CN105838632A - Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof - Google Patents

Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof Download PDF

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CN105838632A
CN105838632A CN201610333309.1A CN201610333309A CN105838632A CN 105838632 A CN105838632 A CN 105838632A CN 201610333309 A CN201610333309 A CN 201610333309A CN 105838632 A CN105838632 A CN 105838632A
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succinic acid
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CN105838632B (en
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徐国强
蒋伶活
苏珂
李佳雨
刘维瑾
范欧洋
赵祯
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Jiangnan University
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Abstract

The invention discloses saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof, and belongs to the fields of genetic engineering and fermentation engineering. Malate dehydrogenase gene RoMDH from rhizopus oryzae is adopted to replace PDC1, the yield of ethanol obtained by fermentation of wild saccharomyces cerevisiae reaches the maximum value 13.55 plus or minus 1.062g/L, and the yield of improved strain MP ethanol is 11.09 plus or minus 0.539g/L, and compared with the yield obtained by adopting the wild type, the yield is reduced by 18.15 percent. On the basis, SDH2 gene is knocked out. After culture medium optimization, MP Delta S deleted strain can accumulate succinic acid by 0.698 plus or minus 0.0285g/L, and by contrast, the wild saccharomyces cerevisiae does not accumulate succinic acid. The saccharomyces cerevisiae gene engineering bacteria can effectively reduce the loss of a carbon flow, creates a condition for the engineering yeast to efficiently produce succinic acid, and has a good industrial application value and prospect.

Description

A kind of Saccharomyces cerevisiae gene engineering bacteria producing succinic acid and application thereof
Technical field
The present invention relates to a kind of Saccharomyces cerevisiae gene engineering bacteria producing succinic acid and application thereof, belong to genetic engineering and fermentation Engineering field.
Background technology
Succinic acid (succinic acid) is a kind of four-carbon dicarboxylic acid, and formal name used at school is succinic acid (butanedioic acid). Succinic acid has more than a 30 kind of important derivatives chemicals: oxolane, BDO, gamma-butyrolacton, maleic acid and anti-fourth Enedioic acids etc., have the huge market space.At the main acidic flavoring agent of food service industry, flavoring agent etc., can be used in pharmaceuticals industry Synthesis tranquilizer, pH adjusting agent, contraceptive, cancer therapy drug etc., be mainly used as ion chelating agent, surface activity at chemical industries Agent, detergent additive, foaming agent and lubricant etc..It is noted that succinic acid is as biodegradable plastic polybutadiene The primary raw material of acid butanediol ester (can be degradable), is with a wide range of applications.Important application in view of above-mentioned succinic acid Being worth and many advantages, USDOE set up Isosorbide-5-Nitrae-dicarboxylic acids group specially in 2004, studies succinic acid, malic acid and prolongs The production technology of fumarate and application.
The production method of succinic acid mainly has chemical synthesis and microbe fermentation method at present.Compared with chemical synthesis, Microbe fermentation method has a lot of advantage: raw material sources are wide, and low cost is renewable；Reaction condition is gentle, and energy consumption is little, environment friend Good.Therefore, Production by Microorganism Fermentation succinic acid enjoys the concern of people, and in recent years, microbial method produces the research of succinic acid Progress is rapidly.Saccharomyces cerevisiae (Saccharomyces cerevisiae) is biological, because having as a kind of eucaryon mode trickle: heredity Abundant information, Metabolically engineered easy to operate；Nutritional need is simple, and separation-extraction technology is with low cost；At low ph conditions (even PH < 3.0) well-grown；It is resistant to the substrate of high concentration；It is GRAS (General Regarded As by FDA certification Safe) microorganism, fermented product has the advantages such as safety and becomes fermenting and producing carboxylic acid and (lactic acid, acetone acid, malic acid, prolong Fumarate, succinic acid, α-ketoglutaric acid etc.) potential the suitableeest microorganism.But, utilize fermentation by saccharomyces cerevisiae to produce succinic acid face Face problems with: (1) produces substantial amounts of ethanol at the condition batch fermentation of high concentration sugar and ventilation, for producing with carboxylic acid for target For thing, a large amount of accumulation of ethanol make the substantial amounts of loss of carbon stream；(2) mesostate during succinic acid is TCA circulation, makes Brewer yeast itself does not have the metabolic pathway of excess accumulation succinic acid, needs to build the route of synthesis of succinic acid, but succinic acid has The highest reduction potential, if by TCA reduction approach accumulation succinic acid, needing two to go back protohydrogen, relatively difficult accumulation；(3) realize Reduce ethanol to produce, after acetone acid is accumulated, how to make acetone acid be further converted to target product succinic acid, need to increase Strong carboxylase reaction, is allowed to be converted into oxaloacetic acid, enters TCA circulation, and then is converted into succinic acid, this strategy succinum The key that acid yield improves.
Summary of the invention
First technical problem that the invention solves the problems that is to provide a kind of Saccharomyces cerevisiae gene engineering bacteria producing succinic acid, for Metabolic engineering saccharomyces cerevisiae efficiently produces succinic acid and lays the first stone.
Described engineering bacteria is with saccharomyces cerevisiae as starting strain, substituted for alcohol metabolism approach with malate dehydrogenase gene In Pyruvate Decarboxylase Gene PDC1, on this basis, knocked out the succinate dehydrogenase gene in succinic acid metabolic pathway SDH2。
In one embodiment of the invention, described malate dehydrogenase gene derives from Rhizopus oryzae.
In one embodiment of the invention, the nucleotide sequence such as Gene of described succinate dehydrogenase gene SDH2 Shown in ID:1360234.
In one embodiment of the invention, described saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1C, is purchased from EUROSCARF(http://web.uni-frankfurt.de/fb15/mikro/euroscarf/data/cen.html)；Its Genotype is: MATa；ura3-52；trp1-289；leu2-3,112；his3Δ1；MAL2-8C；SUC2.
Second technical problem that the invention solves the problems that be to provide build described Saccharomyces cerevisiae gene engineering bacteria method, Sequence PDC1 as shown in SEQ ID NO.5 is replaced and knocks out box, and SDH2 knocks out box, transformed saccharomyces cerevisiae respectively CEN.PK2-1C, builds the Saccharomyces cerevisiae gene engineering bacteria obtaining producing succinic acid.
It is with plasmid pUG27 as template that described SDH2 knocks out box, enters with primer shown in SEQ ID NO.3, SEQ ID NO.4 Performing PCR amplification obtains.
The construction method of described pUG27 plasmid sees " Guldener U, Heck S, Fielder T, et al.A new efficient gene disruption cassette for repeated use in budding yeast.Nucleic Acids Res, 1996,24 (13): 2519-2524 " and " Guoqiang Xu, Qiang Hua, et al.Regulation ofthiamine synthesis in Saccharomyces cerevisiae for improved pyruvate production.Yeast,2012,29:209-217”。
3rd technical problem to be solved by this invention is to provide the described Saccharomyces cerevisiae gene engineering bacteria of a kind of application and sends out Ferment produces the method for carboxylic acid.
Described carboxylic acid includes lactic acid, acetone acid, malic acid, Fumaric acid, succinic acid, α-ketoglutaric acid etc..
In one embodiment of the invention, said method comprising the steps of: by 30 DEG C, cultivate 24h under 220rpm Genetic engineering bacterium seed with the inoculum concentration of 5% proceed to fermentation culture based on 30 DEG C, cultivate 96h under the conditions of 220rpm.
Seed culture medium is YPD culture medium: glucose 20g/L, peptone 20g/L, yeast powder 10g/L.
Fermentation medium: without amino yeast nitrogen 1.7g/L, (NH4)2SO45g/L, glucose 20g/L, add calcium carbonate 5g/L。
Use saccharomyces cerevisiae CEN.PK2-1C when being starting strain, fermentation medium also needs add leucine 100mg/L, Tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L.
Present invention malate dehydrogenase gene RoMDH replaces PDC1, and the ethanol production of wild type Saccharomyces cerevisiae fermentation reaches To maximum 13.55 ± 1.062g/L, the ethanol production of transformation bacterial strain is that 11.09 ± 0.539g/L, relatively wild type have dropped 18.15%.On this basis, knock out SDH2 gene, after medium optimization, the dual-gene Saccharomyces cerevisiae gene engineering bacteria lacked Can accumulate succinic acid 0.698 ± 0.0285g/L, by contrast, wild type Saccharomyces cerevisiae does not accumulate succinic acid.The present invention can have The loss of effect minimizing carbon stream, and realize the accumulation of succinic acid, gained genetic engineering bacterium can be used for building other carboxylic acid superior strains.
Accompanying drawing explanation
The replacement of Fig. 1 PDC1 is expressed and is knocked out box, and size is 2724bp
Fig. 2 SDH2 knocks out box, and size is 1544bp.
Fig. 3 PDC1 gene knockout or the replacement impact on alcohol fermentation.(a) thalli growth；(b) glucose utilization； (c) acetone acid；(d) coproduct ethanol.
The impact on succinate fermentative of Fig. 4 SDH2 gene delection.(a) thalli growth；(b) glucose utilization；(c) succinum Acid；(d) coproduct ethanol.
Detailed description of the invention
High-performance liquid chromatogram determination ethanol, the method for residual sugar content: in fermentation liquid, the mensuration of ethanol and residual sugar content uses High performance liquid chromatograph (HPLC) detects.Fermentation liquor processes and supernatant is after 0.22 μm filtering with microporous membrane, utilizes RID (differential refraction detector) detects, and liquid-phase chromatography method is as follows: chromatographic column: BIO-RAD organic acid post；Column temperature: 35 DEG C；Stream Dynamic phase: 0.0275% (v/v) dilute sulfuric acid, through 0.22 μm membrane filtration degasification；Flow velocity: 0.6mL/min；The detection time: 25min；Sample size: 20 μ L.
The assay method (Bio-Rid nucleic acid instrument) of Biomass: with the 0.1M HCl suitable multiple of dilution, set wavelength as 600nm, takes 200 μ L and measures its light absorption value.
The assay method (HPLC method) of ethanol: high performance liquid chromatograph is Waters, US's product, and model is 1515, Chromatographic column is Aminex HPX-87H column (Bio-Rad).Ethanol RID detector.
Seed culture medium forms: glucose 2%, yeast extract 1%, peptone 2%, deionized water constant volume, and pH is natural, Autoclaving (115 DEG C, 20min).
Fermentation medium forms: without amino yeast nitrogen 3.4g/L, ammonium sulfate 5g/L, glucose 40g/L, distinguish on request Add leucine 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L.Add calcium carbonate 5g/L, fill liquid Amount is 40mL/250mL.
The low producing and ethanol of embodiment 1 and the structure of accumulation succinic acid saccharomyces cerevisiae engineered yeast
Pyruvic carboxylase base is substituted for the malate dehydrogenase gene RoMDH coming from Rhizopus oryzae on the basis of wild type Because of PDC1.On this basis, the key gene SDH2 in succinic acid metabolic pathway is knocked out.
1, bacterial strain and plasmid
Saccharomyces cerevisiae (S.cerevisiae) CEN.PK2-1C is purchased from Europe EUROSCARF (http://web.uni- Frankfurt.de/fb15/mikro/euroscarf/data/cen.html), its genotype is MATa；ura3-52；trp1- 289；leu2-3,112；his3Δ1；MAL2-8C；SUC2.Knock out box template pUG27 plasmid (comprising HIS3 marker gene), Cre The construction method of expression plasmid pSH47 (reclaiming for labelling) sees document " Guldener U, Heck S, Fielder T, et al.A new efficient gene disruption cassette for repeated use in budding yeast [J] .Nucleic Acids Res, 1996,24 (13): 2519-2524 " and " Guoqiang Xu, Qiang Hua, et al.Regulation of thiamine synthesis in Saccharomyces cerevisiae for improved pyruvate production.Yeast,2012,29:209-217”。
2, the structure of gene knockout box
With plasmid ST+RoMDH+TDHt+HIS as template, carry out PCR amplification with MP-F, MP-R primer, it is thus achieved that sequence is such as Shown in SEQ ID NO.5 with MDH replace PDC1 HIS knock out box, its partial sequence with on gene PDC1 coding region to be knocked out The DNA fragmentation of downstream homology, its nucleic acid electrophoresis figure is as shown in Fig. 1 swimming lane 1 and 2.With plasmid pUG27 as template, with SDH2-F, SDH2-R primer carries out PCR amplification, it is thus achieved that SDH2 knocks out box, and its nucleic acid electrophoresis figure is as shown in Fig. 2 swimming lane 1.Used draws Thing sequence such as table 1:
Table 1. is replaced with RoMDH and is knocked out PDC1 gene and knock out the primer used in SDH2
3, convert
By LiAc method, PDC1 replacement is knocked out box transformed saccharomyces cerevisiae CEN.PK2-1C, be coated with SD-HIS flat board, obtain Positive transformant carry genome and carry out PCR checking, correct transformant protects bacterium, is pdc1 single-gene gene-deleted strain CEN.PK2- 1CMP.And with plasmid pSH47, HIS labelling is removed.On this basis, SDH2 gene knockout box is transformed into saccharomyces cerevisiae CEN.PK2-1CMP, is coated with SD-HIS flat board, and the positive transformant obtained carries genome and carries out PCR checking, correct transformant Protect bacterium, be sdh2 gene-deleted strain.By transformation metabolic pathway, obtain reducing the saccharomyces cerevisiae engineered yeast of alcohol accumulation.
Embodiment 2 fermentation method produces succinic acid
By 30 DEG C, cultivate the genetic engineering bacterium seed of 24h with initial OD under 220rpm600The inoculum concentration of=0.2 proceeds to fermentation Cultivate based on 30 DEG C, cultivate 96h under the conditions of 220rpm, survey OD every certain period of time sampling, sampling 1mL is centrifugal simultaneously preserves It is used for surveying the yield of ethanol and succinic acid.
The assay method of cellular biomass:
Take a certain amount of bacteria suspension to be placed in 1.5mL EP, add the carbonic acid in the diluted hydrochloric acid dissolution bacteria suspension of appropriate 0.1M Calcium is also diluted to suitable multiple simultaneously, and vortex mixes, and with Bio-Rid nucleic acid instrument, at 600nm, colorimetric surveys OD value.
The assay method (HPLC method) of ethanol and succinic acid content:
The fermentation liquid 12000rpm of each time point is centrifuged 2min, takes supernatant 500 μ L, process by trichloroacetic acid equimultiple, Place more than 3h in 4 DEG C, settle albumen.Then, after filtering by the hydrophilic film of 0.22 μm, the sample obtained carries out HPLC analysis.
Control strain Saccharomyces cerevisiae CEN.PK2-1C accumulate when 36h ethanol reach 13.55 ± 1.062g/L, after replacing PDC1 with RoMDH, ethanol production drops to 11.09 ± 0.539g/L, relatively have dropped before transformation 18.15%.On this basis, after SDH2 knocks out, succinic acid production reaches 0.698 ± 0.0285g/L, and before knocking out, yeast Strain is not accumulate succinic acid.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be with being as the criterion that claims are defined.

Claims (9)

1. the Saccharomyces cerevisiae gene engineering bacteria producing succinic acid, it is characterised in that with saccharomyces cerevisiae as starting strain, use Fructus Mali pumilae Dehydrogenase gene substituted for Pyruvate Decarboxylase Gene PDC1 in alcohol metabolism approach, and has knocked out succinic acid metabolic pathway In succinate dehydrogenase gene SDH2.
A kind of Saccharomyces cerevisiae gene engineering bacteria producing succinic acid the most according to claim 1, it is characterised in that described Fructus Mali pumilae Dehydrogenase gene derives from Rhizopus oryzae.
A kind of Saccharomyces cerevisiae gene engineering bacteria producing succinic acid the most according to claim 1, it is characterised in that described succinum The nucleotide sequence of dehydrogenase gene SDH2 is as shown in Gene ID:1360234.
A kind of Saccharomyces cerevisiae gene engineering bacteria producing succinic acid the most according to claim 1, it is characterised in that described wine brewing Yeast is saccharomyces cerevisiae CEN.PK2-1C；Its genotype is: MATa；ura3-52；trp1-289；leu2-3,112；his3Δ1； MAL2-8C；SUC2.
5. one kind build the arbitrary described Saccharomyces cerevisiae gene engineering bacteria of claim 1-4 method, it is characterised in that by sequence Row PDC1 as shown in SEQ ID NO.5 replaces and knocks out box, and SDH2 knocks out box, transformed saccharomyces cerevisiae CEN.PK2-respectively 1C, builds the Saccharomyces cerevisiae gene engineering bacteria obtaining producing succinic acid.
6. the method applying the arbitrary described Saccharomyces cerevisiae gene engineering bacteria fermenting and producing carboxylic acid of claim 1-4, its feature Being, described carboxylic acid includes lactic acid, acetone acid, malic acid, Fumaric acid, succinic acid, α-ketoglutaric acid.
Method the most according to claim 6, it is characterised in that comprise the following steps: by 30 DEG C, cultivate 24h under 220rpm Genetic engineering bacterium seed with the inoculum concentration of 5% proceed to fermentation culture based on 30 DEG C, cultivate 96h under the conditions of 220rpm；Fermentation training Support base: without amino yeast nitrogen 1.7g/L, (NH4)2SO45g/L, glucose 20g/L, add calcium carbonate 5g/L.
8. according to the method described in claim 6 or 7, it is characterised in that using saccharomyces cerevisiae CEN.PK2-1C is starting strain Time, fermentation medium also needs add leucine 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L.
9. claim 1-4 arbitrary described Saccharomyces cerevisiae gene engineering bacteria application in building carboxylic acid superior strain.
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WO2021163780A1 (en) * 2020-02-21 2021-08-26 Braskem S.A. Production of ethanol with one or more co-products in yeast
CN114164127A (en) * 2021-11-03 2022-03-11 山东阜丰发酵有限公司 Biological process for improving succinic acid fermentation efficiency

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