solution after dissolving is transferred to constant volume in 100ml volumetric flask, and its concentration is 100mg/L.50,20,10,5 and 1mg/L is diluted to respectively again with deionized water.
The structure of embodiment 1 recombination microzyme Pichia pastoris GS115/pGAPZB-MIOX
With pichia spp (Pichia pastoris GS115) genome for template, pcr amplification MIOX gene
Primers F (sequence is as shown in SEQ ID NO.3), R (sequence is as shown in SEQ ID NO.4) are as follows, and wherein underscore part is primer restriction enzyme site:
F:CCG
CTCGAGATGTCAGTACAAAAAAGCACGAAG
R:TGC
TCTAGATCAAAACTTTACCAGCTTTTGAGG
XhoI, XbaI double digestion is carried out to the inositol oxygenase gene that amplification obtains, be connected to and there is (carrier pGAPZB is through XhoI, XbaI double digestion) on the expression vector of respective cut, be converted in E.coil Top10, guaranteeing to identify recombinant expression plasmid pGAPZB-MIOX under the prerequisite that reading frame is correct, through DNA sequencing comparison, recombination sequence is correct.Recombinant plasmid electricity after BspHI linearizing proceeds in expressive host Pichia pastoris GS115, and recombinant clone is correct through PCR checking, verifies correct recombinant bacterium called after Pichia pastoris GS115/pGAPZB-MIOX.
The structure of embodiment 2 recombination microzyme Pichia pastoris GS115/pPIC9K-GAP-MIOX
With pGAPZB plasmid for template, pcr amplification GAP promotor, primer is F1 (sequence is as shown in SEQ ID NO.5), R1 (sequence is as shown in SEQ ID NO.6), and underscore part is restriction enzyme site
F1：C
GAGCTCAGATCTTTTTTGTAGAAATGTCTTG
R1：CTTCGTGCTTTTTTGTACTGACATCGTTTCGAAATAGTTGTTCAATT
With pichia spp (Pichia pastoris GS115) genome for template, amplification MIOX gene, primer is F2 (sequence is as shown in SEQ ID NO.7), R2 (sequence is as shown in SEQ ID NO.8) is as follows, and dash area is restriction enzyme site
F2：AATTGAACAACTATTTCGAAACGATGTCAGTACAAAAAAGCACGAAG
R2：ATAAGAAT
GCGGCCGCTCAAAACTTTACCAGCTTTTGAGG
Use the method for fusion DNA vaccine, by GAP promotor and MIOX gene fusion, and introduce restriction enzyme site Sacl at fusion fragment two ends, Notl, double digestion is carried out to fusion fragment, be connected to and there is (pPIC9K is through Sac1, Not1 double digestion) on the expression vector of respective cut, be GAP promotor by the AOX promoter replacement on pPIC9K, Transformed E .coli JM109, guaranteeing to identify recombinant expression plasmid pPIC9K-GAP-MIOX under the prerequisite that reading frame is correct, through DNA sequencing comparison, recombination sequence is correct.Recombinant plasmid point after SalI linearizing proceeds in expressive host Pichia pastoris GS115, and recombinant clone is correct through PCR checking, verifies correct recombinant bacterium called after Pichia pastoris GS115/pPIC9K-GAP-MIOX.
Embodiment 3 recombinant yeast pichia pastoris fermentative production glucuronic acid
Restructuring Pichia pastoris GS115/pPIC9K-GAP-MIOX clone is used to carry out fermentation culture.Mono-clonal is inoculated in the YPD substratum (yeast extract 10g/L, peptone 20g/L, glucose 20g/L) of 25ml, 30 DEG C, 200rpm cultivates 24h.Be seeded in 50ml (shaking flask capacity is 500ml) fermention medium by 10% inoculum size and ferment, cultivate 60 hours.Fermention medium is divided into YPD substratum and YPD-MI substratum (adding 60mM inositol in YPD substratum).After cultivation terminates, get 1ml fermented liquid centrifugal 10min under 8000rpm, get supernatant through 0.22um membrane filtration, product is detected by LC-MS, Fig. 2 is the LC-MS detected result of product glucuronic acid, A is glucuronic acid standard specimen, and B is restructuring bacterial strain fermentation liquor, can be found out [M-1] of glucuronic acid by standard specimen
+be 193.03, the glucuronic acid with same molecular amount (m/z) can be detected in recombinant bacterial strain fermented liquid.
Embodiment 4 compares the amount of the glucuronic acid that recombinant yeast pichia pastoris generates in different fermentations substratum
With YPD-MI substratum (adding 60mM inositol in YPD substratum) in YPD fermention medium, fermentation recombinant yeast pichia pastoris, cultivated after 48 hours, and detect product, result as shown in Figure 1 and Figure 2.As seen from Figure 1, control strain Pichia pastoris GS115 not malaga uronic acid, recombinant bacterium generates glucuronic acid 20mg/L in YPD substratum, generates glucuronic acid 50mg/L in YPD-MI substratum.The precursor substance inositol adding inositol oxygenase (MIOX) is in the medium conducive to the output improving glucuronic acid.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a recombination microzyme inositol oxygenase gene M IOX is cloned into the recombination microzyme built in the yeast host bacterium that sets out.
2. recombination microzyme according to claim 1, is characterized in that, described inositol oxygenase gene M IOX derive from following any one: mouse, pichia spp, Candida albicans.
3. recombination microzyme according to claim 1, is characterized in that, the aminoacid sequence of described inositol oxygenase gene is the sequence shown in SEQ ID NO.2.
4. recombination microzyme according to claim 1, is characterized in that, the nucleotide sequence of described inositol oxygenase gene is the sequence shown in SEQ ID NO.1.
5. recombination microzyme according to claim 1, is characterized in that, described in set out yeast host bacterium be following any one: yeast saccharomyces cerevisiae, Candida lipolytica, pichia spp.
6. recombination microzyme according to claim 1, is characterized in that, described in the yeast host bacterium that sets out be pichia spp.
7. recombination microzyme according to claim 1, is characterized in that, described in regulating and expressing, the promotor of inositol oxygenase gene M IOX is GAP, Gal1, AOX1 or TEF1.
8. a construction process for recombination microzyme described in claim 1, is characterized in that, is to be cloned on expression vector by inositol oxygenase gene M IOX to form recombinant vectors, then proceeds to after recombinant vectors linearizing in yeast host bacterium, obtains recombination microzyme.
9. utilizing a method for recombination microzyme synthesis of glucose aldehydic acid described in claim 1, is be inoculated in fermention medium by seed culture fluid with the inoculum size of 5%-10%, 25 DEG C-30 DEG C, 180rpm-220rpm, cultivates 24h-96h.
10. method according to claim 9, is characterized in that, the carbon source of described substratum be following any one or multiple: glucose, inositol, glycerine, sucrose.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929459A (en) * | 2017-03-30 | 2017-07-07 | 华南理工大学 | A kind of method of recombination bacillus coli and its construction method with glucaric acid is produced by metabolic engineering |
| CN107022514A (en) * | 2017-03-30 | 2017-08-08 | 华南理工大学 | A kind of recombination bacillus coli and its construction method and the method that glucuronic acid is produced by metabolic engineering |
| CN109423469A (en) * | 2017-09-05 | 2019-03-05 | 中国科学院微生物研究所 | A kind of method producing glucuronic acid and its dedicated engineering bacteria |
| CN109706199A (en) * | 2019-02-28 | 2019-05-03 | 江南大学 | A method of glucuronic acid yield is improved using Lu Shi Zygosaccharomyces |
| CN110923247A (en) * | 2019-12-27 | 2020-03-27 | 甘肃农业大学 | Barley stripe disease pathogenic gene Pgmimox and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102066552A (en) * | 2008-04-04 | 2011-05-18 | 麻省理工学院 | Cellular production of glucaric acid |
| CN104080918A (en) * | 2012-02-24 | 2014-10-01 | 旭化成化学株式会社 | Method for producing glucaric acid |
-
2014
- 2014-10-22 CN CN201410568261.3A patent/CN104312934A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102066552A (en) * | 2008-04-04 | 2011-05-18 | 麻省理工学院 | Cellular production of glucaric acid |
| CN104080918A (en) * | 2012-02-24 | 2014-10-01 | 旭化成化学株式会社 | Method for producing glucaric acid |
Non-Patent Citations (3)
| Title |
|---|
| DE SCHUTTER,K.等: "NCBI Reference Sequence: XM_002490887.1", 《GENBANK》 * |
| SHUXIANG ZHENG等: "Efficient Expression of Myo-inositol Oxygenase in Escherichia coli and Application for Conversion of Myo-inositol to Glucuronic Acid", 《FOOD SCIENCE AND BIOTECHNOLOGY》 * |
| TAE SEOK MOON等: "Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929459A (en) * | 2017-03-30 | 2017-07-07 | 华南理工大学 | A kind of method of recombination bacillus coli and its construction method with glucaric acid is produced by metabolic engineering |
| CN107022514A (en) * | 2017-03-30 | 2017-08-08 | 华南理工大学 | A kind of recombination bacillus coli and its construction method and the method that glucuronic acid is produced by metabolic engineering |
| CN109423469A (en) * | 2017-09-05 | 2019-03-05 | 中国科学院微生物研究所 | A kind of method producing glucuronic acid and its dedicated engineering bacteria |
| CN109706199A (en) * | 2019-02-28 | 2019-05-03 | 江南大学 | A method of glucuronic acid yield is improved using Lu Shi Zygosaccharomyces |
| CN110923247A (en) * | 2019-12-27 | 2020-03-27 | 甘肃农业大学 | Barley stripe disease pathogenic gene Pgmimox and application thereof |
| CN110923247B (en) * | 2019-12-27 | 2023-04-11 | 甘肃农业大学 | Barley stripe disease pathogenic gene Pgmiox and application thereof |
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