CN103923847A - Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate - Google Patents

Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate Download PDF

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CN103923847A
CN103923847A CN201410093771.XA CN201410093771A CN103923847A CN 103923847 A CN103923847 A CN 103923847A CN 201410093771 A CN201410093771 A CN 201410093771A CN 103923847 A CN103923847 A CN 103923847A
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yeast
acid
hph
pda1
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陈坚
周景文
郭洪伟
堵国成
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Jiangnan University
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Abstract

The invention discloses Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate, belonging to the technical field of metabolic engineering. According to the invention, catalytic activity of a pyruvate dehydrogenase E1 component in a cell is improved through overexpression of PDA1, so the accumulation of an intermediate metabolite pyruvic acid is decreased, and supply of intracellular acetyl-CoA is reinforced; moreover, supply of intracellular acetyl-CoA is increased for accumulation of alpha-oxoglutarate for cells, which enables extracellular accumulation of alpha-oxoglutarate to be obviously increased. Thus, overexpression of PDA1 enables the content of the intermediate metabolite pyruvic acid to be decreased and accumulation of alpha-oxoglutarate to be reinforced and provides novel thinking for accumulation of organic acid in TCA cycle for cells.

Description

A kind of synthetic sub-Lip river of solution fat yeast of precursor supply enhancing α-ketoglutaric acid of strengthening
Technical field
The present invention relates to a kind of synthetic sub-Lip river of solution fat yeast of precursor supply enhancing α-ketoglutaric acid of strengthening, belong to metabolic engineering field.
Background technology
α-ketoglutaric acid is as one of important intermediate in tricarboxylic acid cycle (TCA) approach, in the metabolism of microorganism cells, play an important role, be not only the key node in tricarboxylic acid cycle, participate in vivo the synthetic and energy metabolism of amino acid, protein, VITAMIN; And be the important node of microorganism regulation and control carbon nitrogen metabolism balance, aspect the research regulatory mechanism relevant to microorganism nitrogen metabolism, there is vital role.Industrial, α-ketoglutaric acid is important fine chemistry industry and the intermediate of medicine industry, is widely used in synthetic multiple amino acids, VITAMIN and other small-molecule substance, has important application prospect in fields such as medicine, organic synthesis, nutrition-fortifying agents.
Due to the special status of α-ketoglutaric acid in extracellular microbial intracellular metabolite, the α-ketoglutaric acid that obtains of screening is produced bacterial strain, in high yield α-ketoglutaric acid, still can accumulate the metabolic by-prodss such as a large amount of pyruvic acid final period in fermentation.Mesostate pyruvic acid is the precursor substance that cell carries out TCA circulation, and the metabolic flux that pyruvic acid enters TCA circulation not only maintains the relevant end product α-ketoglutaric acid accumulation volume that more determines of growth with somatic cells.And, because pyruvic acid and α-ketoglutaric acid have similar physicochemical property, exist in a large number pyruvic acid to increase difficulty and the cost of downstream separation purge process.Therefore strengthen pyruvic acid and enter TCA circulation and not only strengthened the required precursor substance of cell accumulation α-ketoglutaric acid, also for downstream separation purge process has reduced difficulty and cost.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of synthetic sub-Lip river of solution fat yeast gene engineering bacteria of precursor supply enhancing α-ketoglutaric acid of strengthening, by the encoding gene pda1 strengthening pyruvic oxidase vigor of overexpression pyruvic oxidase E1 component α subunit, promote pyruvic acid dehydrogenation to form acetyl-CoA, strengthening enters the carbon metabolism flow of tricarboxylic acid cycle, for the synthetic α-ketoglutaric acid of sub-Lip river yeast (Yarrowia lipolytica) WSH-Z06 of fat increases precursor supply.
The nucleotides sequence of described gene pda1 is classified the nucleotide sequence shown in GeneID:2908574 in NCBI as.
Described genetic engineering bacterium is that to separate the sub-Lip river yeast WSH-Z06CCTCCNO:M20714 of fat be host, turns phosphoric acid and moves enzyme as the integrated expression vector p0 (hph) of selection markers is as expression vector to contain Totomycin.
The sub-Lip river yeast Yarrowia lipolytica WSH-Z06 of described solution fat obtains from Chinese Typical Representative culture collection center C CTCC, and culture presevation is numbered CCTCCNO:M20714.After overexpression gene pda1, in recombinant bacterial strain cell, acetyl-CoA content improves, and the outer pyruvic acid content of born of the same parents declines, and the α-ketoglutaric acid content of accumulation rises.
A kind of method that builds the sub-Lip river of described solution fat yeast gene engineering bacteria, comprise the following steps: (1) amplification hygromix phosphotransferase hph gene, the nucleotide sequence of hph gene is as shown in SEQ ID NO.1, utilize restriction enzyme StuI and Hind III to process amplification the hph gene and the p0 plasmid that obtain simultaneously, and connect two enzymes and cut product and obtain containing Totomycin and turn phosphoric acid and move the integrated expression vector p0 (hph) that enzyme is selection markers; (2) build recombination and integration type expression plasmid: according to the disclosed sequence of NCBI, the encoding gene of full chemosynthesis pyruvic oxidase E1 component α subunit, and cut open reading frame part and the integrated expression vector p0 (hph) of acquisition by restriction restriction endonuclease Not I and Eco RI simultaneously, connect and obtain recombinant expression plasmid p0 (hph)-PDA1; (3) by gained recombinant plasmid transformed Y.lipolytica WSH-Z06: utilize electroporation method for transformation that the linearizing recombination and integration type of being limited property restriction endonuclease Avr II expression plasmid is transformed to wild-type Y.lipolytica WSH-Z06, checking screening positive transformant.
The construction process of plasmid p0 is referring to document: Swennen D, Paul MF, Vernis L, Beckerich JM, Fournier A, Gaillardin C.Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis.Microbiology-Sgm, 2002.148:41-50.
With a method for described genetic engineering bacterium fermentative production α-ketoglutaric acid, be that gained recombination engineering bacteria is seeded in seed culture medium, 28 DEG C, 200rpm, cultivate 16-18 hour; Inoculum size by 10% is inoculated in 3-L fermentor tank from seed culture medium, and culture temperature is that 28 DEG C, air flow are 1.5vvm, and stirring revolution is 400rpm, cultivates 144-168 hour.
Described seed culture based component is: glucose 20g/L, peptone 10g/L, MgSO 47H 2o0.5g/L, KH 2pO 41g/L, adjusting pH is 5.5.Described fermentation culture based component is: glycerine 100g/L, (NH 4) 2sO 43g/L, KH 2pO 43g/L, MgSO 47H 2o1.2g/L, NaCl0.5g/L, K 2hPO 40.1g/L, vitamin 2 × 10 -7g/L, adjusts pH to add 20g/LCaCO after 5.0 again 3.
Compare with the bacterium that contrasts of overexpression gene pda1 not: in the cell of the recombinant bacterial strain of overexpression GeneID:2908574 gene, the catalysis activity of pyruvic oxidase E1 component improves 10.7%; Intracellular acetyl-CoA content has improved 13.4%, from 2.3nmolmg -1dCW is increased to 2.6nmolmg -1dCW; The pyruvic acid content of now extracellular accumulation has declined 46.3% to 18.7gL -1, α-ketoglutaric acid content is from 37.6gL -1rise to 42.5gL -1.
A kind of method of strengthening the synthetic α-ketoglutaric acid of yeast, it is the encoding gene pda1 strengthening pyruvic oxidase vigor by overexpression pyruvic oxidase E1 component α subunit, promote pyruvic acid dehydrogenation to form acetyl-CoA, strengthening enters the carbon metabolism flow of tricarboxylic acid cycle, for the synthetic α-ketoglutaric acid of yeast increases precursor supply.
Described yeast is preferably separated sub-Lip river yeast (Yarrowia lipolytica) WSH-Z06 of fat.
Usefulness of the present invention: the present invention, by the encoding gene of overexpression pyruvic oxidase E1 component α subunit, not only makes mesostate pyruvic acid accumulation volume decline, has strengthened acetyl in cell-CoA supply; And for cell accumulation α-ketoglutaric acid has improved acetyl in cell-CoA supply, cause the α-ketoglutaric acid of extracellular accumulation obviously to improve.
Brief description of the drawings
Fig. 1 overexpression PDA1 Promote cell's growth, Y.lipolyticaWSH-Z06(■), Y.lipolyticaT1(●).
Fig. 2 overexpression PDA1 increases acetyl-CoA and supplies with.
Fig. 3 overexpression PDA1 improves pyruvic oxidase E1 catalytic vigor.
Fig. 4 recombinant bacterial strain extracellular ketone acid content.
The impact of Fig. 5 overexpression PDA1 on fermentative production α-ketoglutaric acid; A: wild mushroom fermentative production α-ketoglutaric acid; B: recombinant bacterial strain Y.lipolyticaT1 fermentative production α-ketoglutaric acid; Dry cell weight (△), α-ketoglutaric acid (zero), pyruvic acid ().
Embodiment
Materials and methods
YPD substratum (gL -1): peptone 10, yeast extract 5, glucose 10, solid medium separately adds 20gL -1agar.When transformant screening, adding hygromycin B to final concentration is 400mgL -1.
Seed culture medium (gL -1): glucose 20, peptone 10, MgSO 47H 2o0.5, KH 2pO 41.0.Adjust pH to 5.5 with dilute hydrochloric acid, 115 DEG C maintain 15min sterilizing.Solid inclined-plane separately adds 20gL -1agar powder.
Fermention medium (gL -1): glycerine 100, (NH 4) 2sO 43, KH 2pO 43, MgSO 47H 2o1.2, NaCl0.5, K 2hPO 40.1, vitamin 2 × 10 -7, pH=4.5.115 DEG C, sterilizing 15min.Before inoculation, add the CaCO of 121 DEG C of sterilizing 30min 3to content be 20gL -1.
Separate the sub-Lip river yeast Y.lipolytica WSH-Z06 of fat and obtain from Chinese Typical Representative culture collection center C CTCC, bacterium numbering is CCTCC NO:M20714.
Cell acetyl-CoA measures: centrifugal collecting cell, be put into rapidly freezing 120s in liquid nitrogen, and metabolic activity in cells is stopped; Centrifugal 10min under 4 DEG C, 5000rpm condition; Then by centrifugal gained cell suspension in the perchloric acid of 1mL6%, by cytolysis; Add the salt of wormwood of 0.3mL3mol/L that acid is neutralized, centrifugal 20min under 4 DEG C subsequently, 5000rpm condition, removes cell debris; Get supernatant liquor after the micro-filtrate membrane filtration of 0.22 μ m, analyze for HPLC.HPLC condition: StableBond C18 reversed-phase column, column temperature: 28 DEG C, sampling volume: 10 μ L, flow velocity: 1.0mL/min, ultraviolet detection wavelength: 254nm; Moving phase: A is the sodium phosphate (pH5.0) of 0.2mol/L, the sodium phosphate (pH5.0) that B is 800mL0.25mol/L and the mixture of 200mL acetonitrile, and the A with 80%, 20% B are mixed into final moving phase.
The total catalysis activity of pyruvic oxidase E1 catalytic vigor and pyruvic oxidase prozyme is measured: the cell of centrifugal collection in exponential phase of growth, with 0.9% physiological saline washing, use 10mL0.1MKH 2pO 4-K 2hPO 41mMEDTA0.01mMDTTpH7.5 damping fluid suspension cell, under 4 DEG C of conditions, adds quartzite sand grind 5min, the centrifugal 10min of 13000 × g, and supernatant liquor is measured for pyruvic oxidase E1 catalytic vigor and the total catalysis activity of pyruvic oxidase prozyme.
Pyruvic oxidase E1 catalytic vitality test: by 0.5ml cytoclasis supernatant liquor, be added to and contain 50mMKH 2pO 4-K 2hPO 4, 10mMMgCl 2, 2.0mM pyruvic acid, 0.2mM diphosphothiamine, 0.1mM2,6-DCPIP, in the reaction mixture of pH7.0 to cumulative volume be 3mL, at 30 DEG C, under 600nm condition, detect 2,6-DCPIP content, the E1 catalytic vigor of 1U is defined as: 2 of interior reduction of unit time 1 μ mol, the needed enzyme amount of 6-DCPIP.
The total catalysis activity of pyruvic oxidase prozyme is measured: by 0.5ml cytoclasis supernatant liquor, be added to and contain 50mMKH 2pO 4-K 2hPO 4, 10mMMgCl 2, 2.0mM pyruvic acid, 0.2mM diphosphothiamine, 2.5mMNAD +, 0.13mMCoApH8.0 reaction mixture in to cumulative volume be 3mL, at 30 DEG C, under 340nm condition, detect NADH content, the E1 catalytic vigor of 1U is defined as: the needed enzyme amount of NADH that generates 1 μ mol in the unit time.
Extracellular α-ketoglutaric acid and pyruvic acid are measured: by centrifugal fermentation culture 13000 × g, get appropriate supernatant liquor, with 50 times of ultrapure water dilutions, 0.22 μ m filter membrane, analyze for HPLC.HPLC condition: Aminex HPX-87H ion exchange column; Moving phase: 5mmolL -1sulphuric acid soln (550 μ L vitriol oil constant volumes are to 2L), also degassed with the microfiltration membrane suction filtration in 0.22 μ m aperture; Column temperature: 35 DEG C; Sample size: 10 μ L; Flow velocity: 0.6mLmin -1.UV-detector detects: wavelength 210nm.
Y.lipolytica method for transformation: the mono-bacterium colony of fresh Y.lipolytica WSH-Z06 growing on YPD substratum is forwarded in liquid YPD substratum, 28 DEG C, 200rpm, incubated overnight.Be forwarded in fresh liquid YPD substratum 28 DEG C by 10% inoculum size, 200rpm is cultured to OD 600=1.2 left and right, centrifugal collecting cell, in 30 ° DEG C with 8mL100mmolL -1liAc, 10mmolL -1dTT, 0.6molL -1sorbitol10mmolL -1tris-HCL, pH=7.5 damping fluid processes 8 × 10 8cell.Centrifugal collecting cell, with the 1molL of 5mL precooling -1sorbitol washed cell three times, and use 1molL -1sorbitol suspension cell to 10 10cell mL -1.Add 1 μ g in advance linearizing integrating vector to cell suspending liquid, be placed in warm pregnant 5 minutes on ice, this mixture is transferred in the 0.2cm electricity revolving cup of precooling, electric shock, electric shock condition is 2.5KV, 25 μ F, 200 Ω, add the 1molL of 1mL precooling immediately -1sorbitol, room temperature leaves standstill 1 hour, and the 0.2mL product that shocks by electricity is coated and contained 400mgL -1the selection flat board of hygromycin B in, cultivate 48-72 hour for 28 DEG C.
The excessive checking primer of the integrated expression of table 1
Embodiment 1 strengthens acetyl in cell-CoA supply
(1) construction expression goal gene integrated expression vector used: according to this hph gene of gene order design primer amplification of the hygromix phosphotransferase in plasmid pUB4-CRE, the nucleotide sequence of hph gene is as shown in SEQ ID NO.1, utilize restriction enzyme Stu I and Hind III to process amplification the hph gene and the p0 plasmid that obtain simultaneously, and connect two enzymes and cut product and obtain containing Totomycin and turn phosphoric acid and move the integrated expression vector p0 (hph) that enzyme is selection markers.(2) build recombination and integration type expression plasmid: according to the disclosed sequence of NCBI, the encoding gene of full chemosynthesis pyruvic oxidase E1 component α subunit, and cut open reading frame part and the integrated expression vector p0 (hph) of acquisition by restriction restriction endonuclease NotI and EcoRI simultaneously, connect and obtain recombinant expression plasmid p0 (hph)-PDA1.(3) by above-mentioned structure integrated expression vector p0 (hph)-PDA1 transform in wild-type Y.lipolytica WSH-Z06 cell by electric method for transformation, containing 400mgL -1the screening flat board of hygromycin B on select transformant, extract the genomic dna of transformant for the checking of integrated expression.The genomic dna that utilization contains above-mentioned transformant is template, and the encoding gene PDA1 checking to overexpression pyruvic oxidase E1 component α subunit with checking primer pair VBF/E1 α (table 1), by positive transformant called after Y.lipolyticaT1.
The construction process of plasmid pUB4-CRE is referring to document FickersP, LeDallMT, GaillardinC, ThonartP, Nicaud JM.New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica.Journal of Microbiological Methods, 2003.55 (3): 727-737.
The construction process of plasmid p0 is referring to document Swennen D, Paul MF, Vernis L, Beckerich JM, Fournier A, Gaillardin C.Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis.Microbiology-Sgm, 2002.148:41-50.
Wild-type Y.lipolytica WSH-Z06 cell and recombinant bacterial strain Y.lipolyticaT1 are inoculated in YPD substratum simultaneously, at 28 DEG C, under 200rpm condition, cultivate, contrast strain growth situation.Cultivate through 30h, compared with wild-type cell, recombinant bacterial strain Y.lipolyticaT1 biomass has improved 17.0%(Fig. 1).Be cultured to the raji cell assay Raji of 24h, in its cell, acetyl-CoA content shows simultaneously, and overexpression PDA1 has improved acetyl in cell-CoA and supplied with, and intracellular acetyl-CoA content has improved 13.4% compared with control strain, from 2.3nmolmg -1dCW is increased to 2.6nmolmg -1dCW(Fig. 2).
Embodiment 2 impacts of overexpression PDA1 on pyruvic oxidase
Wild-type Y.lipolytica WSH-Z06 cell and recombinant bacterial strain Y.lipolyticaT1 are inoculated in YPD substratum simultaneously, at 28 DEG C, under 200rpm condition, cultivate, the cell of centrifugation in exponential phase of growth, measures pyruvic oxidase E1 catalytic vigor and the total catalysis activity of pyruvic oxidase prozyme in two strain cells by described before method.Compared with wild-type Y.lipolytica WSH-Z06, the intracellular E1 catalytic of recombinant bacterial strain Y.lipolytica T1 vigor has risen to 308.5U/mg protein from 278.5U/mg protein, and overexpression PDA1 successfully makes the intracellular E1 catalytic of the recombinant bacterial strain Y.lipolytica T1 vigor 10.7%(Fig. 3 that risen).Simultaneously, contrasting the total catalysis activity of intracellular pyruvic oxidase prozyme finds, the total catalysis activity impact of the interior pyruvic oxidase prozyme of recombinant bacterial strain Y.lipolytica T1 cell is not obvious, and this prozyme catalysis activity changes not obvious (Fig. 4) in this two strain cell.
Embodiment 3 overexpression PDA1 strengthen α-ketoglutaric acid accumulation
By the method for described production α-ketoglutaric acid before, wild-type Y.lipolytica WSH-Z06 cell and recombinant bacterial strain Y.lipolyticaT1 are inoculated in the 3L fermentor tank that contains 1.5L fermention medium simultaneously, at 28 DEG C, batch fermentation 144h under 200rpm condition, the pyruvic acid of contrast extracellular accumulation and α-ketoglutaric acid accumulation.Contrast is found: the α-ketoglutaric acid of recombinant bacterial strain Y.lipolyticaT1 extracellular accumulation is from 37.6gL -1rise to 43.3gL -1; Now extracellular metabolic intermediate is from 34.2gL -1be reduced to 20.1gL -1(Fig. 5).
Although the present invention with preferred embodiment openly as above; but it is not in order 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, therefore protection scope of the present invention should be with being as the criterion that claims were defined.

Claims (9)

1. strengthen synthetic sub-Lip river yeast (Yarrowia lipolytica) genetic engineering bacterium of solution fat of precursor supply enhancing α-ketoglutaric acid for one kind, it is characterized in that, be at the gene pda1 that separates overexpression coding pyruvic oxidase E1 component α subunit in the yeast of the sub-Lip river of fat.
2. genetic engineering bacterium according to claim 1, is characterized in that, the nucleotides sequence of described gene pda1 is classified the nucleotide sequence shown in GeneID:2908574 in NCBI as.
3. genetic engineering bacterium according to claim 1, is characterized in that, is that to separate the sub-Lip river yeast CCTCCNO:M20714 of fat be host, turns phosphoric acid and moves enzyme as the integrated expression vector p0 (hph) of selection markers is as expression vector to contain Totomycin.
4. one kind builds the method for genetic engineering bacterium described in claim 1, it is characterized in that, step is: the integrated expression vector that (1) construction expression goal gene is used: amplification hygromix phosphotransferase hph gene, the nucleotide sequence of hph gene is as shown in SEQ ID NO.1, utilize restriction enzyme Stu I and Hind III to process amplification the hph gene and the p0 plasmid that obtain simultaneously, and connect two enzymes and cut product and obtain containing Totomycin and turn phosphoric acid and move the integrated expression vector p0 (hph) that enzyme is selection markers; (2) build recombination and integration type expression plasmid: according to the disclosed sequence of NCBI, the open reading frame sequence of the gene of full chemical synthesis coding pyruvic oxidase E1 component α subunit, and cut open reading frame part and the integrated expression vector p0 (hph) of acquisition by restriction restriction endonuclease Not I and Eco RI simultaneously, connect and obtain recombinant expression plasmid; (3) by gained recombinant plasmid transformed Y.lipolytica WSH-Z06: utilize electroporation method for transformation that the linearizing recombination and integration type of being limited property restriction endonuclease AvrII expression plasmid is transformed to wild-type Y.lipolytica WSH-Z06, checking screening positive transformant.
5. application rights requires a method for genetic engineering bacterium fermentative production α-ketoglutaric acid described in 1, is that gained recombination engineering bacteria is seeded in seed culture medium, and 28 DEG C, 200rpm are cultivated 16-18 hour; Inoculum size by 10% is inoculated in 3-L fermentor tank from seed culture medium, and culture temperature is that 28 DEG C, air flow are 1.5vvm, and stirring revolution is 400rpm, cultivates 144-168 hour.
6. method according to claim 5, is characterized in that, described seed culture based component is: glucose 20g/L, peptone 10g/L, MgSO 47H 2o0.5g/L, KH 2pO 41g/L, adjusting pH is 5.5.
7. method according to claim 5, is characterized in that, described fermentation culture based component is: glycerine 100g/L, (NH 4) 2sO 43g/L, KH 2pO 43g/L, MgSO 47H 2o1.2g/L, NaCl0.5g/L, K 2hPO 40.1g/L, vitamin 2 × 10 -7g/L, adjusts pH to add 20g/LCaCO after 5.0 again 3.
8. strengthen the method for the synthetic α-ketoglutaric acid of yeast for one kind, it is characterized in that, it is the encoding gene pda1 strengthening pyruvic oxidase vigor by overexpression pyruvic oxidase E1 component α subunit, promote pyruvic acid dehydrogenation to form acetyl-CoA, strengthening enters the carbon metabolism flow of tricarboxylic acid cycle, for the synthetic α-ketoglutaric acid of yeast increases precursor supply.
9. method according to claim 8, is characterized in that, described yeast is for separating the sub-Lip river yeast of fat (Yarrowia lipolytica).
CN201410093771.XA 2014-03-14 2014-03-14 Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate Pending CN103923847A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104357344A (en) * 2014-11-19 2015-02-18 江南大学 Method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica
CN110499259A (en) * 2019-07-22 2019-11-26 浙江工业大学 A kind of solution ester Ye Shi yeast YW100-1 and its application

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104357344A (en) * 2014-11-19 2015-02-18 江南大学 Method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica
CN110499259A (en) * 2019-07-22 2019-11-26 浙江工业大学 A kind of solution ester Ye Shi yeast YW100-1 and its application
CN110499259B (en) * 2019-07-22 2021-07-27 浙江工业大学 Yarrowia lipolytica YW100-1 and application thereof

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Application publication date: 20140716