CN111635868A - Method for constructing glucaric acid synthetic pathway in saccharomyces cerevisiae cell - Google Patents

Method for constructing glucaric acid synthetic pathway in saccharomyces cerevisiae cell Download PDF

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CN111635868A
CN111635868A CN202010581329.7A CN202010581329A CN111635868A CN 111635868 A CN111635868 A CN 111635868A CN 202010581329 A CN202010581329 A CN 202010581329A CN 111635868 A CN111635868 A CN 111635868A
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saccharomyces cerevisiae
glucaric acid
inositol
udh
miox4
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邓禹
赵运英
徐驰
李�杰
郗洋铭
皮燕
张居旺
许锦可
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Jiangnan University
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Abstract

The invention discloses a method for constructing a glucaric acid synthesis way in a saccharomyces cerevisiae cell, belonging to the technical field of biological engineering. The saccharomyces cerevisiae BY4741 opi1 deletion strain is used as an original strain, and the spatial distance between Inm1 and MIOX4-Udh is shortened through an affibody scaffold, so that the path efficiency is improved. The invention replaces the promoter of the inositol monophosphatase gene INM1 with PTDH3Adding a ZTag label at the N end, then connecting inositol oxygenase MIOX4 and uronate dehydrogenase Udh through a linker, and adding Z at the N end of the fusion proteinIgATag of Anti-ZTag-Anti-ZIgAIntegrated together at the Delta site of the opi deletion genome, by adding glucose twoThe screening of acid yield obtains the glucaric acid high-yield strain, so that the glucaric acid yield of the constructed engineering bacteria is improved by 75 percent.

Description

Method for constructing glucaric acid synthetic pathway in saccharomyces cerevisiae cell
Technical Field
The invention relates to a method for constructing a glucaric acid synthetic pathway in a saccharomyces cerevisiae cell, belonging to the technical field of biological engineering.
Background
Glucaric Acid (GA) is a naturally occurring dibasic acid and is widely used in chemical materials, medical care and food additives, such as synthetic detergents, corrosion inhibitors, adipic acid, ester and polyamide derivatives, etc. 1 or in cholesterol lowering, diabetes treating, tumor treating, etc. Thus, glucaric acid is considered to be one of the "top value-added chemicals from biomass". The method for synthesizing glucaric acid by a glucose chemical oxidation method has the problems of non-selectivity, high cost, low yield, high temperature requirement, many byproducts which are not beneficial to separation and the like. The total biological synthesis of glucaric acid reported so far is mainly carried out in escherichia coli and some fungi, and the yield of the glucaric acid synthesized in escherichia coli is limited by a plurality of factors. Saccharomyces cerevisiae has higher industrial application value and advantages than Escherichia coli, and has been widely used for research of organic acid production. Therefore, the method for biologically synthesizing the glucaric acid by using the saccharomyces cerevisiae has good application prospect.
In recent years, research teams at home and abroad utilize synthetic biology ideas to realize various methods for producing glucaric acid by fermenting microorganisms by taking inositol as a substrate. A glucaric acid synthesis pathway constructed by expressing inositol-1-phosphate synthase Ino1 of Saccharomyces cerevisiae, mouse inositol oxidase MIOX (myo-inositol oxydenase) and uronic acid dehydrogenase Udh of Pseudomonas syringae in Escherichia coli with a yield of 1 g/L; subsequently, after the specific activity of MIOX is improved by using scaffold protein, the yield of glucaric acid is improved to 2.5 g/L. After the stability and the enzyme activity of the MIOX are improved by respectively utilizing protein fusion and directed evolution technologies, the recombinant escherichia coli can synthesize 4.85g/L glucaric acid by utilizing 10.8g/L inositol. The knocking-out of a glucose-6-phosphate dehydrogenase gene zwf in a pentose phosphate pathway of escherichia coli, a glucose phosphoglucomutase gene pgm in a glycogen synthesis pathway and a phosphofructokinase gene pfk in a glycolysis pathway can promote cells to accumulate glucose-6-phosphate and improve the yield of glucaric acid. After co-expressing genes such as cscB, cscA, cscK, ino1, miox, udh and suhB in Escherichia coli E.coli BL21(DE3), the Escherichia coli can synthesize glucaric acid by using sucrose, and through further metabolic modification, the yield of the glucaric acid reaches 1.42g/L and is 0.142g/g of sucrose.
The saccharomyces cerevisiae has the characteristics of strong acid resistance, low temperature resistance, capability of fermenting at low pH, no phage infection, suitability for large-scale fermentation, easiness in separation, high stress resistance and the like, and can be used as single-cell protein for feeds, foods and the like, so that the saccharomyces cerevisiae is widely used for the research of organic acid production, such as rho-hydroxybenzoic acid, rho-aminobenzoic acid, rho-hydroxyphenylacrylic acid, artemisinic acid and the like. The biggest advantage of synthesizing glucaric acid by saccharomyces cerevisiae metabolic engineering is that it can utilize glucose-6-phosphate to synthesize inositol (fig. 1), and the Prather group introduces exogenous MIOX and UDH genes into saccharomyces cerevisiae for heterologous expression, thus realizing the glucaric acid production in saccharomyces cerevisiae by glucose and exogenous inositol, but the yield is only 1.6g/L under the upper tank culture condition. Previous studies found that key enzymes in the glucaric acid synthesis pathway were not expressed in high amounts and activities, and that these factors limited the synthesis of glucaric acid. Therefore, how to enhance the efficiency of the glucaric acid synthesis pathway is a key problem for further improving the glucaric acid production capability of the engineering bacteria.
Disclosure of Invention
The invention aims to improve a saccharomyces cerevisiae gene engineering bacterium for synthesizing D-glucaric acid, which takes opi1 deletion strain of saccharomyces cerevisiae BY4741 as an initial strain, integrates inositol monophosphoryl enzyme gene INM1 on Delta site of genome, and fuses and connects coding genes of inositol oxygenase MIOX4 and uronic acid dehydrogenase Udh and a gene coding an affinity scaffold protein through a linker; the myo-inositol oxygenase MIOX4 was derived from Arabidopsis thaliana (Arabidopsis thaliana) the uronate dehydrogenase Udh was derived from pseudomonas syringae.
In one embodiment, the inositol monophosphate gene INM1 is added with a Ztag tag at the N-terminus and the promoter of the gene INM1 is replaced by P by homologous recombinationTDH3A promoter.
In one embodiment, the myo-inositol oxygenases MIOX4 and Udh are produced by a linker: EAAAAKEAAAAKEAAAAK for fusion expression.
In one embodiment, the N-terminus of the gene fragment MIOX4-Udh fusing myo-inositol oxygenase and uronate dehydrogenase contains an IgA tag.
In one embodiment, the nucleotide sequence encoding the IgA tag is set forth in SEQ ID NO. 4.
In one embodiment, the opi-deleted strain genome further incorporates the DNA fragments ZIgA-MIOX4-udh and Anti-ZTag-Anti-ZIgA at the Delta site.
In one embodiment, said PTDH3The nucleotide sequences of the ZTag and the INM1 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
In one embodiment, Z isIgAThe nucleotide sequence of (A) is shown as SEQ ID NO. 4; Anti-ZTag-Anti-ZIgAThe nucleotide sequence of (A) is shown in SEQ ID NO. 5.
In one embodiment, the nucleotide sequence of Delta1 is shown in SEQ ID NO. 6; the nucleotide sequence of Delta2 is shown in SEQ ID NO. 7.
The second purpose of the invention is to provide a method for constructing the saccharomyces cerevisiae gene engineering bacteria for synthesizing the D-glucaric acid, which comprises the following steps:
(1) constructing the integration frame MIOX4-udh of inositol oxygenase and uronate dehydrogenase; constructing an integration frame Anti-ZTAQ-Anti-ZigA;
(2) integrating the integration frame of the step (1) into the Delta site of the opi deletion strain genome;
(3) transformants with improved yields of glucaric acid relative to the starting strain were screened.
In one embodiment, the Anti-ZTag-Anti-ZIgAThe nucleotide sequence of (A) is shown in SEQ ID NO. 5.
The third method of the invention is to provide a method for fermenting glucaric acid, which is to inoculate the saccharomyces cerevisiae engineering bacteria to a culture medium containing glucose and inositol for fermentation.
In one embodiment, the carbon source of the fermentation medium is glucose at an initial concentration of 20 g/L.
In one embodiment, the fermentation is carried out by inoculating the seed solution into the fermentation medium at an inoculum size of 2%, and culturing at 30 ℃ and 250rpm for 160-240 hours.
In one embodiment, the seed solution is obtained by inoculating a single colony in a YPD medium, culturing at 28-30 ℃ and 200-250 rpm for 16-20 h.
In one embodiment, the fermentation is further supplemented with glucose at 24 hours and 48 hours, respectively.
The invention also claims the application of the saccharomyces cerevisiae gene engineering bacteria or the method in the aspect of preparing glucaric acid or derivative products thereof.
Has the advantages that: in order to improve the efficiency of synthesizing glucaric acid by using glucose and inositol for saccharomyces cerevisiae cells, MIOX4 and Udh are subjected to fusion expression through a linker, then an IgA tag is added at the N end of MIOX4-Udh, a ZTaq tag is added at the N end of inositol monophosphatase Inm1 for overexpression, finally the space distance of a glucaric acid synthesis way is shortened by introducing Anti-ZTag-Anti-ZigA fusion protein, and saccharomyces cerevisiae engineering bacteria for efficiently synthesizing glucaric acid are obtained by screening of integrated copy number. Under the same fermentation condition, the yield of the glucaric acid of the newly constructed engineering bacteria is improved by 75 percent compared with the earlier research result.
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FIG. 1 is a schematic diagram of a construction strategy of the engineering bacteria of the present invention.
FIG. 2 is an agarose gel electrophoresis test of the promoter replacement of the INM1 gene and the ZTaq integrant; wherein,panel A shows LEU2 and PTDH3Detecting the electrophoresis detection result of the PCR product; b is LEU2-PTDH3Detecting the electrophoresis detection result of the PCR product; the C picture is LEU2-PTDH3-results of electrophoretic detection of PCR products of Ztaq; and the D picture is the result of the genotype PCR detection of the transformant.
FIG. 3 is Delta1-TADH1-Anti-ZTaq-anti-ZIgA-PGPD1-HIS3-PTEF1And PTEF1-ZIgA-MIOX4-udh-TCYC1Electrophoretic detection of the-DELTA 2 fusion fragment, wherein lanes 1-3 are PTEF1-ZIgA-MIOX4-udh-TCYC1-Delta2 fusion fragment, lane 4 Delta1-TADH1-Anti-ZTaq-anti-ZIgA-PGPD1-HIS3-PTEF1A fusion fragment.
FIG. 4 shows the results of fermentation in 20 retests of transformants with higher glucaric acid production.
FIG. 5 shows the results of shake flask fermentation of engineering bacteria Bga-5.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
YPD medium comprises peptone 20g, glucose 20g and yeast extract 10g per liter, and yeast extract 1 × 10 after constant volume5Pa sterilizing for 20 min. 20g of agar powder per liter of solid medium was added.
The fermentation medium comprises the following components: YPD medium composition was supplemented with 10.8g/L inositol.
SD-HIS medium: 0.67% Yeast nitrate base, 2% glucose, 1 Xamino acid mixture, 1 Xuracil, 1 Xleucine, autoclaving for use. 100ml of solid culture medium is prepared, and 2.0g of agar powder is added.
SD-LEU medium: 0.67% Yeast nitrate base, 2% glucose, 1 × amino acid mixture, 1 × uracil, 1 × histidine, autoclaving for use. 100ml of solid medium was prepared, and 2.0g of agar was added thereto.
TABLE 11L 10 Xamino acid dosage table
Figure BDA0002552422550000041
Table 2 shows the primers used in the examples
Figure BDA0002552422550000042
Figure BDA0002552422550000051
Example 1: replacement of the INM1 Gene promoter and integration of the ZTaq
To obtain LEU2-PTDH3The ZTaq integration frame is firstly amplified by pHAC181 plasmid template by using primers LEU2-2F and LEU2-2R to obtain a promoter, ORF and terminator fragment of LEU2 gene, and the nucleotide sequence of LEU2 gene is shown as SEQ ID NO. 8. P is obtained BY taking a Saccharomyces cerevisiae BY4741 genome as a template and amplifying BY using a primer PTDH3-F/PTDH3-RTDH3Fragments (fig. 2A); with LEU2 and PTDH3The fragment is taken as a template, and fusion PCR amplification is carried out by a primer LEU2-2F/PTDH3-R to obtain LEU2-PTDH3Fragments (fig. 2B); then, using the synthesized pUC57-ZTAQ plasmid containing the ZTAQ fragment (the synthesized ZTAQ fragment is inserted into the Sma I site of pUC57 plasmid) as a template, amplifying with primers ZTAQ-2F and ZTAQ-2R to obtain a ZTAQ fragment, and finally, LEU2-PTDH3And the two DNA fragments of ZTAQ are fused by primers LEU2-2F and ZTAQ-2R to obtain LEU2-PTDH3ZTAQ (FIG. 2C). Mixing LEU2-PTDH3The ZTAQ fragment was transformed into opi 1-deleted strain, SD-LEU plate was coated, and PCR verification was performed on the extracted genome after the transformant was cultured. The detection results are shown in fig. 2D. The results show that the correct transformants amplified a band of 800 bp.
Example 2: integration of the Delta site of the MIOX4-Udh fusion protein and Anti-ZTAQ-Anti-ZIgA
(1)Delta1-TADH1-Anti-ZTaq-anti-ZIgA-PGPD1-HIS3-PTEF1Amplification of
The genome of Saccharomyces cerevisiae Bga-001 (disclosed in the patent application with publication No. CN 108220176A) was used as a template, and primers were usedDelta1-F and TADH1-R amplification to obtain Delta1-TADH1(572bp) fragment; synthesizing an Anti-ZTAQ-Anti-ZIgA (420bp) fragment by using a synthesized pUC57-Anti-ZTAQ-Anti-ZigA plasmid (inserting a synthesized Anti-ZTAQ-Anti-ZigA fragment shown in SEQ ID NO.5 into the Sma I site of a pUC57 plasmid) containing the Anti-ZTAQ-Anti-ZigA fragment as a template and using primers ZZ-F and ZZ-R; using Bga-001 genome as template, and using PHP-F and PHP-R primer to amplify and obtain PGPD1-HIS3-PTEF1(2200bp) fragment. Finally, the three fragments are subjected to fusion PCR by using primers Delta1-F and PHP-F to obtain Delta1-TADH1-Anti-ZTaq-anti-ZIgA-PGPD1-HIS3-PTEF1And (3) fragment.
(2)PTEF1Amplification of ZIgA-MIOX4-udh-TCYC1-Delta2
Using Bga-001 genome as template and primer PHP-F and PTEF1Amplification of F to obtain PTEF1A fragment; using synthesized pUC57-ZIgA plasmid containing ZIgA fragment (the synthesized ZIgA fragment is inserted into the Sma I site of pUC57 plasmid) as a template, and synthesizing ZIgA (210bp) fragment by using primers ZIgA-F and ZIgA-R; amplifying P by using Bga-001 genome as template and MIOX4-LF/MIOX4-LR as primerTEF1A MIOX4 fragment, a UDH-Delta2 fragment is amplified by using UDH-F11 and UDH-R, and then the two fragments are subjected to fusion PCR by using MIOX4-LF and UDH-R to obtain a MIOX4-UDH-TCYC1-Delta2(2400bp) fragment. Finally, the three fragments are subjected to PCR fusion by using primers ZIgA-F and UDH-R to obtain PTEF1-ZIgAMIOX4-udh-TCYC1-Delta2 fragment.
Finally, the 2 fragments Delta1-T are converted by lithium acetateADH1-Anti-ZTaq-anti-ZIgA-PGPD1-HIS3-PTEF1Fragment and PTEF1-ZIgA-MIOX4-udh-TCYC1The fragment-Delta 2 was incorporated into the strain obtained in example 1, spread on SD-HIS plates, cultured and then fermented in a 5mL tube to preliminarily screen the yield of glucaric acid in the transformant. The results of the yield test of the 20 transformants with the highest glucaric acid among nearly 1000 transformants were selected in total are shown in FIG. 4. From the figure, it can be seen that the yield of glucaric acid of transformant No.5 was the highest, 1.42 times that of control Bga-001.
Example 3: shake flask fermentation synthesis of glucaric acid by No.5 engineering bacteria constructed in example 2
1. Preparing a seed solution: the strain preserved by the glycerol is streaked on a plate, a single colony is selected and inoculated in 10mL YPD medium, the YPD medium is cultured at 30 ℃ and 250rpm for 20h by a shaking table, and the seed solution is obtained.
2. Fermentation conditions are as follows: the seed solution was inoculated at an inoculum size of 2% into a 250mL Erlenmeyer flask containing 50mL of liquid medium, the glucose concentration in the fermentation medium was 20g/L, and 10.8g/L inositol was added. Culturing at 30 deg.C and 250rpm of shaking table for 200 hr.
3. And (3) product detection: taking 1mL of fermentation liquor, centrifuging at high speed for 10min, retaining supernatant, and filtering with a filter membrane of 0.22 μm standard to obtain filtrate as a sample. The detection can be carried out by liquid chromatography-mass spectrometry (LC-MS) or High Performance Liquid Chromatography (HPLC), 5mM sulfuric acid is used as a mobile phase, a chromatographic column is an organic acid column (Aninex Hpx-87H ion exchange column), the column temperature is 55 ℃, a differential refraction detector is adopted, the sample injection amount is 30 mu L, and the flow rate is 0.6 mL/min.
The fermentation result (figure 5) shows that the yield of the glucaric acid produced by the Bga-001 engineering bacteria fermented for 248h is 3.5g/L and the yield of the glucaric acid produced by the newly screened No.5 engineering bacteria is 6.2g/L under the same fermentation condition; compared with the control bacterium Bga-001 engineering bacterium, the yield is improved by 75 percent.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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atgaccattg atctagcttc tatcgagaaa ttcctctgtg aactggctac tgaaaaagta 60
ggaccaatca tcaaatccaa gtccggcact caaaaggatt atgacttgaa gactggctcc 120
aggagcgttg atattgtaac tgctatcgat aaacaggtcg aaaagttaat ttgggaatcg 180
gtaaaaaccc aatatccaac tttcaagttt attggagagg aaagttatgt gaaaggggag 240
accgtgatta ctgatgatcc tacctttatt attgatccaa ttgatgggac tacaaacttt 300
gttcatgatt tcccatttag ctgtacctct cttgggctca cagtaaacaa agagcccgta 360
gtaggcgtta tatataatcc tcacattaat cttctggtat ccgcttctaa aggaaatggg 420
atgagagtta acaacaagga ctatgactat aaatcaaaat tggaatctat gggttctcta 480
atactaaata aatccgtagt ggcattacag ccaggttccg ctagagaggg aaagaatttt 540
cagacaaaaa tggccacata tgaaaaatta ctatcatgtg attacggttt tgttcatgga 600
ttcagaaatt taggatcatc tgcaatgaca atggcatata ttgctatggg gtaccttgat 660
agttattggg atggtggttg ctattcgtgg gacgtgtgtg ctggatggtg tattttgaaa 720
gaagtgggcg gtcgtgtagt aggtgccaat ccaggtgaat ggagtattga tgtcgacaat 780
aggacatatt tggctgtgag gggaacaatt aataacgaaa gtgacgaaca aacaaaatat 840
atcacagact tttggaactg tgttgatggc catttgaaat atgactga 888
<210>4
<211>207
<212>DNA
<213> Artificial sequence
<400>4
atggtagaca acaaattcaa caaagaactg ggttgggcga cctgggagat cttcaactta 60
cctaacttaa acggtgtgca agtgaaggcc ttcatcgata gtttacggga tgacccaagc 120
caaagcgcta acttgctagc agaagctaaa aagctaaatg atgctcaggc gccgaaatct 180
tcctctagtg gttcttcctc ttctgga 207
<210>5
<211>414
<212>DNA
<213> Artificial sequence
<400>5
atggtagaca acaaattcaa caaagaaaga gtgattgcga taggtgagat catgcggtta 60
cctaacttaa acagtctcca agtggtggcc ttcatcaata gtttacggga tgacccaagc 120
caaagcgcta acttgctagc agaagctaaa aagctaaatg atgctcaggc gccgaaatcg 180
tccagctccg gcagctcgtc tagcggcagc agctcttccg gttccagttc cagcggcgta 240
gacaacaaat tcaacaaaga agcacaaacg gcgggggtgg agatcatgga gttacctaac 300
ttaaacaccc ggcaactgct ggccttcatc cagagtttac gagatgaccc aagccaaagc 360
gctaacttgc tagcagaagc taaaaagcta aatgatgctc aggcgccgaa ataa 414
<210>6
<211>258
<212>DNA
<213>Saccharomyces cerevisiae
<400>6
ggctggcaac taatagggac actaccaata tattatcata tacggtgtta gacgatgaca 60
taagatacga ggaactgtca tcgaagttag aggaagctga aatgcaagga ttgataatgt 120
aataggataa tgaaacatat aaaacggaat gaggaataat cgtaatatta gtatatagag 180
ataaagattc cattttgagg attcctatat cctcgaggag aacttctagt atattctgta 240
tacctgatat tatagcct 258
<210>7
<211>222
<212>DNA
<213>Saccharomyces cerevisiae
<400>7
tgttggaata aaaatcaact atcatctact aactagtatt tacgttacta gtatattatc 60
atatacggtg ttagaagatg acgcaaatga tgagaaatag tcatctaaat tagtggaagc 120
tgaaacgcaa ggattgataa tgtaatagga tcaatgaata ttaacatata aaatgatgat 180
aataatattt atagaattgt gtagaattgc agattccctt tt 222
<210>8
<211>1104
<212>DNA
<213> Artificial sequence
<400>8
atgtctgccc ctatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt 60
caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat 120
gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtc 180
ccacttccag atgaggcgct ggaagcctcc aagaaggttg atgccgtttt gttaggtgct 240
gtgggtggtc ctaaatgggg tacaggtagt gttagacctg aacaaggttt actaaaaatc 300
cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt 360
ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga 420
gaattagtgg gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct 480
tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc 540
atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatgttttg 600
gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga atttcctaca 660
ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc 720
cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc 780
tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac 840
aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag 900
aataaggttg accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg 960
aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt 1020
atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc 1080
gaagaagtta agaaaatcct tgct 1104

Claims (10)

1. A Saccharomyces cerevisiae genetic engineering bacterium for synthesizing D-glucaric acid is characterized in that an opi1 deletion strain of Saccharomyces cerevisiae BY4741 is used as an initial strain, and encoding genes of an inositol monophosphate gene INM1, an inositol oxygenase MIOX4 and uronic acid dehydrogenase Udh which are connected BY linker fusion are integrated on a Delta site of a genome, and the encoding genes contain genes for encoding scaffold proteins of an affibody; the myo-inositol oxygenase MIOX4 is derived from arabidopsis; the uronate dehydrogenase Udh is derived from Pseudomonas syringae.
2. The Saccharomyces cerevisiae genetically engineered bacterium of claim 1, wherein the N-terminus of the inositol monophosphatase gene INM1 hasHas a ZTag label; the promoter of the gene INM1 is PTDH3A promoter.
3. The genetically engineered saccharomyces cerevisiae strain of claim 1 or 2, wherein the N-terminus of the gene segment fusing myo-inositol oxygenase and uronate dehydrogenase contains an IgA tag.
4. The genetically engineered bacterium of claim 3, wherein the myo-inositol oxygenases MIOX4 and Udh are produced by a linker: EAAAAKEAAAAKEAAAAK fusion expression.
5. A method for constructing the saccharomyces cerevisiae gene engineering bacteria of any one of claims 1 to 4, which is characterized by comprising the following steps:
(1) constructing the integration frame MIOX4-udh of inositol oxygenase and uronate dehydrogenase; constructing an integration frame Anti-ZTAQ-Anti-ZigA;
(2) integrating the integration frame of the step (1) into the Delta site of the opi deletion strain genome;
(3) transformants with improved yields of glucaric acid relative to the starting strain were screened.
6. The method of claim 5, wherein said Anti-Ztag-Anti-ZIgAThe nucleotide sequence of (A) is shown in SEQ ID NO. 5.
7. The method according to claim 6, wherein step (3) is performed to select transformants having a glucaric acid production increased by 1-fold or more compared to a control.
8. A method for producing glucaric acid, characterized in that the saccharomyces cerevisiae engineering bacteria of any claim 1 to 4 are inoculated into a culture medium containing glucose and inositol and fermented for at least 120h at 28-30 ℃.
9. The method of claim 8, wherein the fermentation is further supplemented with at least one glucose feed during the 24 th to 48 th hours of the fermentation.
10. Use of the genetically engineered Saccharomyces cerevisiae according to any of claims 1 to 4 or the method according to any of claims 8 to 9 for the preparation of glucaric acid or derivatives thereof.
CN202010581329.7A 2020-06-23 2020-06-23 Method for constructing glucaric acid synthetic pathway in saccharomyces cerevisiae cell Pending CN111635868A (en)

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CN104911117A (en) * 2015-06-25 2015-09-16 江南大学 Method for producing glucaric acid by improving yeast fermentation by use of fusion expression
CN107164255A (en) * 2017-04-26 2017-09-15 江南大学 A kind of method for building recombinant Saccharomyces cerevisiae fermenting and producing glucaric acid
CN108220176A (en) * 2018-01-30 2018-06-29 江南大学 A kind of method for improving saccharomyces cerevisiae engineered yeast strain fermenting and producing glucaric acid
CN111019852A (en) * 2019-12-31 2020-04-17 江南大学 Method for improving efficiency of synthesizing glucaric acid by saccharomyces cerevisiae engineering strain

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104911117A (en) * 2015-06-25 2015-09-16 江南大学 Method for producing glucaric acid by improving yeast fermentation by use of fusion expression
CN107164255A (en) * 2017-04-26 2017-09-15 江南大学 A kind of method for building recombinant Saccharomyces cerevisiae fermenting and producing glucaric acid
CN108220176A (en) * 2018-01-30 2018-06-29 江南大学 A kind of method for improving saccharomyces cerevisiae engineered yeast strain fermenting and producing glucaric acid
CN111019852A (en) * 2019-12-31 2020-04-17 江南大学 Method for improving efficiency of synthesizing glucaric acid by saccharomyces cerevisiae engineering strain

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Application publication date: 20200908