CN114045300A

CN114045300A - Construct, vector and cyanobacterium for synthesizing olivinic acid and method for producing olivinic acid in cyanobacterium

Info

Publication number: CN114045300A
Application number: CN202111313584.4A
Authority: CN
Inventors: 吴军华; 邱海燕; 高恶斌
Original assignee: Ningbo Women and Children Hospital
Current assignee: Ningbo Women and Children Hospital
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2022-02-15

Abstract

The invention relates to a construct for the synthesis of olivinic acid, comprising: a promoter which is active in cyanobacteria, and a first gene and a second gene which are under the control of the promoter, wherein the first gene is a TKS gene and OAC gene combined gene TKS/OAC, the sequence of which is shown in SEQ ID NO.2, and the second gene is an acyl activating enzyme gene, the sequence of which is shown in SEQ ID NO. 3. The invention also relates to a vector comprising said construct and to a cyanobacterium comprising said construct or transformed with said vector, and to a method for producing olivinic acid in a cyanobacterium. The combined gene TKS/OAC of TKS gene and OAC gene can catalyze malonyl coenzyme A and hexanoyl coenzyme A generated by blue algae photosynthesis to synthesize olive oil acid. The invention uses blue algae as the chassis organism to reform and synthesize the olive alcohol acid, the production raw materials only need sunlight, moisture and the like, and the construction of production equipment and production environment is simple, thereby greatly reducing the production cost compared with other production methods.

Description

Construct, vector and cyanobacterium for synthesizing olivinic acid and method for producing olivinic acid in cyanobacterium

Technical Field

The invention relates to the technical field of synthetic biology, in particular to a construct for synthesizing olivinic acid, a vector containing the construct, a cyanobacterium containing the construct or transformed by the vector, and a method for producing olivinic acid in the cyanobacterium.

Background

Cyanobacteria (Cyanobacteria) can perform anaerobic photosynthesis, can perform photosynthesis, and can release oxygen by taking water as an electron acceptor. Its development has led to the entire earth's atmosphere going from an anaerobic state to an aerobic state, thus gestating the evolution and development of all aerobic organisms. Blue algae as carbon fixing place for fixing CO in atmosphere₂(CO mitigation to some extent)₂Environmental problems caused by too much). Synechocystis PCC6803(Synechocystis sp. PCC 6803) is a unicellular spherical cyanobacteria that can grow autotrophically with light energy and heterotropically with glucose.

Cyanobacteria (Blue-green algae) is a prokaryotic microorganism capable of carrying out plant-type oxygen-producing photosynthesis, and has the following advantages as a new-generation energy microorganism system: (1) the cyanobacteria can absorb solar energy and fix carbon dioxide as a carbon source for autotrophic growth, and the culture cost is low; (2) cyanobacteria are an ancient microorganism, exist on the earth for billions of years, have strong environmental adaptability and grow rapidly; (3) the cyanobacteria are convenient to operate genetically, the genetic background is clear, and many kinds of genome sequencing work are finished successively, so that the cyanobacteria can be conveniently transformed by utilizing a genetic engineering means. Synechocystis sp.PCC6803 is a representative species of unicellular cyanobacteria, and the whole genome sequencing of the Synechocystis sp.PCC6803 is completed in 1996, is the photosynthetic microorganism which is the earliest to complete the whole genome sequencing, and is one of the most studied cyanobacteria at present.

Olivonic acid (OLA) is a plant secondary metabolite derived from the catalytic action of type III polyketide synthases (PKS IIIs) and has various pharmacological activities together with pentenylated derivatives thereof. The monoaromatic compound, olivolic acid (OLA), is a member of PKS III products and has pharmacological activities such as antibacterial, cytotoxic and photoprotective activities. Furthermore, olivinic acid is a central intermediate in the synthesis of an important class of pharmacological compounds, which are becoming increasingly important due to its numerous pharmacological properties, because it acts as an alkylresorcinol moiety during the biosynthesis of cannabinoids.

At present, the production of olivinic acid and its derivatives is mainly achieved by direct extraction from plants, but since plants grow slowly, the accumulation of these compounds takes at least several months, with longer periods of direct extraction. Although plant biotechnology offers the opportunity to improve the synthesis of natural products from native species, it is difficult to precisely control the expression level of transgenes in plants and adapt to industrial production. Although the chemical synthesis of olivinic acid is another recently reported option, the structural complexity of most natural products determines the inherent inefficiency of total chemical synthesis, which results in low yields and high energy waste. In contrast to these methods, building microbial cell factories to produce these value-added plant natural products is a promising strategy.

Disclosure of Invention

In view of the deficiencies of the prior art, the present application was the first to successfully construct the constructs required for the biosynthesis of cyanomycolic acid. The carrier and the strain are used, and the construction method is proved to have good feasibility, so that the method for producing the olive alcohol acid in the cyanobacteria is provided, and has the advantages of high yield, no pollution, short production period, low cost and environmental protection.

To solve the above problems, the present invention provides a construct for synthesizing olivinic acid, comprising: the gene comprises a promoter which is active in cyanobacteria, and a first gene and a second gene which are under the control of the promoter, wherein the first gene is a butenone synthetase gene and olivine acid cyclase gene combined gene, the sequence of the gene is shown as SEQ ID NO.2, and the second gene is an acyl activating enzyme gene, the sequence of the gene is shown as SEQ ID NO. 3.

Optionally, it comprises a marker gene for screening cyanobacteria transformants upstream of the promoter active in cyanobacteria.

Optionally, the marker gene is a spectinomycin resistance gene fragment, and the sequence of the spectinomycin resistance gene fragment is shown in SEQ ID No. 4.

Optionally, it has at both ends the N-terminal and C-terminal sequences of the slr0168 gene of Synechocystis PCC6803 for homologous recombination.

Optionally, the promoter active in the cyanobacterium is P_cpc560。

Optionally, the cyanobacterium is synechocystis PCC 6803.

A vector comprising any one of the constructs described above for the synthesis of olivinic acid.

A cyanobacterium comprising any one of the constructs described above for use in the synthesis of olivinic acid.

Cyanobacteria transformed with said vectors.

A method of producing olivinic acid in a cyanobacterium, the method comprising: culturing the above cyanobacteria under conditions suitable for synthesizing olivinic acid; and extracting said olivinic acid from the resulting culture.

The synthetic olive alcohol acid of the invention is different from the prior art in that:

1) the invention introduces exogenous key functional genes synthesized by the olivine acid into the synechocystis PCC6803 genome by using a genetic engineering technology, successfully constructs a metabolic pathway for efficiently synthesizing the biological olivine acid in the synechocystis PCC6803 for the first time, and synthesizes the olivine acid in the growth process of cyanobacteria.

2) The invention utilizes the light intensity promoter P with super strong startability_cpc560The crotone synthetase gene (TKS) and the olivine acid cyclase gene (OAC) derived from the hemp trichome are driven to have high gene locus in one or more cyanobacteriaEffectively expresses and improves the yield of the olive alcohol acid.

3) The method has the advantages that cyanobacteria is used as a chassis organism for transformation and synthesis of the olive alcohol acid for the first time, production raw materials only need sunlight, moisture and the like, production equipment and production environment are simple to construct, and a large amount of electricity is not needed, so that compared with other production methods, the production cost is greatly reduced.

4) The synthesis process has no waste liquid discharge, is environment-friendly, has no pollution, and can be used for sustainable production.

Drawings

FIG. 1 is a diagram showing the basic structure of plasmid pMDslr0168 in the example of the present invention;

FIG. 2 is the basic structure diagram of recombinant plasmid pMDslr 0168-omega in the example of the present invention;

FIG. 3 is a diagram showing the basic structure of a recombinant plasmid pMD0168 in the example of the present invention;

FIG. 4 is a diagram showing the basic structure of a recombinant plasmid pMD0168-TKS/OAC in the example of the present invention;

FIG. 5 is a diagram showing the basic structure of the recombinant plasmid pMD-TKS/OAC/AAE in the example of the present invention;

FIG. 6 is a metabolic scheme for biosynthesis of olivinic acid by cyanobacteria in an example of the present invention;

FIG. 7 is an HPLC detection of cyanohydric acid produced by cyanobacteria in an example of the invention;

FIG. 8 is a graph comparing the growth of cyanobacteria of the present examples with wild-type cyanobacteria over a 14 day culture period.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. The procedures of strain culture, molecular biology, molecular genetics and the like referred to herein are all routine procedures in a wide range of corresponding fields. Also, for a better understanding of the present invention, the following definitions and explanations of related terms are provided.

As used herein, "Cyanobacteria" (Cyanobacteria) is a class of photo-and autotrophic prokaryotic microorganisms that are capable of utilizing solar energy to fix carbon dioxide. Cyanobacteria are also known as cyanobacteria, and cyanobacteria can be used interchangeably in the present invention. The cyanobacterium used in the present invention is synechocystis PCC 6803.

As used herein, a "crotonone synthase (TKS)" catalyzes the decarboxylation claisen condensation of a hexanoyl-CoA primer and 3 malonyl-CoA extension units to 3, 5, 7-trioxododecanoyl-CoA.

As used herein, "Olivine Acid Cyclase (OAC)" cyclizes the 3, 5, 7-triiododecanoyl-coenzyme a intermediate to form olivine acid by aldol condensation reaction within the C2-C7 molecule.

As used herein, an "acyl-activating enzyme (AAE 1)" is an enzyme capable of catalyzing hexanoic acid to hexanoyl-CoA.

P as used in the present invention_cpc560The promoter is the promoter (SEQ ID NO.1) P of the operon in the Synechocystis PCC6803 genome encoding Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Ribulose-1, 5-biphosphate carboxylase/oxygenase, Rubisco) which catalyzes the first reaction of the Carlsberg cycle in photosynthesis_cpc560The promoter is active in cyanobacteria.

The slr0168 gene (see, e.g., NC _954899) as used in the present invention is a gene in the genome of synechocystis PCC6803 that encodes an unknown protein. Numerous studies demonstrated that deletion of the slr0168 gene had no effect on the growth activity of cyanobacteria. The gene is located at a neutral site.

The detailed scheme of the invention is as follows:

an object of an embodiment of the present invention is to construct a metabolic pathway for synthesizing biological olivinic acid in cyanobacteria PCC6803, introducing a crotonone synthase gene (TKS), an olivinic acid cyclase gene (OAC) and an acyl activating enzyme gene (AAE1) into cyanobacteria cells to obtain recombinant cyanobacteria cells, and performing photosynthesis using the recombinant cyanobacteria cells to obtain olivinic acid.

Specifically, the cyanobacterium is Synechocystis sp.

Without being bound by any theory, in conjunction with fig. 6, the inventors believe that the mechanism by which cyanobacteria produce olivinic acid in the present application is as follows: carbon fixation in cyanobacteria body generates intermediate pyruvic acid through Kelvin cycle. Pyruvate decarboxylation produces acetyl CoA, which is converted to malonyl-CoA under the catalysis of acetyl-CoA carboxylase, and exogenously added hexanoic acid is catalyzed to hexanoyl-CoA by acyl-activating enzyme I (AAE 1). Crotone synthetase (TKS) catalyzes hexanoyl coenzyme A and 3 malonyl coenzyme A extension units to generate 3, 5, 7-trioxodinodecanoyl-coenzyme A through decarboxylation claisen condensation reaction. This 3, 5, 7-trioxiododecanoyl-coenzyme A intermediate can be cyclized by Olivine Acid Cyclase (OAC) via aldol condensation reaction within the C2-C7 molecule to form olivine acid.

An object of the embodiments of the present invention is to efficiently start and increase the activities of a crotonone synthase, an olivine acid cyclase and an acyl activating enzyme, thereby increasing the yield of olivine acid and achieving the purpose of efficiently synthesizing biological olivine acid using cyanobacteria.

The embodiment of the invention utilizes the light intensity promoter P with super strong startability_cpc560Drives expression of crotone synthase, olive-oil acid cyclase and acyl activating enzyme at one or more gene sites in cyanobacteria. Meanwhile, in order to improve the yield of the olive alcohol acid, a genetic engineering cyanobacterium for synthesizing the biological olive alcohol acid is constructed.

The synthesis method specifically comprises the following steps:

constructing an integration vector pMD0168, selecting slr0168 gene sequence on synechocystis PCC6803 genome as an exogenous gene integration site, wherein the change of the gene sequence does not influence the function of the whole genome of the synechocystis PCC6803, and inserting a Spectinomycin resistance gene (Anti-Spectinomycin, Spe)^R) And a super strong promoter P_cpc560Thus constructing an integration vector pMD0168 for synthesizing olivinic acid;

constructing a gene expression vector pMD-TKS/OAC/AAE: according to the codon preference of Synechocystis sp. PCC6803, the gene sequences of crotone synthetase gene (TKS), olive acid cyclase gene (OAC) and acyl activating enzyme gene (AAE1) from hemp trichomes are subjected to codon optimization, and then the gene sequences after the three codon optimization are inserted into the constructed integration vector;

homologous recombination: transferring the gene expression vector pMD-TKS/OAC/AAE into Synechocystis PCC6803 cell, and performing homologous recombination to obtain crotone synthetase gene (TKS), Olivonic acid cyclase gene (OAC), acyl activating enzyme gene (AAE1), Spectinomycin resistance gene (Anti-Spectinomycin, Spe)^R) And a super strong promoter P_cpc560Integrating the sequences into slr0168 gene locus of synechocystis PCC6803 genome;

screening positive transformants: synechocystis PCC6803 monoclonal cells transformed with pMD-TKS/OAC/AAE vector were selected with spectinomycin, then the cell genome was extracted, and transformants finally carrying the crotone synthase gene (TKS), olivine acid cyclase gene (OAC), acyl activating enzyme gene (AAE1) were determined by PCR molecular characterization.

Example 1: construction of a light intensity super-strong promoter P_cpc560Plasmid pMD-TKS/OAC/AAE

A pMD18-T vector is used as a framework sequence, extracted genomic DNA of Synechocystis PCC6803 is used as a template, primers slr0168Up-F, slr0168Up-R, slr0168Dw-F and slr01 0168Dw-R are respectively used for amplifying 600bp gene sequences on the upstream and the downstream of slr0168 and are respectively connected among HindIII, SphI, KpnI and EcoRI restriction enzyme sites of pMD18-T, and the vector pMDslr0168 shown in figure 1 is obtained.

Plasmid pMDslr 0168-omega is constructed by taking plasmid pJS406 (containing spectinomycin resistance gene) preserved in the experiment as a template, amplifying a spectinomycin resistance gene sequence by adopting primers Spe-F and Spe-R, and inserting a gene fragment obtained by amplification between PstI and XbaI restriction endonuclease sites of a vector pMDslr0168, wherein the basic structure of the plasmid pMDslr 0168-omega is shown in figure 2.

A promoter (Pcpc560) and a terminator (Trbcl) sequence which are obtained by amplifying Pcpc-F/Pcpc-R and Trbcl-F/Trbcl-R by using a primer pair are respectively inserted between XbaI, BamHI and KpnI sites of pMDslr 0168-omega by using the genome DNA of synechocystis PCC6803 as a template to obtain a cyanobacteria integration expression plasmid pMD0168, wherein the basic structure of the plasmid pMD0168 is shown in figure 3.

Nucleotide sequences of a crotone synthase gene (TKS), an olivine acid cyclase gene (OAC) and an acyl activating enzyme gene (AAE1) derived from hemp trichomes were optimized using online software (http:// www.jcat.de /) according to codon preference of Synechocystis PCC6803, and the optimized sequences were synthesized by Biotech corporation (Shanghai, China). The optimized synthetic strain is taken as a template, PCR amplification is carried out on TKS-F/TKS-R and AAE-F/AAE-R by respectively adopting primer pairs, and the amplified gene sequence fragment is inserted into a promoter (P) of a plasmid pMD0168_cpc560) And a terminator (TrbcL), thereby obtaining the cyanobacterial integrated expression plasmid pMD-TKS/OAC/AAE, the basic structure of which is shown in FIG. 4. The specific construction plasmids and primers are shown in tables 1 and 2.

TABLE 1 plasmids used herein

TABLE 2 primers used herein

Example 2: transformation of cyanobacteria and selection of transformants

1. Transformed Synechocystis PCC6803 and resistant passage

Synechocystis PCC6803 has a natural DNA transformation system, can be transformed naturally, can be recombined in a homologous double-crossover manner and can be integrated into a genome, and the purpose of gene knockout can be realized by natural transformation in the embodiment. The specific transformation process and resistance passage flow are as follows:

(1) taking 30mL of fresh cyanobacteria cells in logarithmic growth phase (OD730 is about 0.6-0.8), centrifuging at 4500rpm for 15min, and collecting the cells; washing with fresh BG11 MediumCells were resuspended in 1mL BG11 medium (1.5g L) twice^-1NaNO₃，40mg L^-1K₂HPO₄·3H₂O， 36mg L^-1CaCl₂·2H₂O，6mg L^-1Citric acid, 6mg L^-1Ammonium ferric citrate, 1mg L^-1EDTA disodium salt, 20mg L^-1NaCO₃，2.9mg L^-1H₃BO₃，1.8mg L^-1MnCl₂·4H₂O，0.22mg L^-1ZnSO₄·7H2O，0.39mg L^-1NaMoO₄·2H₂O，0.079mg L^-1CuSO₄·5H₂O and 0.01mg L^- ¹CoCl₂·6H₂O) in (A).

(2) 0.2mL of cell suspension is put into a new EP tube, 2-3 mu g of expression plasmid is added, mixed evenly and placed under the condition of 30 ℃ and 30 mu Em-2s-1 illumination for incubation for 18 hours.

(3) The mixture of cyanobacterial cells and DNA was spread on a nitrocellulose membrane plated on BG11 plates (without antibiotic addition) and incubated at 30 ℃ under 30. mu. Em-2s-1 light for 24 hours. The nitrocellulose membrane was then transferred to a BG11 plate containing the antibiotic corresponding to the strain of interest, and placed upside down in a light incubator.

(4) After about 10 days of culture, a single colony after transformation can be obtained, a transformant is picked out from a plate, and streaked on a fresh BG11 solid medium (containing spectinomycin); after the cells are enriched, the cells are inoculated into liquid BG11 culture medium (containing spectinomycin) for culture.

(5) The transformed cyanobacteria cells were transferred twice to three times in liquid BG11 medium (containing spectinomycin), and after the correct introduction of the construct of interest was verified by genome sequencing, the current strain was determined to be the mutant of interest.

(6) 1mL of target cyanobacteria mutant strain is inoculated and cultured, 4mM sodium caproate is added into BG11 culture solution with corresponding resistance, and the cyanobacteria mutant strain is used for detecting the yield of the olive alcohol acid after the cyanobacteria mutant strain grows vigorously.

2. Strain (algal strain) and plasmid culture

Synechocystis PCC6803 is cultured in BG11 culture medium and is subjected to static culture at 28-30 ℃ under illumination (30 muE/m 2. s). BG11 culture solution additionally contained 10. mu.g mL^-1All solutions of spectinomycin and 5mM glucose were prepared with deionized water, and copper ions of different concentrations were added as required. All the vessels are plastic vessels.

Coli DH 5. alpha. was cultured in LB medium at 37 ℃ and the corresponding antibiotics (50. mu.g/mL for spectinomycin, kanamycin sulfate and ampicillin) were added to the plasmid-containing E.coli strain.

The synechocystis PCC6803 is transformed through the steps to construct the following strains:

cyanobacteria SYN 015: obtained by transforming the plasmid pMD-TKS/OAC/AAE into synechocystis PCC6803, and because the plasmid contains a homologous sequence of 600bp upstream and downstream of slr0168, the Pcpcc 560-Spe between the upstream and downstream fragments of slr0168 in pMD-TKS/OAC/AAE is obtained by double-exchange through homologous recombination^RTKS/OAC/AAE-TrbcL is integrated into Synechocystis PCC6803 at the slr0168 gene locus. Subsequently, by resistance selection, transformants containing spectinomycin resistance were cultured and verified by PCR, and the strain whose correctness was verified was numbered as SYN 015.

Example 3: verification of transgenic cyanobacteria and detection process of products

Selecting about 10 monoclonal cyanobacteria bacterial plaques growing on a solid culture medium containing 10ug/mL spectinomycin BG11, inoculating into 5mL 10ug/mL spectinomycin BG11 liquid culture medium for culture, extracting genomes after cyanobacteria culture, verifying two target genes of TKS/OAC and AAE by using designed primers, sequencing the products by PCR, determining whether the gene sequences are correct, inoculating the PCC6803 cyanobacteria which is verified to be correct and is transferred with the TKS/OAC genes and the AAE genes into 100mL BG11 liquid culture medium for illumination culture, and waiting until OD is reached₇₃₀When the content is more than or equal to 1, adding a substrate sodium caproate. Catalyzing caproic acid into hexanoyl coenzyme A under the action of AAE gene expression, synthesizing olive alcohol acid by using malonyl coenzyme A and hexanoyl coenzyme A generated by cyanobacteria self photosynthesis as substrates through the expression of gene TKS/OAC, and taking out the product after inductionThe cultured transgenic cyanobacteria is subjected to cell disruption by an ultrasonic cell disruption instrument, and after separation and purification of the product, detection is performed by a high performance liquid chromatograph, and the detection result is shown in fig. 7.

Example 4: results of Olivonic acid yield analysis

A fresh mutant strain cyanobacteria sample is subjected to cell disruption, extracted by ethyl acetate, and subjected to high performance liquid chromatography after the sample is concentrated by nitrogen blowing. High performance liquid chromatography conditions: the mobile phases are acetonitrile and 0.1% aqueous formic acid respectively, wherein the phase A is acetonitrile, and the phase B is 0.1% aqueous formic acid. The flow rate is 1.0 ml/min; 0-2min, 10% A and 90% B; 2-20min, 10% A and 90% B; 20-25min, 90% A and 10% B. The detection wavelength was 262nm and the column temperature was 35 ℃. The detection shows that the yield of the synthetic olive alcohol acid of the cyanobacteria mutant strain is 1.52 mg/L.

Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and such changes and modifications will fall within the scope of the present invention.

Sequence listing

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gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc 120

ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa 180

acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc 240

gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt 300

tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt 360

atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa 420

catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag 480

gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct 540

ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc 600

aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat 660

cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc 720

tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta 780

gtcggcaaat aa 792

<210> 5

<211> 5572

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagctcgaat tctagagtgc ttagtgcatc taacgcttga gttaagccgc gccgcgaagc 60

ggcgtcggct tgaacgaatt gttagacatt atttgccgac taccttggtg atctcgcctt 120

tcacgtagtg gacaaattct tccaactgat ctgcgcgcga ggccaagcga tcttcttctt 180

gtccaagata agcctgtcta gcttcaagta tgacgggctg atactgggcc ggcaggcgct 240

ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc 300

aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg ggcggcgagt 360

tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa 420

agagttcctc cgccgctgga cctaccaagg caacgctatg ttctcttgct tttgtcagca 480

agatagccag atcaatgtcg atcgtggctg gctcgaagat acctgcaaga atgtcattgc 540

gctgccattc tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt 600

cgtgcacaac aatggtgact tctacagcgc ggagaatctc gctctctcca ggggaagccg 660

aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca 720

ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact gcggagccgt 780

acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg 840

atagttgagt cgatacttcg gcgatcaccg cttccctcat gatgtttaac tttgttttag 900

ggcgactgcc ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg 960

cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg taccccaaaa aaacagtcat 1020

aacaagccat gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg 1080

accagttgcg tgagcggatc cacctgtaga gaagagtccc tgaatatcaa aatggtggga 1140

taaaaagctc aaaaaggaaa gtaggctgtg gttccctagg caacagtctt ccctacccca 1200

ctggaaacta aaaaaacgag aaaagttcgc accgaacatc aattgcataa ttttagccct 1260

aaaacataag ctgaacgaaa ctggttgtct tcccttccca atccaggaca atctgagaat 1320

cccctgcaac attacttaac aaaaaagcag gaataaaatt aacaagatgt aacagacata 1380

agtcccatca ccgttgtata aagttaactg tgggattgca aaagcattca agcctaggcg 1440

ctgagctgtt tgagcatccc ggtggccctt gtcgctgcct ccgtgtttct ccctggattt 1500

atttaggtaa tatctctcat aaatccccgg gtagttaacg aaagttaatg gagatcagta 1560

acaataactc tagggtcatt actttggact ccctcagttt atccggggga attgtgttta 1620

agaaaatccc aactcataaa gtcaagtagg agattaattc aaaagaggag aaagatctat 1680

gaatcattta cgtgctgaag gtcccgctag tgtgttggct attggcaccg ccaatcccga 1740

aaatattttg ttgcaagatg aatttcccga ttactacttt cgcgtgacca aaagtgaaca 1800

catgacccaa ttgaaagaaa aatttcgtaa aatttgtgat aaatccatga ttcggaaacg 1860

caactgtttt ctgaacgaag aacatttgaa acaaaatccc cggttggtgg aacatgaaat 1920

gcaaaccttg gatgcccgcc aagatatgtt agtggtggaa gtgcccaaat tggggaaaga 1980

tgcctgtgcc aaagccatta aagaatgggg ccaacccaaa tccaaaatta cccatttgat 2040

ttttaccagt gcttccacca ccgatatgcc cggtgctgat tatcattgtg ccaaattgtt 2100

aggcttgagt ccctccgtga aacgtgtgat gatgtatcaa ttaggttgtt atggcggtgg 2160

gaccgtgttg cgtattgcca aagatattgc cgaaaacaac aaaggggccc gggtgttagc 2220

cgtgtgttgt gatattatgg cctgtttatt tcggggtccc agtgaatccg atttggaatt 2280

gttagtgggg caagccattt ttggtgatgg tgctgctgct gtgattgtgg gtgctgaacc 2340

cgatgaaagt gtgggggaac ggcccatttt tgaattagtg agtaccggcc aaaccatttt 2400

gcccaattcc gaaggcacca ttggcggtca tattcgcgaa gccggcttga tttttgattt 2460

gcataaagat gtgcccatgt tgattagtaa caacattgaa aaatgtttga ttgaagcctt 2520

tacccccatt ggtattagtg attggaattc catcttttgg attacccatc ccgggggcaa 2580

agccattttg gataaagtgg aagaaaaatt gcatttgaaa tccgataaat ttgtggatag 2640

tcgtcatgtg ttatccgaac atggcaatat gtccagttcc accgtgttgt ttgtgatgga 2700

tgaattgcgt aaacggagtt tagaagaagg caaatccacc accggcgatg gttttgaatg 2760

gggtgtgtta tttgggtttg gccccggttt gaccgtggaa cgggtggtgg tgcgttccgt 2820

gcccattaaa tattaaggaa ttaggaggta atatatggcc gtgaaacatt tgattgtgtt 2880

gaaatttaaa gatgaaatta ccgaagccca aaaagaagaa tttttcaaaa cctatgtgaa 2940

cttggtgaac attattcccg ccatgaaaga tgtgtattgg gggaaagatg tgacccagaa 3000

aaataaagaa gaaggctaca cccatattgt ggaagtgacc tttgaatccg tggaaaccat 3060

tcaagattac attattcatc ccgcccatgt gggctttggt gatgtgtatc ggagtttttg 3120

ggaaaaattg ttgatttttg attatacccc ccgcaaataa ggatccgcgg taatatatgg 3180

ggaaaaacta caaaagttta gattccgtgg tggccagtga ttttattgcc ttgggcatta 3240

cctccgaagt ggccgaaacc ttgcatggtc gcttagccga aattgtgtgt aattatgggg 3300

ccgccacccc ccaaacctgg attaacattg ccaaccatat tttgagtccc gatttaccct 3360

tttccttgca tcaaatgttg ttttatggct gttacaaaga ttttggtccc gctccccccg 3420

cttggattcc cgatcccgaa aaagtgaaaa gtaccaattt aggcgccttg ttagaaaaac 3480

gtgggaaaga atttttgggc gtgaaataca aagatcccat ttccagtttt agtcattttc 3540

aagaattttc cgtgcggaat cccgaagtgt attggcgcac cgtgttgatg gatgaaatga 3600

aaattagttt ttccaaagat cccgaatgta ttttgcggcg cgatgatatt aataatcccg 3660

gtggttccga atggttgccc ggtggctatt taaacagtgc caaaaactgt ttgaacgtga 3720

actccaacaa aaaattgaat gataccatga ttgtgtggcg cgatgagggt aacgatgatt 3780

tgcccttgaa caaattgacc ttagatcaat tgcggaaacg ggtgtggttg gtggggtatg 3840

ccttagaaga aatggggttg gaaaaaggct gtgccattgc cattgatatg cccatgcatg 3900

tggatgccgt ggtgatttat ttggccattg tgttagccgg ttatgtggtt gtgagtattg 3960

ccgatagttt ttccgccccc gaaattagta cccgtttgcg gttatccaaa gccaaagcca 4020

tttttaccca agatcatatt attcgcggta aaaaacgtat tcccttatat agtcgggtgg 4080

tggaagccaa atcccccatg gccattgtga ttccctgtag tggttccaat attggggccg 4140

aattacggga tggggatatt agttgggatt attttctgga acgcgccaaa gaatttaaaa 4200

actgtgaatt taccgcccgt gaacaacccg tggatgccta taccaatatt ttgttttcca 4260

gtggcaccac cggtgaaccc aaagccattc cctggaccca agccaccccc ttaaaagccg 4320

ccgccgatgg gtggtcccat ttggatattc gcaaaggcga tgtgattgtg tggcccacca 4380

atttaggctg gatgatgggt ccctggttgg tgtatgccag tttgttaaat ggggcctcca 4440

ttgccttata taatgggagt cccttggtgt ccggctttgc caaatttgtg caagatgcca 4500

aagtgaccat gttgggcgtg gtgcccagta ttgtgcggag ttggaaatcc accaattgtg 4560

tgagtgggta tgattggtcc accattcgct gtttttccag ttccggcgaa gcctccaatg 4620

tggatgaata tttgtggtta atgggtcgtg ccaattataa acccgtgatt gaaatgtgtg 4680

gtgggaccga aattggcggt gcctttagtg ccggctcctt tttacaagcc caaagtttga 4740

gttcctttag ttcccaatgt atgggctgta ccttgtacat tttagataaa aacggttatc 4800

ccatgcccaa aaataaaccc ggtattgggg aattggcctt aggtcccgtg atgtttgggg 4860

ccagtaaaac cttgttgaac ggcaaccatc atgatgtgta ctttaagggt atgcccacct 4920

taaatgggga agtgttgcgt cggcatggtg atatttttga attaacctcc aacggctact 4980

atcatgccca tggtcgggcc gatgatacca tgaatattgg gggcattaaa attagttcca 5040

ttgaaattga acgtgtgtgt aatgaagtgg atgatcgggt gtttgaaacc accgccattg 5100

gcgtgccccc cttaggtggt ggtcccgaac aattggtgat tttctttgtg ttgaaagata 5160

gtaacgatac caccattgat ttgaaccaat tgcgtttgtc ctttaacttg ggtttgcaga 5220

aaaaattgaa ccccttgttt aaagtgaccc gcgtggtgcc cttgagttcc ttaccccgca 5280

ccgccaccaa taaaattatg cgccgtgtgt tgcggcaaca attttcccat tttgaataag 5340

gatccgcggc tgcagtcgac cggtgtttgg attgtcggag ttgtactcgt ccgttaagga 5400

tgaacagttc ttcggggttg agtctgctaa ctaattagcc attaacagcg gcttaactaa 5460

cagttagtca ttggcaattg tcaaaaaatt gttaatcagc caaaacccac tgcttactga 5520

tgttcaactt cgacagcaat ttaccaatta ccggaagctt gcatgcctgc ag 5572

Claims

1. A construct for the synthesis of olivinic acid comprising:

a promoter active in cyanobacteria, and

a first gene and a second gene under the control of the promoter, wherein the first gene is a gene combining a crotone synthetase gene and an olive alcohol acid cyclase gene, the sequence of the gene is shown as SEQ ID NO.2, and the second gene is an acyl activating enzyme gene, the sequence of the gene is shown as SEQ ID NO. 3.

2. Construct for the synthesis of olivinic acid according to claim 1, characterized in that: which comprises a marker gene for screening cyanobacteria transformants upstream of the promoter active in cyanobacteria.

3. Construct for the synthesis of olivinic acid according to claim 2, characterized in that: the marker gene is a spectinomycin resistance gene fragment, and the sequence of the spectinomycin resistance gene fragment is shown in SEQ ID NO. 4.

4. Construct for the synthesis of olivinic acid according to claim 1, characterized in that: it has the N-terminal sequence and the C-terminal sequence of the slr0168 gene of Synechocystis PCC6803 at both ends for homologous recombination.

5. Construct for the synthesis of olivinic acid according to claim 1, characterized in that: the promoter active in said cyanobacteria isP _cpc560。

6. Construct for the synthesis of olivinic acid according to any one of claims 1 to 5, characterized in that: the cyanobacterium is synechocystis PCC 6803.

7. A carrier, characterized by: comprising the construct for the synthesis of olivinic acid according to any one of claims 1 to 6.

8. A cyanobacterium transformed with the vector of claim 7.

9. A cyanobacterium, comprising: comprising the construct for the synthesis of olivinic acid according to any one of claims 1 to 6.

10. A method of producing olivinic acid in a cyanobacterium, comprising:

cultivating the cyanobacterium of claim 8 or 9 under conditions suitable for synthesizing olivinic acid; and extracting said olivinic acid from the resulting culture.