CN112662696B

CN112662696B - Engineering cyanobacteria for biosynthesizing p-soyabean aromatic acid and preparation method thereof

Info

Publication number: CN112662696B
Application number: CN202011621035.9A
Authority: CN
Inventors: 高恶斌; 詹诸明
Original assignee: Jiangsu Yongze Biotechnology Co ltd
Current assignee: Jiangsu Yongze Biotechnology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2023-07-07
Anticipated expiration: 2040-12-30
Also published as: CN112662696A

Abstract

The invention belongs to the technical field of bioengineering, and discloses engineering cyanobacteria for synthesizing p-coumaric acid and a preparation method thereof, wherein a carrier for synthesizing p-coumaric acid comprises a phenylalanine enzyme gene and a cinnamic acid-4-hydroxylase gene which are operably connected with a promoter active in cyanobacteria, the phenylalanine enzyme gene codes for a protein with phenylalanine enzyme activity, and the cinnamic acid-4-hydroxylase gene codes for a protein with cinnamic acid-4-hydroxylase activity. The invention utilizes solar energy to fix carbon dioxide to synthesize the p-coumaric acid in the photosynthetic microorganism cyanobacteria, the energy for synthesizing the p-coumaric acid is derived from solar energy, and the carbon source is derived from carbon dioxide, so that compared with the extraction or chemical synthesis of plants, the cost can be effectively reduced, the natural products of the plants prepared by the technology cannot be limited by insufficient raw materials, the microorganism growth speed is high, the genetic operation technology is mature, and the fermentation process cannot pollute and destroy the environment.

Description

Engineering cyanobacteria for biosynthesizing p-soyabean aromatic acid and preparation method thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to engineering cyanobacteria for biosynthesizing p-coumaric acid and a preparation method thereof.

Background

Currently, p-coumaric acid, also known as "4-hydroxycinnamic acid", is one of the natural products that are widely found in plants in nature. The p-soyabean acid is mainly present in fruits, vegetables, grains and fungi, and especially in Chinese herbal medicines, the p-soyabean acid is abundant in content. The p-soyabean aromatic acid is used as an important precursor compound of phenylpropanoids, astragalus and flavonoids, has biological activities beneficial to human bodies such as antioxidation, anti-inflammatory, anti-tumor, anti-platelet aggregation, cardiovascular protection, prevention and improvement of diabetes mellitus and neuroprotection, and has wide application value in biological medicine, cosmetics and food industry. In addition, p-coumaric acid is an intermediate of a secondary metabolic pathway of plant phenylalanine, and is an intermediate for synthesizing high-value health care nutrition products such as flavonoid, isoflavone, stilbene and the like, such as azalea extract, naringenin, anthocyanin, quercetin, catechin, resveratrol and the like. It is also an important organic chemical raw material, and is widely applied to the preparation of fine chemical products such as medicines, essence and spices. At present, the main sources of p-coumaric acid are plant extraction, chemical synthesis and biosynthesis. Plant extraction faces the problems of long growth period, low yield, large environmental impact and the like, while chemical synthesis faces the problems of high energy consumption, low yield, environmental pollution and the like. The method realizes the synthesis of the natural plant product p-coumaric acid by microbial fermentation through a biotechnology method, can effectively reduce the cost, has the advantages of high yield, green environmental protection and the like, and is the most promising production method in the 21 st century.

Cyanobacteria, cyanobacteria (Cyanobacteria), is a prokaryote that performs photosynthesis, also known as blue-green algae or Cyanobacteria. Blue algae has simple structure, no forming cell nucleus, no chloroplast organelle of higher plant and the like, and the photosynthesis of blue algae is carried out on thylakoid membrane. Blue algae are the earliest algae on earth and foster the production of all aerobic organisms. Blue algae as a prokaryote which can photosynthesis as higher plants, many scientists consider blue algae as a "photosynthetic reactor" by fixing CO ₂ Namely, a great amount of chemical products and natural plant products required by human beings can be biosynthesized. Genetic engineering of cyanobacteria for biosynthesis has many advantages, such as simple cell structure, easy genetic manipulation, faster growth rate, and no climate and environmental exposureLand restrictions, etc. Importantly, blue algae has higher solar energy utilization efficiency, has lower nutrition requirement than most microorganisms, and only needs solar energy, water and CO ₂ Inorganic nutrients such as N and P are not dependent on organic carbon sources. In addition, cyanobacteria has many unique advantages inherent in the production of plant natural products. In the synthesis of natural plant products, a number of plant enzymes are involved, of which cytochrome P450 monooxygenases are critical for the synthesis of natural products such as phenylpropanes, alkaloids and terpenes. However, many eukaryotic P450 monooxygenases are membrane-bound proteins and are difficult to heterologously express in most prokaryotic microorganisms such as E.coli, because such microorganisms lack endogenous membrane systems. Blue algae, unlike most prokaryotic microorganisms, possess thylakoid membrane systems within their cells, enabling functional expression of plant-derived P450 monooxygenases. Therefore, the advantages make cyanobacteria a stand platform for photosynthetic production of plant natural products, and have good prospects and wide application values.

Through the above analysis, the problems and defects existing in the prior art are as follows: the prior p-coumaric acid mainly sources are plant extraction, chemical synthesis and biological synthesis, the plant extraction faces the problems of long growth period, low yield, large influence on environment and the like, and the chemical synthesis faces the problems of high energy consumption, low yield, environmental pollution and the like.

The difficulty of solving the problems and the defects is as follows: the blue algae is utilized to synthesize natural plant products, and the plant source enzyme suitability, the reducing power and the energy supply, the optimization of the blue algae photosynthetic efficiency, the discharge of the synthetic products and the like are further required to be studied.

The meaning of solving the problems and the defects is as follows: the photosynthetic blue algae is utilized to synthesize the natural plant products, so that the natural plant products with high value can be more efficiently synthesized, the limitations of limited plant planting area, slow growth, low compound content, multiple molecular analogues and the like are reduced, and technical support is provided for human beings to well develop and utilize the natural plant products and to realize large-scale industrial application.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides engineering cyanobacteria for biologically synthesizing p-coumaric acid and a preparation method thereof.

The invention is realized in such a way that the engineering cyanobacteria for synthesizing the p-bean aromatic acid is cyanobacteria Synechocystis sp.PCC 6803 strain which is preserved in the China general microbiological culture Collection center with the preservation numbers of CGMCC No.5882, CGMCC No.5883 or CGMCC No.5884.

It is another object of the present invention to provide a vector for biosynthesis of p-coumaric acid based on cyanobacteria Synechocystis sp.pcc 6803 strain comprising a phenylalanine enzyme gene and a cinnamic acid-4-hydroxylase gene operably linked to a promoter active in cyanobacteria;

the DNA sequence of the phenylalanine enzyme gene is SEQ ID NO:1, a sequence shown in seq id no; the DNA sequence of the cinnamic acid-4-hydroxylase gene is SEQ ID NO:2, and a sequence shown in seq id no.

Further, the vector has an upstream fragment and a downstream fragment of a cyanobacteria gene at both ends, and a phenylalanine enzyme gene and a cinnamic acid-4-hydroxylase gene are integrated into a cyanobacteria genome at a position where the cyanobacteria gene is located by homologous recombination; the upstream fragment gene DNA sequence is SEQ ID NO. 3, and the downstream fragment gene DNA sequence is SEQ ID NO. 4.

It is another object of the present invention to provide a protein encoded by the vector for biosynthesis of p-coumaric acid, the protein comprising: a protein having phenylalanine enzyme activity encoded by a phenylalanine enzyme gene, and a cinnamic acid-4-hydroxylase gene encoding a protein having cinnamic acid-4-hydroxylase activity;

the PAL gene has the sequence of SEQ ID NO:1 or the PAL gene encodes the nucleotide sequence shown in SEQ ID NO:10, and a polypeptide comprising the amino acid sequence shown in seq id no;

the C4H gene has the sequence of SEQ ID NO:2 or the C4H gene encodes the nucleotide sequence set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.

It is another object of the present invention to provide a kit for detecting p-coumaric acid, comprising the carrier of the bio-synthetic p-coumaric acid.

Another object of the present invention is to provide a first carrier of bio-synthetic p-cyamoxic acid using the carrier of bio-synthetic p-cyamoxic acid, wherein the first carrier of bio-synthetic p-cyamoxic acid is a first carrier constructed by cyanobacteria gene slr 0168;

the slr0168 gene (see NCBI accession number BAA 10047.1) is a gene encoding an unknown functional protein in the genome of synechocystis PCC6803, and previous studies have shown that the deletion of the gene has no effect on the physiological activity of cells, so the position of the gene is considered to be a neutral site in the genome of synechocystis PCC 6803;

the upstream fragment of the slr0168 gene has the sequence of SEQ ID NO:3 and the downstream fragment of the slr0168 gene has the sequence shown in SEQ ID NO: 4;

the first vector comprises a promoter that is copper ion inducible and, if the promoter is a PpetE promoter, has the sequence of SEQ ID NO:5, a sequence shown in seq id no;

the first vector further comprises a marker gene for screening cyanobacterial transformants, if selected from the group consisting of a spectinomycin resistance gene, having the sequence of SEQ ID NO: 8.

Another object of the present invention is to provide a second carrier of bio-synthetic p-coumaric acid using the carrier of bio-synthetic p-coumaric acid, wherein the second carrier of bio-synthetic p-coumaric acid is: constructing a second vector by using the cyanobacteria slr2081 gene;

the slr2081 gene (see NCBI accession number: BAK 50142.1) encodes a prephenate dehydrogenase in the Synechocystis PCC6803 genome, and the deletion of the gene may increase the accumulation of coumaric acid; the upstream fragment of the slr2081 gene has the sequence of SEQ ID NO:6 and the downstream fragment of the slr2081 gene has the sequence shown in SEQ ID NO: 7;

the second vector comprises a promoter which is a strong light-induced promoter, and if the promoter is a cpcB promoter, the promoter has the sequence shown in SEQ ID NO: 9;

the second vector further comprises a marker gene for screening cyanobacterial transformants, if selected from the group consisting of spectinomycin resistance gene, having the sequence of SEQ ID NO: 8.

Another object of the present invention is to provide a method for obtaining p-soyabean aromatic acid, which comprises:

the strain of the engineering cyanobacteria expresses phenylalanine enzyme genes for catalyzing one molecule of phenylalanine to remove one molecule of amino, and deamination is carried out to generate cinnamic acid;

the expressed cinnamic acid-4-hydroxylase gene is used for catalyzing hydroxylation reaction of para position of cinnamic acid benzene ring to generate p-coumaric acid, so that p-coumaric acid is obtained from the culture.

The method for obtaining the p-soyabean aromatic acid further comprises the following steps:

the engineering cyanobacteria obtained by construction can utilize solar immobilized carbon dioxide as a carbon source, and phenylalanine is used as an initial substrate to synthesize the p-coumaric acid.

Another object of the present invention is to provide a microorganism of a second vector for p-stigma nucleic acid, which does not contain or knocks out the slr2081 gene, and highly expresses the phenylalanine enzyme gene PAL and the cinnamic acid-4-hydroxylase gene C4H;

By combining all the technical schemes, the invention has the advantages and positive effects that:

in the present invention, the phenylalanine enzyme gene is derived from ATCC20310 of Rhodotorula glutinis (Rhodotorula glutinis), and has the nucleotide sequence of SEQ ID NO optimized according to the codon usage of Synechocystis sp.PCC 6803: 1, a sequence shown in seq id no; the cinnamic acid-4-hydroxylase gene is derived from Arabidopsis thaliana (Arabidopsis thaliana), and is optimized for its codon usage in accordance with Synechocystis sp.PCC 6803 with the sequence of SEQ ID NO:2, and a sequence shown in seq id no.

And introducing the second vector into engineering cyanobacteria obtained by cyanobacteria, knocking out the slr2081 gene in the strain, and the strain with the gene knocked out is named SYN005. The culture medium is preserved in China center for type culture Collection (CCTCC NO: M2020870) (see a preservation proving annex 1 in detail).

According to the invention, the p-coumaric acid is synthesized by fixing carbon dioxide in the photosynthetic microorganism cyanobacteria by utilizing solar energy, the energy for synthesizing the p-coumaric acid is derived from the solar energy, and the carbon source is derived from the carbon dioxide, so that the cost can be effectively reduced compared with the extraction or chemical synthesis of plants. Therefore, the plant natural product prepared by the technology is not limited by insufficient raw materials, meanwhile, the microorganism growth speed is high, the genetic operation technology is mature, and the fermentation process can not pollute and destroy the environment.

Drawings

FIG. 1 is a diagram of the biosynthesis pathway of p-coumaric acid provided by the example of the present invention.

FIG. 2 is a schematic diagram of the vector pJS407-PAL/C4H provided by an embodiment of the present invention.

FIG. 3 is a schematic diagram of the vector pJS010-PAL/C4H provided by an embodiment of the present invention.

Fig. 4 (a) -4 (b) are schematic diagrams showing the yield of the cyanobacteria SYN005 pair of coumaric acid according to the examples of the present invention.

Fig. 5 (a) shows the peak detection of the p-coumaric acid standard sample by HPLC according to the embodiment of the present invention, and the peak time is 15.726s.

FIG. 5 (b) shows the HPLC detection peak of the Syn0168 product provided by the example of the present invention, wherein peak 1 is p-coumaric acid, and the peak time is 15.685s.

FIG. 5 (c) shows the HPLC detection peak of the Syn2081 product, wherein peak 1 is p-coumaric acid, and the peak time is 15.740s.

Description of the embodiments

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

In view of the problems of the prior art, the present invention provides an engineering cyanobacteria for biosynthesis of p-coumaric acid and a preparation method thereof, and the present invention is described in detail below with reference to the accompanying drawings and examples.

The invention provides engineering cyanobacteria for biosynthesizing p-soyabean aromatic acid, which is cyanobacteria Synechocystis sp.PCC 6803 strain and is preserved in China general microbiological culture Collection center (CGMCC) No.5882, CGMCC No.5883 or CGMCC No.5884.

Example 1: vector construction for engineering cyanobacteria producing p-soyabean aroma

To demonstrate the role of alanine enzymes and cinnamic acid-4-hydroxylase in cyanobacteria synthesis of p-stigmatocystic acid and to increase the yield of p-stigmatocystic acid synthesized in cyanobacteria by increasing the expression of the enzymes, a vector pCA0168 was constructed for integration of the phenylalanine enzyme gene (PAL) and the cinnamic acid-4-hydroxylase gene (C4H) driven by the Pcpc560 promoter into the slr0168 locus of the cyanobacteria genome. To further increase the expression levels of alanine enzymes and cinnamic acid-4-hydroxylase and thus increase the yield of p-bean aromatic acid synthesized by cyanobacteria, a vector pCA2081 for integrating the phenylalanine enzyme gene (PAL) and the cinnamic acid-4-hydroxylase gene (C4H) driven by the Pcpc560 promoter into the slr2081 locus of cyanobacteria genome was constructed.

The above vector converts cyanobacteria PCC6803 to obtain SYN005, and the specific implementation steps are as follows:

1. extraction of cyanobacteria PCC6803 DNA

Centrifuging 1ml algae solution, re-suspending in 200 μl TE, adding appropriate amount of quartz sand, 100 μl saturated phenol and 100 μl chloroform/isoamyl alcohol (24:1), shaking vigorously for 1min, standing on ice for 1min, centrifuging (12,000 rpm, 5 min) after repeating the above three times, and settling supernatant with 1/5 volume of 5M NaCl and 2 times volume of absolute ethanol at-20deg.C for 1 hr; centrifuge (12,000 rpm, 10 min), wash once with 70% ethanol, air dry at room temperature, and then dissolve in 10. Mu.l TE buffer.

2. Construction of vector pJS407-PAL/C4H

Taking the extracted cyanobacteria PCC6803 genome DNA as a template, and taking the DNA of SEQ ID NO:12: the primers Slr0168D1 (5'-cgg ggt acc tcc caa cag tga ata caa CG-3') and Slr0168D2 (5'-tgc gag ctc gac cat tct ctg gat cat tg-3') were PCR amplified, and the obtained PCR product was cloned into the HindIII, ecoRI site of pMD18-T vector (Takara) according to the manufacturer's instructions, thereby obtaining vector pMD0168D. Taking the extracted cyanobacteria PCC6803 genome DNA as a template, and taking the DNA of SEQ ID NO:1 3: slr0168U1 (5'-cgg aag ctt tgt cac cgc tac gtt agg ctt c-3') and Slr0168U2 (5'-ccg ctg cag cca tat aac cat caa agc ca-3') are used as primers for PCR amplification, and the obtained PCR product is cloned to a proper site of pMD0168D to obtain a vector pMD-slr0168.

Plasmid pJS406 (laboratory plasmid, containing spectinomycin resistance gene) was used as template, SEQ ID NO:14: spF (5'-ccg ctg cag ctc acg caa ctg gtc cag aa-3') and SpR (5'-gtg atc tag agt gct tag tgc atc taa cgc t-3') are used as primers for PCR amplification, and a spectinomycin resistance gene fragment is inserted between the two target fragments in a vector pMD0168 to obtain a recombinant plasmid pMD 0168-omega.

Taking the extracted cyanobacteria PCC6803 genome as a template, respectively SEQ ID NOs: 15: pcpcF (5'-agc gtt aga tgc act aag cac tct aga tca cca ttt gga caa aac atc ag-3') and pccr (5'-gct cct agg atc ctt aag acg cgt ttt ctc ctc ttt cta ggt cag tcc tcc ata aac-3');

SEQ ID NO:16: PCR amplification was performed using TrbcF (5'-cgt ctt aag gat cct agg agc tct gca gat cta ccg gtg ttt gga ttg tcg gag ttg-3') and Trbc-R1 (5'-cgt tgt att cac tgt tgg gat aat tgg taa att gct gtc gaa g-3') as primers to obtain 500bp and 200bp products, respectively, and the obtained sequences of the promoter Pcpc560 and TrbcL terminator were inserted into the BamHI site of the vector pMD-slr0168- Ω according to the manufacturer's instructions of the seamless junction kit (Biyun, D7010M) to obtain a recombinant plasmid pJS407.

The sequence represented by SEQ ID NO:17: performing PCR amplification by taking PAL-Bgl (5 'agg aga tta att caa aag agg aga aag atc tat ggc ccc ctc cgt gga ttc cat-3') and PAL-R (5'-tcc aac agc aac aaa tcc atg tcg act gca gtt agg cca tca ttt tca c-3') as primers and taking codon-optimized rhodotorula glutinis (Rhodotorula glutinis) PAL gene as a template, and cutting and recovering a product; the sequence represented by SEQ ID NO:18: C4H-F (5'-atg gat ttg ttg ctg ttg gaa-3') and C4H-Sal (5'-cga caa tcc aaa cac cgg tcg act taa caa ttc cgg ggt ttc atc aca-3') are used as primers, the codon-optimized Arabidopsis thaliana (Arabidopsis thaliana) C4H gene is used as a template for PCR amplification, and the product is cut and recovered. The two PCR amplified products obtained above were cloned between BglII and SalI sites of vector pJS407 according to the manufacturer's instructions of the seamless junction kit (Biyun, D7010M) to obtain recombinant plasmid pJS407-PAL/C4H, and the correct sequence was verified by sequencing, the basic structure is shown in FIG. 2.

3. Construction of vector pJS010-PAL/C4H

Taking the extracted cyanobacteria PCC6803 genome DNA as a template, and taking the DNA of SEQ ID NO:19: slr2081D1 (5'-cgg aag ctt cgc aat gct agc agc cca agA-3') and Slr2081D2 (5'-ccg ctg cag caa gtc tcc cgc caa gga ag-3') primers were PCR amplified and the PCR product obtained was cloned between HindIII and PstI sites of the pMD18-T vector (Takara) according to the manufacturer's instructions to give vector pMD2081D. Taking the extracted cyanobacteria PCC6803 genome DNA as a template, and taking the DNA of SEQ ID NO:20: slr2081U1 (5'-cgg ggt acc tac aac aaa cca acg gcg atc gg-3') and Slr2081U2 (5'-tgc gag ctc act gga gga tag gac cac cag c-3') are used as primers for PCR amplification, and the obtained PCR product is cloned between KpnI and SacI sites of pMD2081D to obtain a vector pMD2081.

Plasmid pJS406 (laboratory plasmid, containing spectinomycin resistance gene) was used as template, SEQ ID NO:21: spF (5'-ccg ctg cag ctc acg caa ctg gtc cag aa-3') and SpR (5'-gtg atc tag agt gct tag tgc atc taa cgc t-3') are used as primers for PCR amplification, and a spectinomycin resistance gene fragment is inserted between the two target fragments in a vector pMD0168 to obtain a recombinant plasmid pMD 0168-omega.

Taking the extracted cyanobacteria PCC6803 genome as a template, respectively taking SEQ ID NOs: 22: pcpcF (5'-agc gtt aga tgc act aag cac tct aga tca cca ttt gga caa aac atc ag-3') and pccr (5'-gct cct agg atc ctt aag acg cgt ttt ctc ctc ttt cta ggt cag tcc tcc ata aac-3');

SEQ ID NO:23: PCR amplification was performed using TrbcF (5'-cgt ctt aag gat cct agg agc tct gca gat cta ccg gtg ttt gga ttg tcg gag ttg-3') and Trbc-R2 (5'-ccg atc gcc gtt ggt ttg ttg tag gta ccg cgg taa ttg gta aat tgc tgt cg-3') as primers to obtain 500bp and 200bp products, respectively, and the obtained sequences of the promoter Pcpc560 and TrbcL terminator were inserted into the BamHI site of the vector pMD2081- Ω according to the manufacturer's instructions of the seamless junction kit (Biyun, D7010M) to obtain a recombinant plasmid pJS010.

The sequence represented by SEQ ID NO:24: performing PCR amplification by taking PAL-Mul (5'-gac tga cct aga aag agg aga aaa cgc gta tgg ccc cct ccg tgg att cca-3') and PAL-R (5'-tcc aac agc aac aaa tcc atg tcg act gca gtt agg cca tca ttt tca c-3') as primers and taking the codon-optimized rhodotorula glutinis (Rhodotorula glutinis) PAL gene as a template, and cutting and recovering a product; the sequence represented by SEQ ID NO:25: C4H-F (5'-atg gat ttg ttg ctg ttg gaa-3') and C4H-Afl (5'-ctg cag agc tcc tag gat cct taa gtt aac aat tcc ggg gtt tca tca c-3') are used as primers, the codon-optimized Arabidopsis thaliana (Arabidopsis thaliana) C4H gene is used as a template for PCR amplification, and the product is cut and recovered. According to the manufacturer's instructions of the seamless connection kit (Biyun Tian, D7010M), the two PCR amplified products obtained above are cloned between MulI and AflII sites of the vector pJS010 to obtain a recombinant plasmid pJS010-PAL/C4H, and the correct sequence is verified by sequencing, and the basic structure is shown in figure 3.

Example 2: transformation of cyanobacteria and selection of transformants

(1) Cyanobacteria PCC6803 was cultured in BG11 medium at 30℃under continuous light with a light intensity of 30. Mu. E m-2 s-1. Glucose was added to BG11 at a final concentration of 5mM during the mixed culture. In the case of passaging or screening on solid plates, the BG11 medium is supplemented with agar powder to a final concentration of 1.4%, TES-NaOH (pH 8.0) to 8mM, na ₂ S ₂ O ₃ To 0.3% and glucose to 5mM.

(2) Taking 10mL of cyanobacteria cells in a logarithmic growth phase (OD 730 is about 0.5-1.0), and centrifugally collecting the cells; the cells were washed twice with fresh BG11 medium and resuspended in 1mL BG11 medium (1.5 g/L NaNO ₃ ，40mg/L K ₂ HPO ₄ ·3H ₂ O，36mg/L CaCl ₂ ·2H ₂ O,6mg/L citric acid, 6mg/L ferric ammonium citrate, 1mg/L EDTA disodium salt, 20mg/L NaCO ₃ ，2.9mg/LH ₃ BO ₃ ，1.8mg/LMnCl ₂ ·4H ₂ O, 0.22mg/LZnSO ₄ ·7H ₂ O，0.39mg/LNaMoO ₄ ·2H ₂ O，0.079mg/L CuSO ₄ ·5H ₂ O and 0.01mg/L CoCl ₂ ·6H ₂ O).

(3) 0.2mL of the cell suspension was placed in a new EP tube, 2-3. Mu.g of the above-mentioned expression plasmid pCA0168 or pCA2081 was added, mixed well, and incubated at 30℃for 5 hours under 30. Mu.Em-2 s-1 illumination.

(4) A mixture of cyanobacterial cells and plasmids was spread on a nitrocellulose membrane spread on a BG11 plate (without antibiotics) and incubated at 30℃for 24 hours under 30. Mu.Em-2 s-1 light. Then, the nitrocellulose membrane was transferred onto BG11 plates containing antibiotics corresponding to the desired strain, and the culture was continued at 30℃under 30. Mu.Em-2 s-1.

(5) After about 5-7 days of culture, transformants were picked from the plates and streaked on fresh BG11 plates (containing the corresponding antibiotics); after the cells are enriched, they are inoculated into liquid BG11 medium (containing the corresponding antibiotics) for culturing.

(6) The transformed cyanobacterial cells were transferred in liquid BG11 medium (containing the corresponding antibiotics) two to three times and used to detect the production of p-coumaric acid after verification of correct introduction of the construct of interest by genome sequencing.

The cyanobacteria were transformed by the above procedure to obtain engineering strains producing p-bean aromatic acid (see Table 1).

TABLE 1 engineering cyanobacteria for the synthesis of p-soyabean acids

Strain	Plasmid(s)	Features (e.g. a character)
			SYN005	pJS010-PAL/C4H	Knockout cyanobacteria PCC6803 endogenous gene slr2081, and expression synthesis of exogenous genes PAL and C4H of p-coumaric acid, has spectinomycin resistance

Example 3: genetically engineered cyanobacteria p-soyabean acid production

1. The experimental steps are as follows: a transparent conical cell culture flask is adopted, 150mL of liquid BG11 culture medium (containing the corresponding resistance) is filled, the initial inoculation concentration is OD730 = 0.1, after continuous illumination culture is carried out for one week under the illumination condition of 30 mu Em-2s-1 at the temperature of 30 ℃, when the OD730 is 1.5, cell sediment and supernatant are collected by centrifugation, and cells are broken by a cell breaker and are respectively used for detection by a high performance liquid chromatograph.

2. Experimental results

The inventors detected p-coumaric acid production in cyanobacteria PCC6803 and SYN005, respectively, and no p-coumaric acid production in wild cyanobacteria PCC6803, and SYN005 detected p-coumaric acid production. FIGS. 4 and 5 show the growth of cyanobacteria PCC6803 and SYN005, respectively, and the production of p-coumaric acid.

The results showed that the wild-type PCC6803 itself was not capable of producing p-coumaric acid, and SYN005 obtained by exogenously introducing phenylalanine enzyme and cinnamic acid-4-hydroxylase was capable of producing p-coumaric acid, and that the yield of p-coumaric acid from SYN005 was 0.197.+ -. 0.032g/L and 0.028.+ -. 0.005gL-1d-1 as accumulated at day 7. The result shows that the phenylalanine enzyme and the cinnamic acid-4-hydroxylase can be expressed in cyanobacteria to effectively synthesize the p-coumaric acid, and the knocking out of the endogenous gene slr2081 can obviously improve the yield of the p-coumaric acid, thereby providing favorable conditions for mass production of plant natural products, i.e. the p-coumaric acid by using cyanobacteria.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Sequence listing

<110> Shanghai spring Biotechnology Co., ltd

<120> engineering cyanobacteria for biosynthesis of p-coumaric acid and preparation method thereof

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<170> SIPOSequenceListing 1.0

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atggccccct ccgtggattc cattgccacc agtgtggcca attccttgag taatggcttg 60

catgccgccg ccgccgccaa tggtggtgat gtgcataaga aaaccgccgg tgccggcagt 120

ttgttgccca ccaccgaaac cacccaattg gatattgtgg aacgcatttt ggccgatgcc 180

ggcgccaccg atcaaattaa attggatggc tataccttga ccttaggcga tgttgtgggt 240

gccgcccggc gtggccggag tgtgaaagtg gccgatagtc cccatattcg cgaaaaaatt 300

gatgcctccg tggaattttt gcgtacccaa ctggataatt ccgtgtatgg tgtgaccacc 360

ggctttggcg gttccgccga tacccggacc gaagatgcca ttagtttgca aaaagcctta 420

ttagaacatc aattgtgtgg tgtgttaccc accagtatgg atggctttgc cttagggcgc 480

ggcttggaaa attccttgcc cttagaagtg gtgcgcggcg ccatgaccat tcgggtgaat 540

agcttaaccc gtggtcatag tgccgtgcgg attgtggtgt tagaagcctt gaccaacttt 600

ttgaatcatg gcattacccc cattgttccc ttacggggca ccatttccgc ctccggcgat 660

ttgtccccct tgagttatat tgccgcctcc attaccgggc atcccgatag taaagtgcat 720

gtggatggca aaattatgtc cgcccaagaa gccattgcct tgaaaggttt acaacccgtg 780

gtgttaggcc ccaaagaagg cttaggcttg gtgaatggta ccgccgtgag tgccagtatg 840

gccaccttag ccttaaccga tgcccatgtt ttaagtttgt tagcccaagc cttaactgcc 900

ctgaccgtgg aagccatggt gggccatgcc ggcagttttc atcccttttt acatgacgtg 960

acccggcccc atcccactca aattgaagtg gcccgtaata ttcggacctt attagaaggt 1020

agtaaatatg ccgtgcacca tgaaaccgaa gtgaaagtga aagatgatga aggcattttg 1080

cggcaagatc gctatccctt acggtgtagt ccccagtggt taggcccctt agtgagtgat 1140

atgattcatg cccatgccgt gttgagttta gaagccggcc aatccaccac cgataatccc 1200

ttaattgatt tggaaaataa aatgacccat catggcggcg cctttatggc ctctagtgtg 1260

gggaatacca tggaaaaaac ccgtttggcc gtggccttga tgggcaaagt gagttttacc 1320

caattaactg aaatgttaaa tgccgggatg aatcgtgccc tgccctcctg tttagccgct 1380

gaagatccct ccctaagtta tcattgtaaa ggcttggata ttgcggccgc cgcctatact 1440

agtgaattag gccatttagc caatcccgtg agcacccatg tgcaacccgc cgaaatgggt 1500

aatcaagcca ttaatagttt agccttgatt agtgcccggc ggaccgccga agctaatgat 1560

gtgttaagtt tattattagc cacccatttg tattgtgtgt tgcaagccgt ggatttacgg 1620

gccatggaat ttgaacatac caaagccttt gaacccatgg tgactgaatt attaaaacaa 1680

cattttggcg ccttagccac cgccgaagtg gaagataaag tgcgtaaaag tatttataaa 1740

cggttacaac aaaataatag ttatgattta gaacaacgtt ggcatgatac ctttagtgtg 1800

gccaccgggg ccgtggtgga agccttagcc ggtcaagaag tgagtttagc ctccttgaat 1860

gcctggaaag tggcctgtgc tgaaaaagcc attgccttga cccggtccgt gcgggatagt 1920

ttttgggccg cccccagtag tagtagtccc gccttgaaat atctgagtcc ccggacccgt 1980

gtgttgtata gttttgtgcg ggaagaagtg ggcgtgaaag cccgccgggg cgatgtgtat 2040

ttaggtaaac aagaagtgac cattggtacc aatgtgagtc ggatttatga agccattaaa 2100

tccggtcgga ttgcccccgt gttggtgaaa atgatggcct aa 2142

<210> 2

<211> 1518

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggatttgt tgctgttgga aaaatccttg attgccgtgt ttgtggccgt gattttagcc 60

accgtgatta gtaaattgcg tggtaaaaaa ttaaaattgc cccccggtcc cattcccatt 120

cccatttttg gcaattggct gcaagtgggg gatgatctga atcatcggaa tttagtggat 180

tatgccaaaa aatttggtga tttgtttttg ttgcggatgg gtcaacgcaa tttagtggtg 240

gtgtcctccc ccgatttaac taaagaagtg ttattaaccc agggtgttga atttggaagc 300

cgtacccgca atgttgtgtt tgatattttt accgggaaag gtcaagatat ggtgtttacc 360

gtgtatggtg aacattggcg caaaatgcgt cggattatga ccgttccctt tttcaccaat 420

aaagtggtgc agcaaaatcg tgaaggttgg gaatttgaag ccgccagtgt ggtggaagat 480

gtgaagaaaa atcccgattc cgccaccaaa ggcatcgtgt tgcggaaacg gttgcaatta 540

atgatgtata ataacatgtt tcggattatg tttgatcgtc gctttgaaag tgaagatgat 600

cccttatttc tgcggctgaa agccctgaat ggcgaacgca gtcgcttagc ccaatccttt 660

gaatataatt atggggattt tattccaatt ttacgtccgt ttctgcgtgg gtacttgaaa 720

atttgtcaag atgtcaaaga tcggcggatt gccttattta aaaaatattt tgtggacgaa 780

cggaaacaaa ttgcctcctc caaacccacc gggagtgaag ggttgaaatg tgccattgat 840

catattttag aagctgaaca aaaaggtgaa attaatgaag ataatgtgtt atatattgtg 900

gaaaatatta atgtggccgc cattgaaacc accttgtggt ccattgaatg gggcattgct 960

gaattggtga atcaccctga aattcaatcc aaattacgga atgaattaga tacggtttta 1020

ggtcctggcg tgcaagtgac tgaacccgat ctgcataaac tgccctattt gcaagccgtt 1080

gtgaaagaaa ctctgcggtt acggatggcc attcccttgt tagtccccca tatgaatctc 1140

catgatgcca aactggccgg ttatgatatt cccgccgaaa gtaaaatttt agtgaatgcc 1200

tggtggttgg ccaataatcc caattcctgg aaaaaacccg aagaatttcg gcccgaacgc 1260

ttctttgaag aagaatccca tgtggaagcc aatggcaatg attttcggta tgttcccttt 1320

ggtgtgggtc gccggagttg tcccggcatt attttagcct tgcccatttt gggcattacc 1380

attggccgca tggtgcaaaa ttttgaattg ttgccccccc ccggtcaaag taaagtggat 1440

acctccgaaa aaggcgggca attttccttg catattttga atcattccat tattgtgatg 1500

aaaccccgga attgttaa 1518

<210> 3

<211> 601

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gagcggcacc acggggcacc accgccactc tccgctgcgc ttgacgcagc ggcacaggat 60

gtcctgaccg ccagcgccct tcagcacctg gaaaaactgc agcaccttct tgctgtagtt 120

cttggcgccg ccctgcactg ccgccccccc tcccgcaccc cgccgccgcc ggcctgaggg 180

cgcctgctcg tcgccctgct tgatccggat gatgccctct acatctgact tagtgagaaa 240

cacattcttg gcgccgtttt ggcgccgttc taaggcataa aggatgacag gttggttcat 300

aattttaagt ttgcgtcagc tgcggggtgc gggaggggag aaatagaagc cttagagact 360

ctgaaaggcg aaggaaaggg agggatcgcg ggagagagcg tgcctgatga gccagacgaa 420

tcgggggagg tgccacatca gcgggcaaca catgacaaga ggaaatcacg gaccggtcac 480

gttcccctcc ggaggtaacg ttacctccgg accacgggag aagacagtga cgaattgagt 540

tgcaatgccg aacagctcta cctgctcgct gcggccacac gtcgcacgct gctgtgcgcc 600

a 601

<210> 4

<211> 601

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

agagccatgc ccgacgggcg gcctgcacgg ctccctcgct ctctatcttg ccactacctg 60

gcgtgccgct acctggcgtg cccaagccat tcaatttttt tctgaccact cctgtgaccg 120

ctccccatga catgtacccg tgacaacccc ttcccaatcc atgcattcca aacttctgtc 180

gggactcccc catggggggg ctgaggaaat gttgcgcatg cgcagtgtgc atactgccat 240

cctcatcttc cccctcccaa gcctggacag agaggcgccc aaacatgact tgtaaagtga 300

ttcgttgcag ctggtggcat gtgcgatccc ggggcacgcc ccaagcagtg cggcaaggca 360

catgcaaaat gagaggcagc aggcacgcac ggcgaagcgt tacgagaagg gaatgctctg 420

catcatgagg ccggctgccg cagccgccgc cccgccctgc tgctgctgct gctgctgcat 480

cgcctgctgg ggcgtcagcg tgggggcggc tcccgcggcg gctggcgccg gcggggcgct 540

gccgacgtac agcttgcgca gggcggggag cagcgccagc tggggcggca gctggtcatg 600

c 601

<210> 5

<211> 309

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cggcgatcgc caaaaacaaa gaaaattcag caattaccgt gggtagcaaa aaatccccat 60

ctaaagttca gtaaatatag ctagaacaac caagcatttt cggcaaagta ctattcagat 120

agaacgagaa atgagcttgt tctatccgcc cggggctgag gctgtataat ctacgacggg 180

ctgtcaaaca ttgtgatacc atgggcagaa gaaaggaaaa acgtccctga tcgccttttt 240

gggcacggag tagggcgtta ccccggcccg ttcaaccaca agtccctata gatacaatcg 300

ccaagaagt 309

<210> 6

<211> 610

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgcaatgcta gcagcccaag atgccaatca gtgaagatct caaacaagct attcggcaac 60

gggcaagata tatctgtgaa tattgtcatt ccccagaacg attgagtgca aacagattca 120

ccattgatca tgtaattccg aaatctttag gtggttctga cgcatttaat aaccttgccc 180

ttgcttgtcg tcgttgtaac gagaggcgct acaactttgt cgccagtata gatccacaaa 240

ctaagactat tgttgccatt tttaaccccc gccaacagaa ttgggcagaa catttcattt 300

ggacaaatag agggataata atacaaggaa ccacgacaac cggtcgtgcg acgtgtatcc 360

gacttgattt aaatgatacc cgttatccag aggaagattc tatccaggaa acacgacgat 420

tttggttgaa aacgggactg catcccccca tagatgatcc ttgcaaaaat tgaatcaatt 480

cggtgatttt cttaaccatt tgaacttctc aatggaattt gataaaaatc aatgaagatg 540

acttgaaatt aaatgaaaat tggtgttgtt ggtttgggtt taattggggc ttccttggcg 600

ggagacttgc 610

<210> 7

<211> 608

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tacaacaaac caacggcgat cgggacaagt atgttgaata aaccaatggc gatcgcctgg 60

agaataaatt taggagacta gacattgatc cagaacaaat ttaggaacaa tggggcccac 120

ggcatggctt tctgttaaaa ataaacaagc actgcccttt cccgctcaga gcgcatgact 180

aaccagccca ccgcatcggt tccctttcaa cgcccctggc acgactatct caaattcagc 240

accgatcaca aagtcattgg cattcaatat ttactgatgt ccttctgctt cttcttagtg 300

gcgggattat tggccatgat tatccgggca gaattactca ccccccaatt ggacgtggtg 360

gaccgtagcc tctacaacgg tctatttacc ctccacggca ccatcatgat tttcctctgg 420

atttttccag ccaatgtggg gttagccaat tacttaattc ccctgatgat tggtgccagg 480

gacgtagcct ttccggtatt gaatgcgatc gccttttggc taatgccagt ggtgggagtg 540

ttattgattg gtagcttttt tctacccaca gggacggccc aggcgggctg gtggtcctat 600

cctccagt 608

<210> 8

<211> 792

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc 60

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> 9

<211> 277

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tcaccatttg gacaaaacat caggaattct aattagaaag tccaaaaatt gtaatttaaa 60

aaacagtcaa tggagagcat tgccataagt aaaggcatcc cctgcgtgat aagattacct 120

tcagaaaaca gatagttgct gggttatcgc agatttttct cgcaaccaaa taactgtaaa 180

taataactgt ctctggggcg acggtaggct ttatattgcc aaatttcgcc cgtgggagaa 240

agctaggcta ttcaatgttt atggaggact gacctag 277

<210> 10

<211> 713

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Met Ala Pro Ser Val Asp Ser Ile Ala Thr Ser Val Ala Asn Ser Leu

1 5 10 15

Ser Asn Gly Leu His Ala Ala Ala Ala Ala Asn Gly Gly Asp Val His

20 25 30

Lys Lys Thr Ala Gly Ala Gly Ser Leu Leu Pro Thr Thr Glu Thr Thr

35 40 45

Gln Leu Asp Ile Val Glu Arg Ile Leu Ala Asp Ala Gly Ala Thr Asp

50 55 60

Gln Ile Lys Leu Asp Gly Tyr Thr Leu Thr Leu Gly Asp Val Val Gly

65 70 75 80

Ala Ala Arg Arg Gly Arg Ser Val Lys Val Ala Asp Ser Pro His Ile

85 90 95

Arg Glu Lys Ile Asp Ala Ser Val Glu Phe Leu Arg Thr Gln Leu Asp

100 105 110

Asn Ser Val Tyr Gly Val Thr Thr Gly Phe Gly Gly Ser Ala Asp Thr

115 120 125

Arg Thr Glu Asp Ala Ile Ser Leu Gln Lys Ala Leu Leu Glu His Gln

130 135 140

Leu Cys Gly Val Leu Pro Thr Ser Met Asp Gly Phe Ala Leu Gly Arg

145 150 155 160

Gly Leu Glu Asn Ser Leu Pro Leu Glu Val Val Arg Gly Ala Met Thr

165 170 175

Ile Arg Val Asn Ser Leu Thr Arg Gly His Ser Ala Val Arg Ile Val

180 185 190

Val Leu Glu Ala Leu Thr Asn Phe Leu Asn His Gly Ile Thr Pro Ile

195 200 205

Val Pro Leu Arg Gly Thr Ile Ser Ala Ser Gly Asp Leu Ser Pro Leu

210 215 220

Ser Tyr Ile Ala Ala Ser Ile Thr Gly His Pro Asp Ser Lys Val His

225 230 235 240

Val Asp Gly Lys Ile Met Ser Ala Gln Glu Ala Ile Ala Leu Lys Gly

245 250 255

Leu Gln Pro Val Val Leu Gly Pro Lys Glu Gly Leu Gly Leu Val Asn

260 265 270

Gly Thr Ala Val Ser Ala Ser Met Ala Thr Leu Ala Leu Thr Asp Ala

275 280 285

His Val Leu Ser Leu Leu Ala Gln Ala Leu Thr Ala Leu Thr Val Glu

290 295 300

Ala Met Val Gly His Ala Gly Ser Phe His Pro Phe Leu His Asp Val

305 310 315 320

Thr Arg Pro His Pro Thr Gln Ile Glu Val Ala Arg Asn Ile Arg Thr

325 330 335

Leu Leu Glu Gly Ser Lys Tyr Ala Val His His Glu Thr Glu Val Lys

340 345 350

Val Lys Asp Asp Glu Gly Ile Leu Arg Gln Asp Arg Tyr Pro Leu Arg

355 360 365

Cys Ser Pro Gln Trp Leu Gly Pro Leu Val Ser Asp Met Ile His Ala

370 375 380

His Ala Val Leu Ser Leu Glu Ala Gly Gln Ser Thr Thr Asp Asn Pro

385 390 395 400

Leu Ile Asp Leu Glu Asn Lys Met Thr His His Gly Gly Ala Phe Met

405 410 415

Ala Ser Ser Val Gly Asn Thr Met Glu Lys Thr Arg Leu Ala Val Ala

420 425 430

Leu Met Gly Lys Val Ser Phe Thr Gln Leu Thr Glu Met Leu Asn Ala

435 440 445

Gly Met Asn Arg Ala Leu Pro Ser Cys Leu Ala Ala Glu Asp Pro Ser

450 455 460

Leu Ser Tyr His Cys Lys Gly Leu Asp Ile Ala Ala Ala Ala Tyr Thr

465 470 475 480

Ser Glu Leu Gly His Leu Ala Asn Pro Val Ser Thr His Val Gln Pro

485 490 495

Ala Glu Met Gly Asn Gln Ala Ile Asn Ser Leu Ala Leu Ile Ser Ala

500 505 510

Arg Arg Thr Ala Glu Ala Asn Asp Val Leu Ser Leu Leu Leu Ala Thr

515 520 525

His Leu Tyr Cys Val Leu Gln Ala Val Asp Leu Arg Ala Met Glu Phe

530 535 540

Glu His Thr Lys Ala Phe Glu Pro Met Val Thr Glu Leu Leu Lys Gln

545 550 555 560

His Phe Gly Ala Leu Ala Thr Ala Glu Val Glu Asp Lys Val Arg Lys

565 570 575

Ser Ile Tyr Lys Arg Leu Gln Gln Asn Asn Ser Tyr Asp Leu Glu Gln

580 585 590

Arg Trp His Asp Thr Phe Ser Val Ala Thr Gly Ala Val Val Glu Ala

595 600 605

Leu Ala Gly Gln Glu Val Ser Leu Ala Ser Leu Asn Ala Trp Lys Val

610 615 620

Ala Cys Ala Glu Lys Ala Ile Ala Leu Thr Arg Ser Val Arg Asp Ser

625 630 635 640

Phe Trp Ala Ala Pro Ser Ser Ser Ser Pro Ala Leu Lys Tyr Leu Ser

645 650 655

Pro Arg Thr Arg Val Leu Tyr Ser Phe Val Arg Glu Glu Val Gly Val

660 665 670

Lys Ala Arg Arg Gly Asp Val Tyr Leu Gly Lys Gln Glu Val Thr Ile

675 680 685

Gly Thr Asn Val Ser Arg Ile Tyr Glu Ala Ile Lys Ser Gly Arg Ile

690 695 700

Ala Pro Val Leu Val Lys Met Met Ala

705 710

<210> 11

<211> 505

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Met Asp Leu Leu Leu Leu Glu Lys Ser Leu Ile Ala Val Phe Val Ala

1 5 10 15

Val Ile Leu Ala Thr Val Ile Ser Lys Leu Arg Gly Lys Lys Leu Lys

20 25 30

Leu Pro Pro Gly Pro Ile Pro Ile Pro Ile Phe Gly Asn Trp Leu Gln

35 40 45

Val Gly Asp Asp Leu Asn His Arg Asn Leu Val Asp Tyr Ala Lys Lys

50 55 60

Phe Gly Asp Leu Phe Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val

65 70 75 80

Val Ser Ser Pro Asp Leu Thr Lys Glu Val Leu Leu Thr Gln Gly Val

85 90 95

Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly

100 105 110

Lys Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys

115 120 125

Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln

130 135 140

Gln Asn Arg Glu Gly Trp Glu Phe Glu Ala Ala Ser Val Val Glu Asp

145 150 155 160

Val Lys Lys Asn Pro Asp Ser Ala Thr Lys Gly Ile Val Leu Arg Lys

165 170 175

Arg Leu Gln Leu Met Met Tyr Asn Asn Met Phe Arg Ile Met Phe Asp

180 185 190

Arg Arg Phe Glu Ser Glu Asp Asp Pro Leu Phe Leu Arg Leu Lys Ala

195 200 205

Leu Asn Gly Glu Arg Ser Arg Leu Ala Gln Ser Phe Glu Tyr Asn Tyr

210 215 220

Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Gly Tyr Leu Lys

225 230 235 240

Ile Cys Gln Asp Val Lys Asp Arg Arg Ile Ala Leu Phe Lys Lys Tyr

245 250 255

Phe Val Asp Glu Arg Lys Gln Ile Ala Ser Ser Lys Pro Thr Gly Ser

260 265 270

Glu Gly Leu Lys Cys Ala Ile Asp His Ile Leu Glu Ala Glu Gln Lys

275 280 285

Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn

290 295 300

Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala

305 310 315 320

Glu Leu Val Asn His Pro Glu Ile Gln Ser Lys Leu Arg Asn Glu Leu

325 330 335

Asp Thr Val Leu Gly Pro Gly Val Gln Val Thr Glu Pro Asp Leu His

340 345 350

Lys Leu Pro Tyr Leu Gln Ala Val Val Lys Glu Thr Leu Arg Leu Arg

355 360 365

Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys

370 375 380

Leu Ala Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala

385 390 395 400

Trp Trp Leu Ala Asn Asn Pro Asn Ser Trp Lys Lys Pro Glu Glu Phe

405 410 415

Arg Pro Glu Arg Phe Phe Glu Glu Glu Ser His Val Glu Ala Asn Gly

420 425 430

Asn Asp Phe Arg Tyr Val Pro Phe Gly Val Gly Arg Arg Ser Cys Pro

435 440 445

Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Ile Gly Arg Met

450 455 460

Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Val Asp

465 470 475 480

Thr Ser Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Asn His Ser

485 490 495

Ile Ile Val Met Lys Pro Arg Asn Cys

500 505

<210> 12

<211> 58

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cggggtacct cccaacagtg aatacaacgt gcgagctcga ccattctctg gatcattg 58

<210> 13

<211> 60

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cggaagcttt gtcaccgcta cgttaggctt cccgctgcag ccatataacc atcaaagcca 60

<210> 14

<211> 60

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccgctgcagc tcacgcaact ggtccagaag tgatctagag tgcttagtgc atctaacgct 60

<210> 15

<211> 107

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

agcgttagat gcactaagca ctctagatca ccatttggac aaaacatcag gctcctagga 60

tccttaagac gcgttttctc ctctttctag gtcagtcctc cataaac 107

<210> 16

<211> 100

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cgtcttaagg atcctaggag ctctgcagat ctaccggtgt ttggattgtc ggagttgcgt 60

tgtattcact gttgggataa ttggtaaatt gctgtcgaag 100

<210> 17

<211> 103

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

aggagattaa ttcaaaagag gagaaagatc tatggccccc tccgtggatt ccattccaac 60

agcaacaaat ccatgtcgac tgcagttagg ccatcatttt cac 103

<210> 18

<211> 69

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

atggatttgt tgctgttgga acgacaatcc aaacaccggt cgacttaaca attccggggt 60

ttcatcaca 69

<210> 19

<211> 59

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cggaagcttc gcaatgctag cagcccaaga ccgctgcagc aagtctcccg ccaaggaag 59

<210> 20

<211> 63

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cggggtacct acaacaaacc aacggcgatc ggtgcgagct cactggagga taggaccacc 60

agc 63

<210> 21

<211> 60

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

ccgctgcagc tcacgcaact ggtccagaag tgatctagag tgcttagtgc atctaacgct 60

<210> 22

<211> 107

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

agcgttagat gcactaagca ctctagatca ccatttggac aaaacatcag gctcctagga 60

tccttaagac gcgttttctc ctctttctag gtcagtcctc cataaac 107

<210> 23

<211> 110

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

cgtcttaagg atcctaggag ctctgcagat ctaccggtgt ttggattgtc ggagttgccg 60

atcgccgttg gtttgttgta ggtaccgcgg taattggtaa attgctgtcg 110

<210> 24

<211> 100

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gactgaccta gaaagaggag aaaacgcgta tggccccctc cgtggattcc atccaacagc 60

aacaaatcca tgtcgactgc agttaggcca tcattttcac 100

<210> 25

<211> 70

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

atggatttgt tgctgttgga actgcagagc tcctaggatc cttaagttaa caattccggg 60

gtttcatcac 70

Claims

1. A vector for the biosynthesis of p-coumaric acid, characterized in that it is based on the cyanobacteria Synechocystis sp.pcc 6803 strain, comprising a phenylalanine enzyme gene and a cinnamic acid-4-hydroxylase gene operably linked to a promoter active in cyanobacteria, the phenylalanine enzyme gene and the cinnamic acid-4-hydroxylase gene being inserted into the cyanobacteria genome slr2081 site by homologous recombination; the two ends of the phenylalanine enzyme gene and the cinnamic acid-4-hydroxylase gene are respectively provided with an upstream fragment and a downstream fragment of the cyanobacteria gene, the DNA sequence of the upstream fragment gene is SEQ ID NO. 6, and the DNA sequence of the downstream fragment gene is SEQ ID NO. 7;

2. A microorganism containing the vector for biosynthesizing p-coumaric acid according to claim 1, wherein the microorganism does not contain or knocks out the slr2081 gene, and highly expresses the phenylalanine enzyme gene PAL and the cinnamic acid-4-hydroxylase gene C4H;

the PAL gene has the sequence of SEQ ID NO:1 or the PAL gene encodes the nucleotide sequence shown in SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

3. The microorganism that biosynthesizes a vector for p-coumaric acid according to claim 2, wherein the C4H gene has the amino acid sequence of SEQ ID NO:2 or the C4H gene encodes the nucleotide sequence set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.