CN105754963A - Method for improving yield of fumaric acid - Google Patents
Method for improving yield of fumaric acid Download PDFInfo
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- CN105754963A CN105754963A CN201610332689.7A CN201610332689A CN105754963A CN 105754963 A CN105754963 A CN 105754963A CN 201610332689 A CN201610332689 A CN 201610332689A CN 105754963 A CN105754963 A CN 105754963A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
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- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01001—Pyruvate carboxylase (6.4.1.1)
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Abstract
The invention discloses a method for improving the yield of fumaric acid and belongs to the fields of genetic engineering and fermentation engineering. The method comprises the following steps: performing codon optimization on a pyruvate carboxylase gene RoPYC of rhizopus oryzae to obtain a gene RoPYC* and then mutating a R485 site of the gene RoPYC* into proline. In comparison with the yield after the expression of a wild type pyruvate carboxylase before codon optimization, the yield is improved by 24.6 percent; 10 percent of carbon dioxide is additionally introduced when genetically-engineered bacteria of an expression mutant is fermented for 36 hours in a fermentation tank of 7L, so that the yield of the fumaric acid reaches 453.6 mg/Lm and is improved by 20 percent through comparison. According to the method disclosed by the invention, a synthetic route through which a carbon metabolic flow enters the fumaric acid from pyruvic acid is effectively enhanced, so that conditions are created for the construction of engineering yeasts to efficiently produce the fumaric acid and other dicarboxylic acid, therefore, the industrial application value and the prospect are good.
Description
Technical field
The present invention relates to a kind of method improving fumaric acid yield, belong to genetic engineering and field of fermentation engineering.
Background technology
Saccharomyces cerevisiae (Saccharomyces cerevisiae) is biological, because having as a kind of eucaryon mode trickle: hereditary information is enriched,
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 RegardedAs Safe) microorganism by FDA certification,
Fermented product has the advantages such as security and becomes fermenting and producing carboxylic acid (lactic acid, pyruvic acid, malic acid, fumaric acid, amber
Acid, KG etc.) potential the suitableeest microorganism.But, saccharomyces cerevisiae is sent out under conditions of high concentration sugar and ventilation in batches
Ferment can produce substantial amounts of ethanol, and for the fermentation with carboxylic acid as target product, a large amount of accumulation of ethanol make carbon stream substantial amounts of
Loss.By weakening the activity of the key enzyme in ethanol pathway, it is possible to reduce towards the carbon metabolism flow of ethanol, thus reduce carbon stream
Loss；On this basis, the synthesis way of objective carboxylic acid can by stoping or weaken the further metabolism of objective carboxylic acid, be built
Footpath.
The effect of pyruvate carboxylase is to convert pyruvic acid into oxaloacetic acid, and then carbon stream can be incorporated into the synthesis of objective carboxylic acid
Approach, therefore, the effect of pyruvate carboxylase can vividly describe as " biological valve ", how to strengthen carboxylase reaction,
Carbon stream is more effectively incorporated into the route of synthesis of objective carboxylic acid, becomes metabolic engineering saccharomyces cerevisiae and produce a pass of carboxylic acid
Key problem.Meanwhile, carboxylase reacts can fix carbon dioxide, thus the problem solving environment.There are some researches show thin
In born of the same parents, the height of pyruvate carboxylase activity is to malic acid, butanedioic acid, the accumulation important role of glutamic acid.Any with binary
Carboxylic acid is the fermentation by saccharomyces cerevisiae technique of target product, all will face a same problem: how to strengthen carboxylase reaction,
Promote that carbon stream is flowed to the route of synthesis of objective carboxylic acid by pyruvic acid？How to improve the activity of pyruvate carboxylase？
Summary of the invention
First purpose of the present invention is to provide a kind of carboxylase enzyme mutant.
Described mutant is on the basis of amino acid sequence parent's Rhizopus oryzae pyruvate carboxylase as shown in SEQ ID NO.1,
The arginine of the 485th is suddenlyd change and becomes proline.
In one embodiment of the invention, the gene of described parent's Rhizopus oryzae pyruvate carboxylase is encoded after codon optimized
Nucleotide sequence as shown in SEQ ID NO.2.
Second object of the present invention is to provide a kind of genetic engineering bacterium expressing described mutant.
In one embodiment of the invention, described genetic engineering bacterium is with saccharomyces cerevisiae as host.
In one embodiment of the invention, described genetic engineering bacterium be lacked simultaneously encoding pyruvate decarboxylases PDC1,
Alcohol dehydrogenase ADH1, the saccharomyces cerevisiae of gene of fumarase FUM1 are host.
In one embodiment of the invention, with pY15TEF1 as expression vector.
In one embodiment of the invention, the nucleotide sequence of described Pyruvate Decarboxylase Gene PDC1 such as Gene ID:
Shown in 850733, the nucleotide sequence of alcohol dehydrogenase gene ADH1 as shown in Gene ID:854068, fumarase base
Because the nucleotide sequence of FUM1 is as shown in Gene ID:855866.
In one embodiment of the invention, the gene order encoding described carboxylase enzyme mutant is at SEQ ID
The arginic codon of coding the 485th is revised as encoding in sequence basis shown in NO.2 the codon CCA of proline.
Third object of the present invention is to provide and a kind of utilizes the engineering bacteria fermentation expressing described mutant to produce dicarboxylic acids
Method, is to be passed through carbon dioxide during the fermentation.
Described dicarboxylic acids, including fumaric acid, malic acid, butanedioic acid, KG etc..
In one embodiment of the invention, described dicarboxylic acids is fumaric acid.
In one embodiment of the invention, the intake of carbon dioxide is 0.1vvm.
In one embodiment of the invention, by three gene deletion strains of process LAN carboxylase enzyme mutant
The seed liquor of (Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1), is seeded to equipped with 4L
In the 7L fermentation tank of fermentation medium, in 30 DEG C, cultivate under the conditions of 220rpm, initial OD600=0.2, throughput is
1vvm, speed of agitator is 300rpm, when fermentation proceeds to 36h, is passed through the carbon dioxide of 0.1vvm.
In one embodiment of the invention, fermentation medium contains: without amino yeast nitrogen 3.4g/L, ammonium sulfate 5g/L,
Glucose 40g/L, leucine 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L.As
Fruit is to ferment in shaking flask, adds calcium carbonate 5g/L, if in 7L fermentation cylinder for fermentation, with KOH and 2N of 2N
Dilute sulfuric acid control pH.
The invention still further relates to described mutant and fumaric acid that described mutant obtains is in food, feed, chemical industry, medicine system
The application of the aspect such as standby.
The mutant name of the present invention: be on the basis of amino acid sequence shown in SEQ ID NO.1, use " Original amino position
Put the amino acid of replacement " represent mutant.Such as R485P represent by the amino acid of position 485 by parent arginine (Arg,
R) proline (Pro, P) is replaced with.
Beneficial effects of the present invention: the gene of the pyruvate carboxylase of Rhizopus oryzae is carried out codon optimized by (1), is optimized
After gene RoPYC*, after being expressed, fumaric acid yield reaches 426.4 ± 8.9mg/L, ratio wild type pyruvate carboxylase
Output increased during expression 32.2%；(2) saccharomyces cerevisiae of process LAN carboxylase enzyme mutant is constructed, it is possible to effectively
Improving the yield of dicarboxylic acids, create condition for efficiently production fumaric acid and other dicarboxylic acids, having well industry should
With being worth and prospect；Lack at pyruvate decarboxylase PDC1, alcohol dehydrogenase ADH1 and fumarase FUM1 simultaneously
Expressing pyruvate carboxylase RoPYC*R485P in saccharomycete, fumaric acid yield further increases 24.6%；(3) pass through
Fermentation 36h is additionally passed through the carbon dioxide of 0.1vvm, and fumaric acid output increased can be made to 453.6mg/L, and relatively comparison is (whole
Process is not passed through carbon dioxide) improve 20%.
Accompanying drawing explanation
The codon optimized impact on fumaric acid fermentation of Fig. 1: pyruvate carboxylase gene；
Fig. 2: the RoPYC*R485P impact on fumaric acid fermentation；
Fig. 3: CO2Be passed through on engineered strain pdc1adh1fum1 ↑ RoPYC*R485P fumaric acid fermentation impact.
Detailed description of the invention
Ethanol, residual sugar content and the assay method of fumaric acid: use high performance liquid chromatograph (HPLC) detection.Fermentation liquor
Process and supernatant is after 0.22 μm filtering with microporous membrane, utilize RID (differential refraction detector) detection ethanol and residual sugar to contain
Amount, utilizes VWD (UV-detector) to detect fumaric acid content, and liquid-phase chromatography method is as follows: high performance liquid chromatograph is the U.S.
Waters Products, model is 1515, and chromatographic column is Aminex HPX-87H column (Bio-Rad).Column temperature: 35 DEG C；
Flowing 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, sets wavelength as 600nm,
Take 200 μ L and measure its light absorption value.
Seed culture medium: yeast nitrogen 1.7g/L, (NH4)2SO45g/L, glucose 20g/L, add bright ammonia the most respectively
Acid 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L, the deionization water capacity, pH is natural,
Autoclaving (115 DEG C, 20min).
Fermentation medium: without amino yeast nitrogen 3.4g/L, ammonium sulfate 5g/L, glucose 40g/L, add bright the most respectively
Propylhomoserin 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L, add calcium carbonate 5g/L, dress
Liquid measure is first 40mL/250mL (shaking flask), after change 4L/7L (fermentation tank) into.
Yeast conversion method (plasmid): (1) access in 3mL seed culture medium flat board activation saccharomyces cerevisiae list bacterium colony, 30 DEG C,
220rpm overnight incubation；(2) fill EP pipe bacterium solution, under the conditions of 4000rpm, carry out room temperature 1min be centrifuged；(3) appropriate
Sterilized water is washed, and carries out room temperature 1min and be centrifuged under the conditions of 4000rpm；(4) 1.0M LiAc 36 μ L it is sequentially added into, 10
Mg/mL ssDNA 10 μ L (ssDNA shifts to an earlier date boiling water bath 5min, places 5min on ice), plasmid 500ng, 50%PEG 240
μ L, gentle mixing；(5) 42 DEG C of heat shocks 30 minutes；(6) under the conditions of 4000rpm, carry out room temperature 1min be centrifuged, add 1mL
Sterilized water is washed；(7) 4000rpm is centrifuged 1min, stays suitable quantity of water pressure-vaccum cell, the selective flat board of coating, cultivates 3-5 for 30 DEG C
d。
The embodiment 1 RoPYC pyruvate carboxylase of source (Rhizopus oryzae) gene codon optimized and the expression in saccharomyces cerevisiae
1, the acquisition of the codon optimized rear gene of RoPYC
The RoPYC nucleotide sequence of Rhizopus oryzae will be derived from, be submitted to the sequence of codon optimized website (http://www.jcat.de/)
In row frame, then selecting expressive host is saccharomyces cerevisiae, after submitting available codon optimized after gene order, by this sequence
The unnamed gene that row are corresponding is RoPYC*.
The sequence of RoPYC* being delivered to Shanghai bioengineering limited company and carries out gene chemical synthesis, the gene after synthesis is connected to T
On carrier.On this basis, we carry out PCR amplification with RoPYC*+T load as masterplate, and the primer is as shown in table 1.
Table 1 expands the primer of RoPYC* gene
The PCR primer obtained, carries out double digestion with SpeI and SalI, then with through the plasmid pY15TEF1 of same double digestion
Being attached, construction method sees (Xu et al.Fumaric acid production in Saccharomyces cerevisiae by
Silico aided metabolic engineering, 2012), converting large intestine competent cell, coating is added with the LB of ampicillin
Flat board.Bacterium colony PCR verifies the transformant grown and proposes plasmid enzyme restriction checking, carries out correct transformant protecting bacterium and checking order,
The named pY15TEF1-RoPYC* of plasmid designations.
2, RoPYC* expression in saccharomyces cerevisiae and the impact on fumaric acid fermentation
Build three gene deletion strains Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1 (structures
Construction method refers to the patent application of Application No. 201410340560.1), by the recombinant plasmid of above-mentioned structure
PY15TEF1-RoPYC* is transformed into three gene deletion strains Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △
ADH1 △ FUM1, is the RoPYC* of SEQ ID NO.1 to process LAN amino acid sequence in this bacterium, and in shaking flask condition
Lower investigated RoPYC gene codon optimize before and after on fumaric acid fermentation impact, result is as shown in Figure 1.
Embodiment 2 RoPYC* rite-directed mutagenesis and expression
Carry out rite-directed mutagenesis with PCR method, N315F, R485P, N1078F site of RoPYC* carried out rite-directed mutagenesis,
F and R with pY15TEF1-RoPYC plasmid as template, containing mutational site as primer, Takara company high-fidelity enzyme
PrimeSTAR GXL carries out PCR and amplifies whole plasmid.The system of being digested comprises 1 μ L PCR primer and 1 μ LDpn I enzyme,
Cumulative volume 20 μ L, 37 DEG C are digested overnight.Digestion products carries out fragment purification.Take purified product 5 μ L and convert 30 μ L competence
Cell Trans1-T1, is coated with LA flat board, the transformant grown inoculation LA culture medium, and upgrading grain sends to Shanghai raw work order-checking.
For RoPYC*R485P sudden change primers F (Pro), (sequence is respectively such as SEQ ID NO.5, SEQ ID NO.6 for R (Pro)
Shown in), as shown in table 2.
Table 2 rite-directed mutagenesis primer
Note: lowercase for mutating alkali yl, the amino acid of they correspondences is on right side.
Select the mutant that order-checking is correct, at three gene deletion strains Saccharomyces cerevisiae
This mutant of process LAN in CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1, has obtained series of genes engineering bacteria.By gene
Engineering bacterial strain, under the conditions of same, carried out fermenting experiment, has compared through codon optimized N315F, R485P, N1078F
Saccharomyces cerevisiae is produced the impact of fumaric acid by the rite-directed mutagenesis in three sites, and result, as in figure 2 it is shown, result shows, is wherein expressed
The engineering bacteria yield of R485P has reached 465.5 ± 6.5mg/L, ratio the 373.5 ± 13.4 of the engineering bacteria expressing unmutated RoPYC*
Mg/L improves 24.6%.
Embodiment 3 carbon dioxide be passed through on fumaric acid fermentation impact
That has investigated carbon dioxide on 7L fermentation tank level is passed through the impact on fumaric acid fermentation, and result is as shown in Figure 3.
Condition of culture: by 30 DEG C, the genetic engineering bacterium seed of cultivating 24h under 220rpm proceed to fermentation medium so that it is initial
OD600=0.2, and cultivate 96h in 30 DEG C, throughput is 1vvm, and speed of agitator is 300rpm, when fermentation proceeds to 36h, logical
Enter the carbon dioxide of 0.1vvm.
As seen from Figure 3, fermentation proceeds to 36h, after being passed through the carbon dioxide of 0.1vvm, and compares (whole sweat
The most it is not passed through carbon dioxide) to compare, being gradually increased of fumaric acid yield, when fermentation proceeds to 120h, fumaric acid yield reaches
To 453.6mg/L, relatively comparison improves 25.7%.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to 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
What claims were defined is as the criterion.
Claims (9)
1. a carboxylase enzyme mutant, it is characterised in that described mutant is at amino acid sequence such as SEQ ID NO.1
On the basis of shown pyruvate carboxylase, the arginine of the 485th is suddenlyd change and becomes proline.
2. the gene of the coding a kind of carboxylase enzyme mutant described in claim 1, it is characterised in that at SEQ ID NO.2
The arginic codon of coding the 485th is revised as encoding in shown sequence basis the codon CCA of proline.
3. the genetic engineering bacterium expressing mutant described in claim 1, it is characterised in that to have lacked encoding pyruvate simultaneously
Acid decarboxylase PDC1, alcohol dehydrogenase ADH1, the saccharomyces cerevisiae of gene of fumarase FUM1 are host.
Genetic engineering bacterium the most according to claim 3, it is characterised in that with pY15TEF1 as expression vector, expresses power
Profit requires the gene described in 2.
5. the engineering bacteria fermentation that a kind utilizes described in claim 3 or 4 produces the method for dicarboxylic acids, it is characterised in that
It is to be passed through carbon dioxide during the fermentation.
Method the most according to claim 5, it is characterised in that described dicarboxylic acids includes fumaric acid, malic acid, amber
Amber acid, KG.
Method the most according to claim 5, it is characterised in that described dicarboxylic acids is fumaric acid, by genetic engineering bacterium
Seed liquor, be seeded to equipped with in the 7L fermentation tank of 4L fermentation medium, in 30 DEG C, cultivate under the conditions of 220rpm,
Initial OD600=0.2, throughput is 1vvm, and speed of agitator is 300rpm, when fermentation proceeds to 36h, is passed through 0.1vvm
Carbon dioxide.
Method the most according to claim 7, it is characterised in that fermentation medium contains: without amino yeast nitrogen 3.4
G/L, ammonium sulfate 5g/L, glucose 40g/L, leucine 100mg/L, tryptophan 20mg/L, histidine 20mg/L,
Uracil 20mg/L.
Method the most according to claim 8, it is characterised in that in shaking flask during fermentation, adds calcium carbonate 5g/L；
When 7L fermentation cylinder for fermentation, control pH with the dilute sulfuric acid of KOH and 2N of 2N.
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Cited By (2)
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| CN108641970A (en) * | 2018-04-16 | 2018-10-12 | 江南大学 | It is a kind of to reduce the aspergillus oryzae of byproducts build-up and its application in malic acid building-up process |
| CN109609486A (en) * | 2019-01-30 | 2019-04-12 | 浙江华睿生物技术有限公司 | A kind of fumaric acid enzyme mutant and its application |
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