CN113122486B

CN113122486B - Method for total biosynthesis of malonic acid

Info

Publication number: CN113122486B
Application number: CN201911420851.0A
Authority: CN
Inventors: 邓禹; 赵运英; 李诗韵; 李国辉; 毛银; 周胜虎
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-06-13
Anticipated expiration: 2039-12-31
Also published as: CN113122486A

Abstract

The invention discloses a method for total biosynthesis of malonic acid, and belongs to the technical field of bioengineering. The invention takes escherichia coli BL21 (DE 3) as a host, and the modules overexpress phosphoenolpyruvate carboxylase gene (ppc) from escherichia coli, succinate dehydrogenase gene (sdhC), succinic semialdehyde dehydrogenase gene (yne 1), asparaginase gene (aspa), heterologous gene aspartic acid-alpha-dehydrogenase gene (panD) from corynebacterium glutamicum, heterologous gene beta-alanine pyruvate transaminase gene (pa 0123) from pseudomonas aeruginosa, thus constructing a total biosynthesis path of malonic acid and enabling engineering bacteria to successfully accumulate malonic acid. The biological preparation of the malonic acid and the precursor thereof has the advantages of less pollution, high product quality and the like, and has great development prospect.

Description

Method for total biosynthesis of malonic acid

Technical Field

The invention relates to a method for total biosynthesis of malonic acid, and belongs to the field of bioengineering.

Background

Malonic acid (malonic acid; propanedioic acid; propane diacid) is also known as carrot acid, apple acid or beet acid. The molecular structure has two functional groups, namely active methylene and carboxyl, so that the molecular structure can participate in various chemical reactions, and is an important organic synthesis intermediate. It is mainly used in the aspects of perfume, adhesive, resin additive, medical intermediate, electroplating polishing agent, explosion control agent, heat welding fluxing additive, etc.

At present, the hydrolysis method of cyanoacetic acid and malonate is commonly used in industry to prepare malonic acid. However, the hydrolysis of cyanoacetic acid uses NaCN, while cyanide ions are extremely toxic, have great environmental hazard and have complex reaction process. The malonic ester is hydrolyzed mainly by directly hydrolyzing dimethyl (ethyl) malonate, wherein the malonic ester is also prepared into cyanoacetic acid through chloroacetic acid neutralization, cyanidation and other reactions, and then the malonic ester is produced by esterification, so that the method has great environmental hazard and complex reaction process. And the malonic acid obtained by the two methods needs to be subjected to complex and complicated purification procedures, so that the industrial production is limited.

In order to solve the above problems, people focus their eyes on the roads where malonic acid is biosynthesized. However, the study of the bio-production of malonic acid has progressed slowly due to lack of knowledge of suitable enzymes and metabolic pathways. Malonate semialdehyde is an important precursor of malonate, but this conversion is difficult due to the deletion of malonate semialdehyde dehydrogenase, and thus malonate bio-production proceeds slowly.

Disclosure of Invention

In order to solve the above problems, the present invention provides a recombinant E.coli producing malonic acid, which is based on E.coli BL21 (DE 3), and in which phosphoenolpyruvate carboxylase gene (ppc), succinate dehydrogenase gene (sdhC), succinic semialdehyde dehydrogenase gene (yne 1), asparaginase gene (aspA), heterologous gene aspartic acid-alpha-dehydrogenase gene (panD) from Corynebacterium glutamicum, and heterologous gene beta-alanine pyruvate transaminase gene (pa 0123) from Pseudomonas aeruginosa are expressed in a split manner.

In one embodiment of the invention, the split-module overexpression is to fuse and express genes yne1 and pa0123, fusion and express genes aspa and panD, and fusion and express genes sdhC and ppc.

In one embodiment, the nucleotide sequence of ppc is shown in SEQ ID NO.1 and the nucleotide sequence of sdhC is shown in SEQ ID NO. 2; the nucleotide sequence of aspA is shown as SEQ ID NO.3, and the nucleotide sequence of panD is shown as SEQ ID NO. 4; the nucleotide sequence of pa0123 is shown as SEQ ID NO.5, and the nucleotide sequence of yne1 is shown as SEQ ID NO. 6.

In one embodiment of the present invention, gene yne1, pa0123 uses pRSFDuet-1 as expression vector; the genes aspa and panD take pTrc99a as expression vectors; the gene fragments sdhC and ppc are expressed by pCDFDuet-1.

The invention also provides a method for constructing the recombinant escherichia coli, which comprises the following steps:

(1) The plasmid pRSFDuet-1 is used as a skeleton vector, and gene segments yne1 and pa0123 are connected to obtain a recombinant plasmid pRSF-yne1-pa0123;

(2) The plasmid pTrc99a is used as a skeleton vector, and gene fragments aspa and panD are connected to obtain a recombinant plasmid pTrc99a-aspa-panD;

(3) The plasmid pCDFDuet-1 is used as a skeleton vector to connect gene fragments sdhC and ppc to obtain recombinant plasmid pCDF-sdhC-ppc;

(4) pRSF-yne1-pa0123, pTrc99a-aspa-panD and pCDF-sdhC-ppc are transferred into escherichia coli BL21 (DE 3) together to obtain recombinant escherichia coli.

In one embodiment of the present invention, step (1) after the gene fragment pa0123 and the plasmid pRSFDuet-1 are digested with both Nco I and Hind III, they are digested with T ₄ DNA ligase is connected to obtain recombinant plasmid pRSF-pa0123; treating both yne1 and recombinant plasmid pRSF-pa0123 with XhoI and Nde I, and then T ₄ DNA ligase is used for connection to obtain recombinant plasmid pRSF-yne1-pa0123.

In one embodiment of the present invention, the gene fragment aspa and the plasmid pTrc99a are both digested with Sal I and Xho I, and then digested with T in step (2) ₄ DNA ligase is connected to obtain recombinant plasmid pTrc99a-aspa; the panD and recombinant plasmid pTrc99a-aspa were treated with Xho I and Hind III double cleavage, followed by T ₄ The DNA ligase was ligated to obtain the recombinant plasmid pTrc99a-aspa-panD.

In one embodiment of the present invention, step (3) after the gene fragment sdhC and the plasmid pCDFDuet-1 are both digested with Nco I and EcoR I, they are digested with T ₄ DNA ligase is connected to obtain recombinant plasmid pCDF-sdhC; the ppc and recombinant plasmid pCDF-sdhC were treated with EcoR I and Sal I double cleavage and then T was used ₄ The DNA ligase was ligated to obtain the recombinant plasmid pCDF-sdhC-ppc.

The third object of the invention is to provide a method for producing malonic acid by using the recombinant escherichia coli fermentation, which takes SOB culture medium as fermentation culture medium,culturing recombinant colibacillus at 35-37 deg.C until OD ₆₀₀ 1mM IPTG was added at 0.6-0.8 and the temperature was lowered to 30℃for induction culture for 48 hours.

In one embodiment of the invention, the SOB medium has a composition of 2g/100ml tryptone, 0.5g/100ml yeast powder, 0.05g/100ml NaCl,2.5mM KCl,10mM MgCl ₂ 0.8g/100ml glucose, 50. Mu.g/ml kanamycin sulfate, 50. Mu.g/ml ampicillin, 50. Mu.g/ml streptomycin.

In one embodiment of the invention, the seed solution is prepared by streaking the glycerol-deposited strain on a plate, picking a single colony, inoculating into a 250ml conical flask containing 50ml of LB liquid medium, shaking the flask overnight at 37℃at 250 rpm/min. Transferring 1ml of bacterial liquid into 50ml of LB liquid culture medium, culturing at 37 ℃ and 250rpm until reaching OD ₆₀₀ When reaching 0.6-0.8, the strain is transferred to 50ml of SOB fermentation medium.

The invention also claims the use of said method for the preparation of malonic acid or its derivative products.

The beneficial effects are that: compared with a chemical method, the method for synthesizing malonic acid by using the escherichia coli full biological method firstly takes glucose as a precursor, can be developed in a sustainable way, and greatly reduces the pollution degree to the environment. The method for producing malonic acid by glucose fermentation is a high-yield way, provides environmental benefits by eliminating cyanide, chloroacetic acid and the like, does not need to use other compounds as a premise, can synthesize malonic acid by glucose by over-expressing 2 genes, namely a heterologous gene aspartic acid-alpha-dehydrogenase gene (panD) from corynebacterium glutamicum and a heterologous gene beta-alanine pyruvic transaminase gene (pa 0123) from pseudomonas aeruginosa, has simple method, mature fermentation process, enables escherichia coli BL21 (DE 3) to produce malonic acid at high yield, and has convenient operation, low cost and fermentation time of 48 hours of 0.23g/L.

Drawings

FIG. 1 shows the malonic acid synthesis pathway.

FIG. 2 is a pRSF-yne1-pa0123 plasmid map.

FIG. 3 is a map of pTrc99a-aspa-panD plasmid.

FIG. 4 is a map of pCDF-sdhC-ppc plasmid.

FIG. 5 is a map of pRSF-yne1-pa0123 plasmid colony pcr validation, 1: marker,2-5: colonies pcr, which were pRSF-yne1-pa0123, were validated for pa0123.

FIG. 6 is a graph showing the fermentation results of recombinant E.coli in SOB medium, IPTG 1 mM.

FIG. 7 is a liquid-phase mass spectrum detection spectrum of malonic acid; wherein (A) is a liquid phase diagram of a malonic acid standard sample and a liquid phase diagram only showing the malonic acid standard sample respectively; (B) Respectively a mass spectrum of a malonic acid standard sample and a mass spectrum which only displays malonic acid; (C) Respectively a liquid phase diagram of a fermentation broth sample and a liquid phase diagram only showing malonic acid in the fermentation broth; (D) Respectively a mass spectrum of a fermentation broth sample and a mass spectrum which only displays malonic acid in the fermentation broth; STANDARD is 1g/L STANDARD malonic acid sample, and PA is fermentation broth sample.

Detailed Description

Malonic acid liquid phase mass spectrum detection and result analysis:

pretreatment: and (3) centrifuging the fermentation sample at 12,000r/min for 2min to separate the fermentation liquor from the thalli, and treating the fermentation liquor by a 0.22 mu m filter membrane for detection by liquid phase mass spectrometry.

Liquid phase mass spectrometry conditions: detection wavelength: 200- - -400n, analytical column: BEH C18 (2.1x150mm 1.7um), column temperature 45 ℃, flow rate: sample injection amount of 0.3 ml/min: 5 μl, detector: waters Acquity PDA (200-400 nm).

The mobile phase conditions were as follows:

TABLE 1 primer sequence listing according to the following examples

Example 1: recombinant plasmid construction and recombinant escherichia coli acquisition.

The nucleotide sequences of ppc, sdhC, aspA, panD, pa0123 and yne1 are respectively shown in SEQ ID NO. 1-6.

The plasmid pRSFDuet-1 is digested with Nco I and Hind III, the target gene fragment pRSF-1 (3761 bp) is recovered by cutting gel, the plasmid pUC57-pa0123 synthesized by Jin Wei intelligent company is used as template, the primer pa0123-F/R is used for PCR amplification to obtain target gene fragment pa0123 (sequence is shown as SEQ ID NO. 5), then the two target fragments pRSF-1 and pa0123 are used for T ₄ DNA ligase is connected, JM109 is transformed, positive transformants are picked up by colony PCR, plasmid digestion is extracted, PCR verification is carried out, the primer is veri-pRSF-F/R, and the plasmid after verification is named pRSF-pa0123.

The plasmid pRSF-pa0123 is digested with Xho I and Nde I, the target gene fragment pRSF-2 (5101 bp) is recovered by cutting, the same enzyme is used for cutting, the large intestine genome is used as template, the primer yne1-F/R is used for PCR amplification to obtain the yne1 fragment (shown as SEQ ID NO. 6), and then the two target fragments pRSF-2 and yne1 are used for T ₄ DNA ligase is connected, JM109 is transformed, positive transformants are picked up by colony PCR, plasmid digestion and PCR verification are carried out, the primer is verified to be veri-pRSF-F/R, and the plasmid after verification is named pRSF-yne1-pa0123 (the plasmid diagram is shown in FIG. 2).

The plasmid pTrc99A is digested with Sal I and Xho I, the target vector DNA fragment pTrc99A-1 (4170 bp) is recovered by digestion, the same digestion is performed with the large intestine genome as template, the aspa fragment obtained by PCR amplification (as shown in SEQ ID NO. 3) is amplified with the primers aspa-F/R, and then the target fragments pTrc99A-1 and aspa are subjected to T ₄ DNA ligase is connected, JM109 is transformed, positive transformants are picked up by colony PCR, plasmid digestion is extracted, PCR verification is carried out, the primer is veri-pTrc-F/R, and the plasmid after verification is named pTrc99a-aspa.

The plasmid pTrc99A-aspa is digested with Xho I and Hind III, the vector DNA fragment pTrc99A-2 (5622 bp) is recovered by cutting, the plasmid pUC57-panD synthesized by Jin Wei intelligent company is digested with the same enzyme as the template, the primers panD-F/R are used for PCR amplification to obtain the target gene fragment panD (shown as SEQ ID NO. 4),then T is used for two target fragments of pTrc99A-2 and panD ₄ DNA ligase is connected, JM109 is transformed, positive transformants are picked up by colony PCR, plasmid digestion is extracted, PCR verification is carried out, the primers are veri-pTrc-F/R, and the plasmid after verification is named pTrc99a-aspa-panD (the plasmid diagram is shown in FIG. 3).

The plasmid pCDFDuet-1 was digested with Nco I and EcoRI, the target vector DNA fragment pCDF-1 (3744 bp) was recovered by digestion with the same enzyme, the large intestine genome was used as a template, the ppc fragment obtained by PCR amplification with the primer ppc-F/R (as shown in SEQ ID NO. 1) was amplified, and then the two target fragments pCDF-1 and ppc were subjected to T ₄ DNA ligase is connected, JM109 is transformed, positive transformants are picked up by colony PCR, plasmid digestion and PCR verification are carried out, the primer is verified to be veri-pCDF-F/R, and the plasmid after verification is correctly named pCDF-ppc.

The target DNA fragment pCDF-2 (6379 bp) was recovered by double digestion of the plasmid pCDF-ppc with EcoR I and Sal I, digested with the same enzymes using the large intestine genome as a template, amplified by PCR with the primer sdhC-F/R to give the sdhC fragment (as shown in SEQ ID NO. 2), and then subjected to T-ligation with the two target fragments ₄ DNA ligase is connected, JM109 is transformed, positive transformants are picked up by colony PCR, plasmid digestion and PCR verification are carried out, the primer is verified to be veri-pCDF-F/R, and the plasmid after verification is named pCDF-ppc-sdhC (the plasmid diagram is shown in FIG. 4).

pRSF-yne1-pa0123, pTrc99a-aspa-panD and pCDF-sdhC-ppc are transferred into competent cells of escherichia coli BL21 (DE 3) together to prepare recombinant escherichia coli.

Example 2: shake flask fermentation and result analysis of recombinant E.coli.

Fermentation medium: SOB culture medium with components of 20g/L tryptone, 5g/L yeast powder and 0.5g/LNaCl,2.5mM KCl,10mM MgCl ₂ 4g/L glucose, 50. Mu.g/ml kanamycin sulfate, 50. Mu.g/ml ampicillin, 50. Mu.g/ml streptomycin.

Seed liquid preparation: the glycerol-deposited strain was streaked on a plate, and single colonies were picked and inoculated into a 250ml Erlenmeyer flask containing 50ml of LB liquid medium, and shake flask at 37℃and 250rpm/min overnight.

Fermentation conditions:2% inoculum size (1 ml) inoculated into shake flask fermentation medium to give initial OD ₆₀₀ 0.1. Culturing at 37deg.C and 250r/min to OD ₆₀₀ 1.0mM IPTG was added at 0.8-1.0, and the temperature was changed to 30℃and the rpm/min for culturing.

Analysis of results: taking samples every 4H for the first 12H and every 12H for the last 60H, centrifuging at 12,000r/min for 5min to separate the fermentation liquid from thallus, and treating the fermentation liquid with 0.22 μm filter membrane for HPLC (high performance liquid chromatography) detection with mobile phase of 5mM H ₂ SO ₄ The column temperature was 30 ℃, differential detector. From the results of the liquid phase, no malonic acid production was detected for the first 24 hours, malonic acid production was detected from 36 hours, and the yield reached a maximum of 0.23g/L at 48 hours.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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

<110> university of Jiangnan

<120> a method for total biosynthesis of malonic acid

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<170> PatentIn version 3.3

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gaattttccc acggttatac atattcagcg cacccagtgg catgtgctgc gggattagca 1020

gcactggaca tcttggcgcg cgataactta gtacagcagt cagcagagtt agctccacac 1080

ttcgagaagg gattgcatgg ccttcaaggt gccaagaatg ttatcgacat tcgcaactgc 1140

ggcttagcag gcgcgatcca gatcgctccc cgtgatgggg atccgacagt tcgccccttt 1200

gaagccggga tgaaactgtg gcaacaaggg ttttacgtcc gtttcggcgg cgacactctg 1260

cagtttgggc caacatttaa tgcacgccca gaggaattgg accgtctttt tgacgctgta 1320

ggtgaggctc tgaacggaat tgcctga 1347

<210> 6

<211> 1149

<212> DNA

<213> Escherichia coli

<400> 6

aaaccaatca accaggcgcg cgctgaagtg gcgaaatcgg cgaatttgtg tgactggtat 60

gcagaacatg gtccggcaat gctgaaggcg gaacctacgc tggtggaaaa tcagcaggcg 120

gttattgagt atcgaccgtt ggggacgatt ctggcgatta tgccgtggaa ttttccgtta 180

tggcaggtga tgcgtggcgc tgttcccatc attcttgcag gtaacggcta cttacttaaa 240

catgcgccga atgtgatggg ctgtgcacag ctcattgccc aggtgtttaa agatgcgggt 300

atcccacaag gcgtatatgg ctggctgaat gccgacaacg acggtgtcag tcagatgatt 360

aaagactcgc gcattgctgc tgtcacggtg accggaagtg ttcgtgcggg agcggctatt 420

ggcgcacagg ctggagcggc actgaaaaaa tgcgtactgg aactgggcgg ttcggatccg 480

tttattgtgc ttaacgatgc cgatctggaa ctggcggtga aagcggcggt agccggacgt 540

tatcagaata ccggacaggt atgtgcagcg gcaaaacgct ttattatcga agagggaatt 600

gcttcggcat ttaccgaacg ttttgtggca gctgcggcag ccttgaaaat gggcgatccc 660

cgtgacgaag agaacgctct cggaccaatg gctcgttttg atttacgtga tgagctgcat 720

catcaggtgg agaaaaccct ggcgcagggt gcgcgtttgt tactgggcgg ggaaaagatg 780

gctggggcag gtaactacta tccgccaacg gttctggcga atgttacccc agaaatgacc 840

gcgtttcggg aagaaatgtt tggccccgtt gcggcaatca ccattgcgaa agatgcagaa 900

catgcactgg aactggctaa tgatagtgag ttcggccttt cagcgaccat ttttaccact 960

gacgaaacac aggccagaca gatggcggca cgtctggaat gcggtggggt gtttatcaat 1020

ggttattgtg ccagcgacgc gcgagtggcc tttggtggcg tgaaaaagag tggctttggt 1080

cgtgagcttt cccatttcgg cttacacgaa ttctgtaata tccagacggt gtggaaagac 1140

cggatctga 1149

Claims

1. A recombinant escherichia coli for producing malonic acid is characterized in that escherichia coli BL21 (DE 3) is taken as a host, and phosphoenolpyruvate carboxylase gene, succinic dehydrogenase gene, succinic semialdehyde dehydrogenase gene and asparaginase gene from escherichia coli, heterologous gene aspartic acid-alpha-dehydrogenase gene from corynebacterium glutamicum and beta-alanine pyruvic transaminase gene from pseudomonas aeruginosa are subjected to modular overexpression;

the split-module overexpression is to fuse and express a succinic semialdehyde dehydrogenase gene and a beta-alanine pyruvic transaminase gene, fuse and express an asparaginase gene and an aspartic acid-alpha-dehydrogenase gene, and fuse and express a succinic dehydrogenase gene and a phosphoenolpyruvic carboxylase gene;

the nucleotide sequence of the phosphoenolpyruvate carboxylase gene is shown as SEQ ID NO.1, and the nucleotide sequence of the succinate dehydrogenase gene is shown as SEQ ID NO. 2; the nucleotide sequence of the asparaginase gene is shown as SEQ ID NO.3, and the nucleotide sequence of the aspartic acid-alpha-dehydrogenase gene is shown as SEQ ID NO. 4; the nucleotide sequence of the beta-alanine pyruvic transaminase gene is shown as SEQ ID NO.5, and the nucleotide sequence of the succinic semialdehyde dehydrogenase gene is shown as SEQ ID NO. 6;

the plasmid pRSFDuet-1 is used as skeleton carrier to connect gene segment succinic semialdehyde dehydrogenase geneyne1Beta-alanine pyruvic transaminase genepa0123The method comprises the steps of carrying out a first treatment on the surface of the The plasmid pTrc99a is used as skeleton carrier to connect gene segment asparaginase geneaspaAspartic acid-alpha-dehydrogenase genepanDThe method comprises the steps of carrying out a first treatment on the surface of the The plasmid pCDFDuet-1 is used as a skeleton carrier to connect gene segment succinic dehydrogenase genesdhCPhosphoenolpyruvate carboxylase geneppc。

2. A method of constructing the recombinant escherichia coli of claim 1, comprising the steps of:

(1) The plasmid pRSFDuet-1 is used as a skeleton vector to connect gene fragmentsyne1、pa0123Obtaining recombinant plasmid pRSF-yne1-pa0123;

(2) The plasmid pTrc99a is used as skeleton carrier to connect gene fragmentaspa、panDObtaining recombinant plasmid pTrc99a-aspa-panD;

(3) The plasmid pCDFDuet-1 is used as a skeleton vector to connect gene fragmentssdhC、ppcObtaining recombinant plasmid pCDF-sdhC-ppc;

3. A method for producing malonic acid, wherein the recombinant escherichia coli of claim 1 is inoculated into a fermentation medium containing glucose for fermentation to produce malonic acid.

4. A method according to claim 3, characterized in that the recombination is performedE.coli is cultivated at 35-37 ℃ until OD ₆₀₀ At 0.6-0.8, IPTG was added for induction.

5. The method of claim 4, wherein the induction is at 28-30 ℃ for 36-60 hours.

6. The method according to any one of claims 3 to 5, wherein SOB medium is used as fermentation medium,

7. use of the recombinant escherichia coli of claim 1 or the method of any one of claims 3-6 for preparing malonic acid.