CN113416748A

CN113416748A - Expression vector for synthesizing cannabidiol, heterologous expression method and application

Info

Publication number: CN113416748A
Application number: CN202110656198.9A
Authority: CN
Inventors: 曹应龙; 虞沂; 邹小林
Original assignee: Hubei Carbon Yuan Materia Medica Biotechnology Co ltd
Current assignee: Hubei Carbon Yuan Materia Medica Biotechnology Co ltd
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-09-21

Abstract

The invention discloses an expression vector for synthesizing cannabidiol, a heterologous expression method and application thereof, wherein 5 gene expression cassette vectors are constructed through in vitro de novo design and through five enzymes of hexanoyl-CoA synthase, polyketide peptide synthase, olive acid synthase, isopentenyl transferase and cannabidiolic acid synthase, and instantaneous cotransformation experiments in tobacco show that the high yield of CBD can be realized through the method. According to the invention, the CBD can be efficiently produced by using a synthetic biology technical means, the synthetic product does not contain THC, and the content of the measured CBD is high; in addition, the tobacco is used as a conventional economic crop in agricultural production, the planting technology, the field management, the yield of harvested leaves and the like are all advantageous, and the planting cost can be greatly reduced.

Description

Expression vector for synthesizing cannabidiol, heterologous expression method and application

Technical Field

The invention relates to the technical field of cannabidiol plant expression, in particular to an expression vector for synthesizing cannabidiol, a heterologous expression method and application.

Background

Cannabidiol (CBD) is the major active ingredient in industrial cannabis. Industrial hemp, also known as hemp or hemp, has been used by china for some textile products in the past 6000 years. Unlike psychoactive Tetrahydrocannabinol (THC), CBD is a multitarget drug with pharmacological activities of anti-inflammatory, analgesic, anxiolytic, anticancer, etc., without addiction, but rather to counteract the hallucinogenic effects of THC, and is called "antidote compound". CBD has wide application, and has the effects of repairing basal cells, resisting oxidation and aging in cosmetics; the food additive can increase food nutrition; meanwhile, CBD can be used as one of the treatment medicines for epilepsy. In summary, CBD has penetrated into various industries of textiles, pharmaceuticals, cosmetics, foods, and the like. Because of the enormous industrial value of CBD, more and more countries and regions begin to release medical hemp legalization, and CBD has huge commercial value and broad development prospect.

CBD can be obtained by extraction and chemical synthesis. Chemical synthesis, whether chemical or semi-chemical, is limited by its particular chemical structure, e.g., chiral and asymmetric centers, and is used in a relatively limited number of applications. The main current source of CBD is the direct extraction from industrial cannabis. After the synthesis pathway of CBD has been resolved, there are also methods for producing CBD by fermentation using heterologous hosts of microorganisms, which are not yet available on a large scale due to high oxygen and energy consumption. More and more natural products are beginning to be synthesized by plants, and if synthesized by photosynthesis of plants, only CO is used₂And water as raw material, and can be used for synthesizing various complex natural products by means of photosynthesis, and has no need of high energy consumption and high oxygen consumptionAnd (4) fermenting.

Therefore, it is necessary to construct engineering bacteria for synthesizing pathway in vitro, achieve heterologous expression by infecting plants to obtain CBD product, which is free of THC and has high yield, to promote large-scale application.

Disclosure of Invention

In view of the above, the present invention provides an expression vector, a heterologous expression method and applications of synthesized cannabidiol, which can achieve heterologous expression by infecting tobacco to obtain cannabidiol product and obtain high yield to promote large-scale application.

In view of this, the scheme of the invention is as follows:

an expression vector for the synthesis of cannabidiol comprising enzymes required for the expression of cannabidiol, said enzymes being AAE1, TKS, OAC, CsPT4 and CBDAS.

The invention also provides tobacco cells comprising a vector as described above.

The invention also provides the application of the tobacco cell in the production of cannabidiol.

The invention also provides a method for constructing the engineering bacteria for synthesizing cannabidiol, which adopts a mode of combining gene expression cassettes to construct recombinant vectors of coding genes AAE1, TKS, OAC, CsPT4 and CBDAS, and the recombinant vectors are transformed into agrobacterium to obtain the engineering bacteria.

According to the embodiment of the invention, the recombinant vector is obtained by adding the genes encoding the target genes OAC, CsPT4 and CBDAS through pUC19-TKS respectively to obtain an intermediate vector, then connecting a pC2300S vector and adding the gene AAE1 through homologous recombination.

According to an embodiment of the invention, in constructing the intermediate vector, the coding genes OAC and CsPT4 are added with 15-20 degenerate bases N.

The invention also provides a method for producing cannabidiol through heterologous expression, which comprises the step of infecting the engineering bacteria on tobacco leaves to obtain an expression product. Preferably, the engineering bacteria are cultured by using an LB culture medium, and the tobacco is infected after being resuspended; the content of cannabidiol in the tobacco leaves is 0.69054 mg/mL.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides the method for synthesizing the CBD by utilizing plant biology, the CBD can be efficiently produced, the tobacco is used as the conventional cash crop for agricultural production, the planting technology, the field management, the yield of harvested leaves and the like of the tobacco have advantages over industrial hemp, and the planting cost can be greatly reduced.

2. The invention synthesizes CBD by using plants, the synthesized product does not contain THC, the plant planting has no limitation any more, and the planting and production can be carried out in the largest scale.

3. The invention optimizes the expression quantity of genes in CBD synthesis, is beneficial to improving the CBD content, and the content measured by the method can reach 1.6 percent and is higher than the variety Yunuman No. 8 with the highest CBD content of industrial hemp in China at present.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic representation of the CBD biosynthetic pathway of the present invention.

FIG. 2 is a construction map of Agrobacterium engineering bacteria pC2300S-AAE1, TKS, OAC, CsPT4 and CBDAS of the present invention.

FIG. 3 is a schematic diagram of the liquid phase conditions of the present invention, olive acid OA and cannabidiol CBD.

FIG. 4 is an HPLC plot of transient expression in tobacco of the present invention.

FIG. 5 is a schematic diagram showing the identification of mass spectrum result of the tobacco heterologous expression no-load pC2300s of the present invention.

FIG. 6 is a schematic representation of the mass spectrum results of a standard CBD of the present invention.

FIG. 7 is the mass spectrum result of CBD of the sample of the present invention.

FIG. 8 is a diagram showing the quantitative results of the tobacco heterologously expressed CBD of the present invention.

FIG. 9 is a standard concentration curve of CBD according to the present invention.

Detailed Description

The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

The sources of the test materials of the invention are as follows:

nicotiana benthamiana (Nicotiana benthamiana) was cultivated in the laboratory. Plasmid pC2300S (Kana resistance), purchased from Wuhan Nature Biotech, Inc. Agrobacterium competent EHA105, purchased from Shanghai Only organisms.

The RNA electrophoresis buffer is generally TAE, and the formula is as follows: the formulation of 50 XTAE buffer was prepared as follows: the following reagents, tris, were weighed: 242 g; na (Na)₂EDTA.2H₂O: 37.2g, then adding 800mL of deionized water, and fully stirring for dissolving; adding 57.1mL of acetic acid, and fully and uniformly mixing; adding deionized water to constant volume of 1L, and storing at room temperature.

The formulation of LB medium was as follows: tryptone: 10g/L, Yeast extract: 5g/L, NaCl: 10 g/L.

The biosynthetic pathway of CBD's of the invention in tobacco is shown in FIG. 1. hexanoyl-CoA formation is catalyzed by hexanoyl-CoA synthetase (AAE1), followed by polyketide synthase (TKS) to form 3,5, 7-trioxadecanyl-CoA, the second enzymatic step being the formation of olivolic acid by olivo-late cyclase (OAC); the next step is the prenylation of the olivinic acids by an aromatic prenyltransferase to form cannabigerolic acid (CBGA). CBGA is a central branch point intermediate in the biosynthesis of different major classes of cannabinoids.

Terpenoids are the most abundant compounds among plant metabolites and are composed of isoprene as a structural unit. Plays an important role in the respiration, photosynthesis, and growth, development, reproduction, signal transduction, and defense of plants. In tobacco, terpenoid biosynthesis occurs in the cytoplasm and plastids, the precursor substance is IPP with the structure C5, and IPP and DMAPP synthesized in plastids by the methylerythritol phosphate pathway are precursors for the production of geranyl diphosphate (GPP). Cannabis sativa fruit acid synthase (CsPT4, also known as CBGAS) is C-alkylated by geranyl diphosphate (GPP) with CBGA. Cannabidiol CBD is obtained from CBGA in the lipo-exosomes by cannabidiol synthase (CBDAS).

The related coding genes and amino acid sequences of the invention are as follows:

the nucleotide sequence of the coding gene AAE1 is shown as SEQ ID NO: 1, the encoded amino acid sequence is shown as SEQ ID NO: 6.

the nucleotide sequence of the coded gene TKS is shown as SEQ ID NO: 2, the coded amino acid sequence is shown as SEQ ID NO: 7.

the nucleotide sequence of the coding gene OAC is shown as SEQ ID NO: 3, the coded amino acid sequence is shown as SEQ ID NO: 8.

the nucleotide sequence of the coding gene CsPT4 is shown as SEQ ID NO: 4, the coded amino acid sequence is shown as SEQ ID NO: 9.

the nucleotide sequence of the coding gene CBDAS is shown in SEQ ID NO: 5, the coded amino acid sequence is shown as SEQ ID NO: 10.

the invention provides an agrobacterium engineering bacterium which can simultaneously express hexanoyl-CoA synthase AAE1, polyketide synthase TKS, olive acid cyclase OAC, cannabidiolic acid synthase CsPT4 and cannabidiol synthase CBDAS required for synthesizing cannabidiol.

The embodiment also provides a construction method of the agrobacterium engineering bacteria, which comprises the following steps: the vector pUC19-TKS is used to construct recombinant vectors pUC19-AAE1, TKS, OAC, CsPT4 and CBDAS containing the coding target gene OAC, the coding target gene CsPT4 and the coding target gene CBDAS. Because the genes TKS and OAC have the same 35S promoter and the same poly A terminator, the genes CsPT4 and CBDAS have the same NOS promoter and the same NOS terminator, in order to avoid mismatching during construction and generate an incomplete vector only connected with one or two, the invention adds an arbitrary DNA fragment with 15-20 base sequences behind the gene OAC and also carries out the same treatment on the gene CsPT 4. Thus, a complete pUC19-AAE1, TKS, OAC, CsPT4, CBDAS vector was obtained during construction. The pC2300S vector and the intermediate vector pUC19-TKS, OAC, CsPT4, CBDAS were then digested separately by using HinDIII and EcoRI. TKS, OAC, CsPT4 and CBDAS are then ligated to pC2300S vector using T4 ligase. And then, selecting a HindIII enzyme cutting site at the upstream of the vector by utilizing pC2300S-TKS, OAC, CsPT4 and CBDAS vectors, cutting the vector, and constructing the gene AAE1 on the vector pC2300S-AAE1, TKS, OAC, CsPT4 and CBDAS by a homologous recombination method, thereby obtaining a gene expression frame containing five gene fragments.

Preferably, the method for obtaining each coding gene is: the respective gene sequences derived from cannabis were synthesized into vectors pUC 19. Thus, pUC19-AAE1, pUC19-TKS, pUC19-OAC, pUC19-CspT4, pUC19-CBDAS were obtained.

Specifically, the method for constructing the vectors pUC19-TKS, OAC, CsPT4 and CBDAS comprises the following steps: selecting EcoRI and HindIII enzyme cutting sites from the multiple cloning sites of pUC19-TKS for enzyme cutting, purifying and recovering a linear vector, adding genes OAC, CsPT4 and CBDAS by using an In-Fusion cloning technology to obtain pUC19-TKS, OAC, CsPT4 and CBDAS; and selecting two enzyme cutting sites of HindIII and EcoRI from the multiple cloning site of pC2300S for enzyme cutting, purifying and recovering a vector framework, and connecting the TKS, OAC, CsPT4 and CBDAS gene cassettes cut from pUC19-TKS, OAC, CsPT4 and CBDAS to a vector pC2300S through T4 ligase. A HindIII restriction site is selected at the upstream of the vector, the vector is cut, and the gene AAE1 is constructed on the vector pC2300S-AAE1, TKS, OAC, CsPT4 and CBDAS by a homologous recombination method, so that a gene expression frame containing five gene segments is obtained. AAE1 gene for synthesizing hexanoyl-CoA selected the promoter pAtUB10 and the terminator tAdh, which can promote high expression of the gene in tobacco. That is, when the AAE1 gene was ligated together with the promoter pAtUB10 and the terminator tAdh, ligation of a three-gene four-fragment was completed again.

Preferably, the primers used for constructing the vectors pUC19-TKS, OAC, CsPT4, CBDAS include:

pUC19-OAC-For：ctaaaatccagatcccccgatgattacgccaaccttcca(SEQ ID NO：11)

pUC19-OAC-Rev：aattggcaatgcatcggagccgggggatctggattttag(SEQ ID NO：12)

pUC19-CsPT4-For：gctccgatgcattgccaattgatcatgagcggagaattaa(SEQ ID NO：13)

pUC19-CsPT4-Rev：acgagctccccaaaaccctccccgatctagtaacatagat(SEQ ID NO：14)

pUC19-CBDAS-For：gagggttttggggagctcgtgatcatgagcggagaattaa(SEQ ID NO：15)

pUC19-CBDAS-Rev：taaaacgacggccagtgaattccccgatctagtaacatagat(SEQ ID NO：16)

preferably, the primers used for constructing the recombinant vector pC2300S-AAE1 comprise:

pC2300S-AAE1-For：

tacccggggatcctctagagtcgacatgggtaagaactacaagtccttggattccgttg(SEQ ID NO：17)；

pC2300S-AAE1-Rev：

actcgagcttgcatgcctgcaggtcgactcattcgaagtgggagaattgttgtctcaag(SEQ ID NO：18)。

the primers used for constructing the promoter pAtUB10 of the recombinant vector pC2300S-AAE1 comprise:

pCB-pAtUB10-For：

tgtaaaacgacggccagtgccaagcttgacgtcgttgtggttggtgctttccttacattc(SEQ ID NO：19)

pCB-pAtUB10-Rev：

aatccaaggacttgtagttcttacccataagatctgcatctgttaatcagaaaaactcag(SEQ ID NO：20)

the primers used for constructing the terminator tADh of the recombinant vector pC2300S-AAE1 comprise:

pCB-tAdh-For：

aacaattctcccacttcgaatgaagccattctctcgcagatgatgttcactttctg(SEQ ID NO：21)

pCB-tAdh-Rev：

gagtgtcgtgctccaccatgttggaagcttcgagtcactacggttagtccgtttgcattc(SEQ ID NO：22)

example 1 transient transformation of tobacco with Agrobacterium engineering bacteria to produce cannabidiol

1. Selecting agrobacterium engineering bacteria (strain EHA105) transformed with expression plasmids in LB culture medium containing corresponding antibiotics, fermenting and culturing at 28 + -0.5 deg.C for 24 hr.

2. To 5mL of fresh LB containing the corresponding antibiotic, 100. mu.L of 0.5M MES and 2. mu.L of 100mM AS were added, followed by inoculation of 50. mu.L of Agrobacterium solution and cultivation at 28 ℃ with shaking at 250rpm until OD600 became 1.0 (approximately 12-18 hours).

The cells were centrifuged at 3.4000rpm at room temperature for 10 minutes to collect the cells, resuspended with 10mM MgCl2 to OD600 of 1.0, and then 100mM AS was added at a rate of 2. mu.L/ml of the suspension, and the mixture was allowed to stand for 3 hours. Specifically, the medium is selected from an LB medium, a TB medium or a SOB medium, preferably a TB medium.

4. Taking Nicotiana benthamiana (Nicotiana benthamiana) in a vigorous growth period (about one month and without flowering), sucking the bacterial liquid by using an injector, removing a needle head, abutting the front surface of the leaf with a finger, penetrating the bacterial liquid from the back surface of the leaf, and if the injection is not smooth, making a tiny wound on the leaf by using the needle head and then injecting the bacterial liquid from the wound.

5. Under normal conditions, cannabidiol can be detected by sampling for 24-48 hours.

Example 2 extraction of cannabidiol

Tobacco leaves 3-7 days after infection were taken and the product was analyzed. Tobacco leaves were taken and weighed to ensure that the leaf wet weight was the same for both the unloaded and sample. The leaves were ground in liquid nitrogen, ground to a powder, added to a 50mL centrifuge tube and approximately 10mL of 20% methanol mass spectrometer was added. The 50mL centrifuge tube containing the sample was placed on a shaker at 37 ℃ and 250rpm for 1 hour. And then placing the centrifuge tube into an instrument by using a dynamic immersion method at room temperature, and reacting for 45 minutes. The tube was removed and centrifuged at 4500rpm for 10-15 minutes. The supernatant was collected, filtered through a 0.45 μm organic phase filter, and the sample was spin-dried using a rotary evaporator.

Dissolving the sample with about 100 μ L of mass spectrum methanol, placing the re-dissolved sample in a 1.5mL centrifuge tube, centrifuging at 12000rpm for 5-10 min, and taking the supernatant through a 0.45 μm organic phase membrane. The sample is filled in a sample bottle for HPLC analysis.

Example 3 cannabidiol CBD liquid phase conditions and tobacco biosynthetic products liquid phase conditions were investigated

The positive cloning results were examined using the empty vector pC2300S (Kana resistance) directly transformed into Agrobacterium EHA105 as a blank as shown in FIG. 2, where pAtUB10 is the promoter of the gene AAE1 and tAdh is the terminator of the gene AAE 1. Both the genes TKS and OAC use the 35S promoter and 35S poly A as terminators. The genes CsPT4 and CBDAS use the NOS promoter and NOS terminator.

In addition, HPLC analysis of samples from the fermentation broth of the engineered bacteria and mass spectrometric identification of LC-MS (FIGS. 5-7) showed that the compound had an M/z value of 313.21730[ M-H ] and the secondary fragment was identical to the standard for cannabidiol, indicating that the engineered bacteria are capable of de novo production of cannabidiol.

In FIG. 5, the mass spectrum of the result of the transient expression of the pC2300s in the tobacco under the condition of no load of the negative control of P19 in FIG. 5 shows that the molecular ion peak of CBD 313.21730 within the effective error can not be found. A-2 is a fragmentation mode 1 of CBD 313 molecules which are bombed under mass spectrum conditions, and under normal conditions, two molecules 245 and 71 can be obtained. A-3 is a fragmentation mode 2 of CBD 313 molecules which are bombed under mass spectrum conditions, and under normal conditions, 135 molecules and 177 molecules can be obtained.

In fig. 6, B-1 is the standard CBD mass spectrum, and it can be seen that the molecular ion peak of CBD 313.21730 within the valid error can be seen at RT 26.85. B-2 is 245 molecular ion peak obtained under fragmentation mode 1 of CBD 313 molecule bombed under mass spectrum condition. B-3 is a 135-molecule ion peak obtained under the fragmentation mode 2 that CBD 313 molecules are bombed under the mass spectrum condition.

In FIG. 7, C-1 is vector pC2300s-AAE1, TKS, OAC, CsPT4, CBDAS is expressed in tobacco transiently to obtain mass spectrum, and it can be seen that molecular ion peak of CBD 313.21730 in effective error is observed at RT 26.85, and the time of CBD peak is consistent with that of standard product. C-2 is 245 molecular ion peak obtained under fragmentation mode 1 of CBD 313 molecule bombed under mass spectrum condition. C-3 is a 135 molecular ion peak obtained under the fragmentation mode 2 that CBD 313 molecules are bombed under the mass spectrum condition. 135 and 245 are consistent with the time-to-peak of standard CBD.

Wherein, the liquid phase analysis conditions of the fermentation product are as follows:

column size Kromasil 5. mu.L 100A C18250X 4.6mm (Dikama corporation). The column temperature is 25 ℃, and the DAD ultraviolet detection wavelength is 254nm and 275 nm.

Mobile phase, mobile phase a: h₂O, mobile phase B: acetonitrile (0.1% formic acid), flow rate 0.6mL/min, injection volume: 5 μ L.

The elution conditions were as follows:

0.0-0.5min 5％B

0.5-5min 5-33％B

5-11min 33-35％B

11-20min 35-100％B

20-22min 100％B

22-23min 100-5％B

23-30min 5％B。

the HPLC detection profile is shown in FIG. 3.

Compared with a blank control group, in the whole terpenoid biosynthesis pathway, the agrobacterium engineering bacteria can generate a new product peak only when five genes of AAE1, TKS, OAC, CsPT4 and CBDAS exist; HPLC analysis and LC-MS mass spectrometric identification of a sample separated from engineering bacteria fermentation liquor show that the M/z value of the compound is 313.21730[ M-H ], and secondary fragments 135, 245 are the same as fragment ions generated by a cannabidiol standard product, which indicates that the engineering bacteria can generate cannabidiol CBD de novo.

Example 4 determination of the content of heterologously expressed CBD in tobacco

The embodiment provides a process method for producing cannabidiol by using agrobacterium engineering bacteria, which comprises the following steps: the agrobacterium engineering bacteria are cultured by using an LB culture medium, the tobacco is infected after the bacteria are re-suspended, and the cannabidiol content is detected by sampling after 3-7 days.

As shown in FIGS. 4 and 8, data 1 in FIG. 4 is CBD, data 2 is CBGA, data 3 is unloaded pC2300s, and data 4-6 are HPLC results of transient expression of the vectors pC2300s-AAE1, TKS, OAC, CsPT4, CBDAS in tobacco. Data 7-9 are HPLC results of transient expression of pC2300s-TKS, OAC, CsPT4, CBDAS in tobacco. As can be seen from fig. 4, in data 5, CBGA was accumulated in a large amount, and in data 8, CBD was accumulated to some extent. Data 1 in FIG. 8 is an empty pC2300s, and data 2 is a transient expression HPLC map of pC2300s-AAE1, TKS, OAC, CsPT4, CBDAS in tobacco. Data 3-9 are HPLC plots of standard CBD at different concentration gradients under the same liquid phase conditions, respectively. This gave the data as given in Table 1.

Table 1: CBD Standard concentration Curve

And fitting the obtained standard curve diagram 9, and substituting the effluent area of the sample into the linear relation obtained by the standard substance to obtain the content of the CBD in the sample. The final cannabidiol yield is calculated to be 0.69054mg/mL when the LB culture medium is used, and the yield is about 1.6%.

This expression also indicates that the use of genes from the same species for Agrobacterium recombination overall would have a high fitness, more enabling the stimulation of the potential productivity of the genes.

The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative apparatus, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.

Sequence listing

<110> Hubei carbon element and herbal Biotech Co., Ltd

<120> expression vector for synthesizing cannabidiol, heterologous expression method and application

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2163

<212> DNA

<213> AAE

<400> 1

atgggtaaga actacaagtc cttggattcc gttgttgcct ctgattttat tgctttgggt 60

atcacttctg aagttgccga aactttacat ggtagattgg ctgaaattgt ctgtaattat 120

ggtgctgcta ctccacaaac ctggattaac attgctaacc atatcttgtc tccagacttg 180

ccattttcct tgcatcaaat gttgttctac ggttgctaca aggattttgg tccagctcca 240

ccagcatgga ttccagatcc agaaaaagtt aagtctacca acttgggtgc tttgttggaa 300

aagagaggta aagaattttt gggtgtcaag tacaaggacc caatctcttc attttcccac 360

ttccaagaat tctccgttag aaacccagaa gtttattgga gaactgtctt gatggacgaa 420

atgaagatct cattctctaa ggatccagaa tgcatcttga gaagagatga tattaacaac 480

ccaggtggtt ctgaatggtt gcctggtggt tatttgaatt ctgctaagaa ctgcttgaac 540

gtcaactcca acaaaaagtt gaacgacact atgatcgttt ggagagatga aggtaatgac 600

gatttgccat tgaacaagtt gaccttggat caattgagaa agagagtttg gttagttggt 660

tacgccttgg aagaaatggg tttggaaaaa ggttgtgcta ttgctatcga tatgccaatg 720

catgttgatg ccgttgttat ctatttggct atagttttgg ctggttacgt cgttgtttca 780

attgctgatt cattttctgc cccagaaatc tctactagat tgagattgtc taaggccaag 840

gctattttca cccaagatca tattatcaga ggtaagaaga gaatcccatt atactccaga 900

gttgttgaag ctaaatctcc aatggctatc gttattccat gttccggttc taatattggt 960

gccgaattga gagatggtga tatctcttgg gattacttct tggaaagagc caaagaattc 1020

aagaactgcg aattcactgc tagagaacaa ccagttgacg cttacactaa tatcttgttc 1080

tcttctggta ctaccggtga acctaaagct attccatgga ctcaagctac tccattgaaa 1140

gctgctgctg atggttggtc acatttggat attagaaagg gtgatgttat cgtctggcca 1200

actaatttgg gttggatgat gggtccttgg ttggtttatg cttctttgtt gaatggtgct 1260

tccattgcct tgtataatgg ttctccattg gtttctggtt tcgccaagtt tgttcaagat 1320

gctaaggtta ctatgttggg tgttgttcca tctatcgtta gatcttggaa atccaccaat 1380

tgtgtctctg gttatgattg gtctactatc agatgcttct catcttctgg tgaagcttct 1440

aacgttgatg aatacttgtg gttgatgggt agagctaatt acaagccagt tattgaaatg 1500

tgcggtggta ctgaaattgg tggtgctttt tctgcaggtt cattcttgca agctcaatcc 1560

ttgtcatcct tttcttctca atgtatgggt tgcaccttgt acatcttgga taagaatggt 1620

tatccaatgc caaagaacaa accaggtatt ggtgaattgg ctttgggtcc agttatgttt 1680

ggtgcttcta aaaccttgtt gaacggtaac catcacgatg tttactttaa gggtatgcca 1740

actttgaacg gtgaagtttt gagaagacac ggtgacattt tcgaattgac ttctaacggt 1800

tactaccatg ctcacggtag agctgatgat acaatgaata tcggtggtat caagatctcc 1860

tccatcgaaa ttgaaagagt ctgcaacgaa gttgacgaca gagtttttga aactaccgct 1920

attggtgttc caccattggg tggtggtcca gaacaattgg ttattttctt cgttttgaag 1980

gactccaacg ataccaccat cgatttgaat caattgagat tgtccttcaa cttgggttta 2040

caaaagaagt tgaacccttt gtttaaggtc actagagttg tcccattgtc atctttgcca 2100

agaacagcta ccaacaagat catgagaaga gtcttgagac aacaattctc ccacttcgaa 2160

tga 2163

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atgaaccact tgagagctga aggtccagct tctgttttgg ctatcggtac tgccaatcca 60

gaaaacatct tattacaaga tgaatttcca gattattact tcagagtcac caagtctgaa 120

catatgaccc aattgaagga aaagtttcgt aaaatttgtg acaagtccat gatcagaaag 180

cgtaactgtt tcttaaacga agagcacttg aaacaaaacc caagattggt tgaacacgag 240

atgcaaactt tggacgccag acaagatatg ttggttgttg aagttccaaa attgggtaag 300

gacgcctgtg ccaaggctat caaggaatgg ggtcaaccaa agtctaagat cactcatttg 360

atcttcacct ctgcctctac taccgatatg ccaggtgctg attaccattg tgctaagttg 420

ttgggtttgt ctccatccgt caaaagagtc atgatgtacc aattaggttg ttacggtggt 480

ggtaccgttt tgagaattgc taaggacatc gctgaaaaca acaagggtgc tagagttttg 540

gctgtttgtt gtgacatcat ggcctgctta ttcagaggtc catctgaatc tgacttggaa 600

ttattggttg gtcaagctat tttcggtgat ggtgctgccg ctgtcatcgt cggtgctgag 660

ccagatgaat ccgtcggtga aagaccaatt ttcgaattgg tttccaccgg tcaaactatc 720

ttgccaaact ctgaaggtac tattggtggt cacatcagag aagctggttt gatcttcgat 780

ttgcacaagg acgttcctat gttgatctcc aacaatattg aaaagtgttt aatcgaagcc 840

ttcaccccaa ttggtatttc cgactggaac tctattttct ggattactca tccaggtggt 900

aaggccattt tagataaggt cgaagaaaag ttgcacttaa aatccgacaa gttcgttgat 960

tctagacatg tcttgtccga gcacggtaac atgtcctctt ctaccgtctt gttcgtcatg 1020

gatgaattga gaaagagatc tttggaagaa ggtaaatcca ctaccggtga tggttttgaa 1080

tggggtgttt tgtttggttt cggtccaggt ttgactgttg aaagagtcgt cgtcagatct 1140

gttccaatta agtactaa 1158

<210> 3

<211> 306

<212> DNA

<213> OAC

<400> 3

atggccgtta agcacttaat tgtcttgaag ttcaaggatg aaatcactga agctcaaaaa 60

gaggaattct tcaagaccta cgttaacttg gttaacatta ttccagctat gaaggacgtc 120

tactggggta aggacgttac ccaaaaaaac aaggaagagg gttacactca catcgtcgaa 180

gttaccttcg aatctgtcga aactattcaa gactacatca ttcacccagc tcacgttggt 240

ttcggtgatg tctacagatc tttctgggaa aagttgttga tcttcgatta cacccctaga 300

aagtaa 306

<210> 4

<211> 1197

<212> DNA

<213> CsPT4

<400> 4

atgggactat ctcttgtctg tacgttctca tttcaaacca actaccatac acttttaaat 60

ccccataata aaaacccgaa aaactcctta ttgtcatatc aacaccccaa aacgccaata 120

atcaagagta gttacgacaa tttccccagc aaatactgtt tgacgaaaaa ttttcatctt 180

ttaggcttaa attcacacaa taggatatct tctcaatccc gttccataag ggcaggatct 240

gaccaaatcg aggggtctcc gcaccatgag tcagacaact ccattgctac caaaatactt 300

aatttcggtc atacgtgttg gaaacttcaa cgtccgtatg tcgtgaaagg catgatatct 360

atcgcctgcg gtctgtttgg gagagagtta tttaataata ggcacttatt ttcctggggg 420

cttatgtgga aggccttttt cgctctagtt cctatccttt cattcaactt ctttgcagcc 480

attatgaacc aaatttacga cgtagacata gacagaatca acaaacctga tctgcctcta 540

gtatccggcg agatgtctat agagacggct tggattttgt ctataatagt ggccctaact 600

ggcttgatcg ttacaattaa actgaagtcc gctccactgt tcgtgttcat atacatattt 660

gggattttcg caggtttcgc ttactcagtg ccccccataa gatggaagca gtatcctttc 720

actaacttct taatcactat ttcttcacac gtggggttgg catttacctc atatagcgcc 780

actacatccg ctctgggcct gccctttgtc tggagacctg cgttttcttt tataatagcg 840

tttatgacag tcatgggcat gactatcgct tttgcaaaag acataagtga tatagaagga 900

gatgccaaat acggggtatc cactgtcgct actaagctgg gtgctagaaa catgactttt 960

gtggtgagcg gggttctact tcttaattat ctggtgagca tctccatcgg gataatatgg 1020

ccacaagtgt tcaaatccaa tataatgatt ctgtcacacg ctatactagc cttttgccta 1080

attttccaga cgagagaact ggctctagca aattatgcca gtgctcccag cagacagttc 1140

ttcgaattta tatggctttt atactatgct gaatacttcg tgtatgtttt catctaa 1197

<210> 5

<211> 1374

<212> DNA

<213> CBDAS

<400> 5

atggcgcatg tctctcatat ccaaggcact attctatgct ccaagaaagt tggcttgcag 60

attcgaactc gaagtggtgg tcatgattct gagggcatgt cctacatatc tcaagtccca 120

tttgttatag tagacttgag aaacatgcgt tcaatcaaaa tagatgttca tagccaaact 180

gcatgggttg aagccggagc tacccttgga gaagtttatt attgggttaa tgagaaaaat 240

gagaatctta gtttggcggc tgggtattgc cctactgttt gcgcaggtgg acactttggt 300

ggaggaggct atggaccatt gatgagaaac tatggcctcg cggctgataa tatcattgat 360

gcacacttag tcaacgttca tggaaaagtg ctagatcgaa aatctatggg ggaagatctc 420

ttttgggctt tacgtggtgg tggagcagaa agtttcggaa tcattgtagc atggaaaatt 480

agactggttg ctgtcccaaa gtctactatg tttagtgtta aaaagatcat ggagatacat 540

gagcttgtca agttagttaa caaatggcaa aatattgctt acaagtatga caaagattta 600

ttactcatga ctcacttcat aactaggaac attacagata atcaagggaa gaataagaca 660

gcaatacaca cttacttctc ttcagttttc cttggtggag tggatagtct agtcgacttg 720

atgaacaaga gttttcctga gttgggtatt aaaaaaacgg attgcagaca attgagctgg 780

attgatacta tcatcttcta tagtggtgtt gtaaattacg acactgataa ttttaacaag 840

gaaattttgc ttgatagatc cgctgggcag aacggtgctt tcaagattaa gttagactac 900

gttaagaaac caattccaga atctgtattt gtccaaattt tggaaaaatt atatgaagaa 960

gatataggag ctgggatgta tgcgttgtac ccttacggtg gtataatgga tgagatttca 1020

gaatcagcaa ttccattccc tcatcgagct ggaatcttgt atgagttatg gtacatatgt 1080

agttgggaga agcaagaaga taacgaaaag catctaaact ggattagaaa tatttataac 1140

ttcatgactc cttatgtgtc caaaaatcca agattggcat atctcaatta tagagacctt 1200

gatataggaa taaatgatcc caagaatcca aataattaca cacaagcacg tatttggggt 1260

gagaagtatt ttggtaaaaa ttttgacagg ctagtaaaag tgaaaaccct ggttgatccc 1320

aataactttt ttagaaacga acaaagcatc ccacctcttc cacggcatcg tcat 1374

<210> 6

<211> 720

<212> PRT

<213> AAE

<400> 6

Met Gly Lys Asn Tyr Lys Ser Leu Asp Ser Val Val Ala Ser Asp Phe

1 5 10 15

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

20 25 30

Leu Ala Glu Ile Val Cys Asn Tyr Gly Ala Ala Thr Pro Gln Thr Trp

35 40 45

Ile Asn Ile Ala Asn His Ile Leu Ser Pro Asp Leu Pro Phe Ser Leu

50 55 60

His Gln Met Leu Phe Tyr Gly Cys Tyr Lys Asp Phe Gly Pro Ala Pro

65 70 75 80

Pro Ala Trp Ile Pro Asp Pro Glu Lys Val Lys Ser Thr Asn Leu Gly

85 90 95

Ala Leu Leu Glu Lys Arg Gly Lys Glu Phe Leu Gly Val Lys Tyr Lys

100 105 110

Asp Pro Ile Ser Ser Phe Ser His Phe Gln Glu Phe Ser Val Arg Asn

115 120 125

Pro Glu Val Tyr Trp Arg Thr Val Leu Met Asp Glu Met Lys Ile Ser

130 135 140

Phe Ser Lys Asp Pro Glu Cys Ile Leu Arg Arg Asp Asp Ile Asn Asn

145 150 155 160

Pro Gly Gly Ser Glu Trp Leu Pro Gly Gly Tyr Leu Asn Ser Ala Lys

165 170 175

Asn Cys Leu Asn Val Asn Ser Asn Lys Lys Leu Asn Asp Thr Met Ile

180 185 190

Val Trp Arg Asp Glu Gly Asn Asp Asp Leu Pro Leu Asn Lys Leu Thr

195 200 205

Leu Asp Gln Leu Arg Lys Arg Val Trp Leu Val Gly Tyr Ala Leu Glu

210 215 220

Glu Met Gly Leu Glu Lys Gly Cys Ala Ile Ala Ile Asp Met Pro Met

225 230 235 240

His Val Asp Ala Val Val Ile Tyr Leu Ala Ile Val Leu Ala Gly Tyr

245 250 255

Val Val Val Ser Ile Ala Asp Ser Phe Ser Ala Pro Glu Ile Ser Thr

260 265 270

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

275 280 285

Ile Arg Gly Lys Lys Arg Ile Pro Leu Tyr Ser Arg Val Val Glu Ala

290 295 300

Lys Ser Pro Met Ala Ile Val Ile Pro Cys Ser Gly Ser Asn Ile Gly

305 310 315 320

Ala Glu Leu Arg Asp Gly Asp Ile Ser Trp Asp Tyr Phe Leu Glu Arg

325 330 335

Ala Lys Glu Phe Lys Asn Cys Glu Phe Thr Ala Arg Glu Gln Pro Val

340 345 350

Asp Ala Tyr Thr Asn Ile Leu Phe Ser Ser Gly Thr Thr Gly Glu Pro

355 360 365

Lys Ala Ile Pro Trp Thr Gln Ala Thr Pro Leu Lys Ala Ala Ala Asp

370 375 380

Gly Trp Ser His Leu Asp Ile Arg Lys Gly Asp Val Ile Val Trp Pro

385 390 395 400

Thr Asn Leu Gly Trp Met Met Gly Pro Trp Leu Val Tyr Ala Ser Leu

405 410 415

Leu Asn Gly Ala Ser Ile Ala Leu Tyr Asn Gly Ser Pro Leu Val Ser

420 425 430

Gly Phe Ala Lys Phe Val Gln Asp Ala Lys Val Thr Met Leu Gly Val

435 440 445

Val Pro Ser Ile Val Arg Ser Trp Lys Ser Thr Asn Cys Val Ser Gly

450 455 460

Tyr Asp Trp Ser Thr Ile Arg Cys Phe Ser Ser Ser Gly Glu Ala Ser

465 470 475 480

Asn Val Asp Glu Tyr Leu Trp Leu Met Gly Arg Ala Asn Tyr Lys Pro

485 490 495

Val Ile Glu Met Cys Gly Gly Thr Glu Ile Gly Gly Ala Phe Ser Ala

500 505 510

Gly Ser Phe Leu Gln Ala Gln Ser Leu Ser Ser Phe Ser Ser Gln Cys

515 520 525

Met Gly Cys Thr Leu Tyr Ile Leu Asp Lys Asn Gly Tyr Pro Met Pro

530 535 540

Lys Asn Lys Pro Gly Ile Gly Glu Leu Ala Leu Gly Pro Val Met Phe

545 550 555 560

Gly Ala Ser Lys Thr Leu Leu Asn Gly Asn His His Asp Val Tyr Phe

565 570 575

Lys Gly Met Pro Thr Leu Asn Gly Glu Val Leu Arg Arg His Gly Asp

580 585 590

Ile Phe Glu Leu Thr Ser Asn Gly Tyr Tyr His Ala His Gly Arg Ala

595 600 605

Asp Asp Thr Met Asn Ile Gly Gly Ile Lys Ile Ser Ser Ile Glu Ile

610 615 620

Glu Arg Val Cys Asn Glu Val Asp Asp Arg Val Phe Glu Thr Thr Ala

625 630 635 640

Ile Gly Val Pro Pro Leu Gly Gly Gly Pro Glu Gln Leu Val Ile Phe

645 650 655

Phe Val Leu Lys Asp Ser Asn Asp Thr Thr Ile Asp Leu Asn Gln Leu

660 665 670

Arg Leu Ser Phe Asn Leu Gly Leu Gln Lys Lys Leu Asn Pro Leu Phe

675 680 685

Lys Val Thr Arg Val Val Pro Leu Ser Ser Leu Pro Arg Thr Ala Thr

690 695 700

Asn Lys Ile Met Arg Arg Val Leu Arg Gln Gln Phe Ser His Phe Glu

705 710 715 720

<210> 7

<211> 385

<212> PRT

<213> TKS

<400> 7

Met Asn His Leu Arg Ala Glu Gly Pro Ala Ser Val Leu Ala Ile Gly

1 5 10 15

Thr Ala Asn Pro Glu Asn Ile Leu Ile Gln Asp Glu Phe Pro Asp Tyr

20 25 30

Tyr Phe Arg Val Thr Lys Ser Glu His Met Thr Gln Leu Lys Glu Lys

35 40 45

Phe Arg Lys Ile Cys Asp Lys Ser Met Ile Arg Lys Arg Asn Tyr Phe

50 55 60

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

65 70 75 80

Met Gln Thr Leu Asp Ala Arg Gln Asp Met Leu Val Val Glu Val Pro

85 90 95

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

100 105 110

Pro Lys Ser Lys Ile Thr His Leu Ile Phe Thr Ser Ala Ser Thr Thr

115 120 125

Asp Met Pro Gly Ala Asp Tyr His Cys Ala Lys Leu Leu Gly Leu Ser

130 135 140

Pro Ser Val Lys Arg Val Met Met Tyr Gln Leu Gly Cys Tyr Gly Gly

145 150 155 160

Gly Thr Val Leu Arg Ile Ala Lys Asp Ile Ala Glu Asn Asn Lys Gly

165 170 175

Ala Arg Val Leu Ala Val Cys Cys Asp Ile Met Ala Cys Leu Phe Arg

180 185 190

Gly Pro Ser Asp Ser Asp Leu Glu Leu Leu Val Gly Gln Ala Ile Phe

195 200 205

Gly Asp Gly Ala Ala Ala Val Ile Val Gly Ala Glu Pro Asp Glu Ser

210 215 220

Val Gly Glu Arg Pro Ile Phe Glu Leu Val Ser Thr Gly Gln Thr Ile

225 230 235 240

Leu Pro Asn Ser Glu Gly Thr Ile Gly Gly His Ile Arg Glu Ala Gly

245 250 255

Leu Ile Phe Asp Leu His Lys Asp Val Pro Met Leu Ile Ser Asn Asn

260 265 270

Ile Glu Lys Cys Leu Ile Glu Ala Phe Thr Pro Ile Gly Ile Ser Asp

275 280 285

Trp Asn Ser Ile Phe Trp Ile Thr His Pro Gly Gly Lys Ala Ile Leu

290 295 300

Asp Lys Val Glu Glu Lys Leu His Leu Lys Ser Asp Lys Phe Val Asp

305 310 315 320

Ser Arg His Val Leu Ser Glu His Gly Asn Met Ser Ser Ser Thr Val

325 330 335

Leu Phe Val Met Asp Glu Leu Arg Lys Arg Ser Leu Glu Glu Gly Lys

340 345 350

Ser Thr Thr Gly Asp Gly Phe Glu Trp Gly Val Leu Phe Gly Phe Gly

355 360 365

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

370 375 380

Tyr

385

<210> 8

<211> 100

<212> PRT

<213> OAC

<400> 8

Met Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr

1 5 10 15

Glu Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn

20 25 30

Ile Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln

35 40 45

Lys Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu

50 55 60

Ser Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly

65 70 75 80

Phe Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp

85 90 95

Tyr Thr Pro Arg

100

<210> 9

<211> 114

<212> PRT

<213> CsPT4

<400> 9

Met Gly Met Thr Ile Ala Phe Ala Lys Asp Ile Ser Asp Ile Glu Gly

1 5 10 15

Asp Ala Lys Tyr Gly Val Ser Thr Val Ala Thr Lys Leu Gly Ala Arg

20 25 30

Asn Met Thr Phe Val Val Ser Gly Val Leu Leu Leu Asn Tyr Leu Val

35 40 45

Ser Ile Ser Ile Gly Ile Ile Trp Pro Gln Val Phe Lys Ser Asn Ile

50 55 60

Met Ile Leu Ser His Ala Ile Leu Ala Phe Cys Leu Ile Phe Gln Thr

65 70 75 80

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

85 90 95

Phe Glu Phe Ile Trp Leu Leu Tyr Tyr Ala Glu Tyr Phe Val Tyr Val

100 105 110

Phe Ile

<210> 10

<211> 544

<212> PRT

<213> CBDAS

<400> 10

Met Lys Cys Ser Thr Phe Ser Phe Trp Phe Val Cys Lys Ile Ile Phe

1 5 10 15

Phe Phe Phe Ser Phe Asn Ile Gln Thr Ser Ile Ala Asn Pro Arg Glu

20 25 30

Asn Phe Leu Lys Cys Phe Ser Gln Tyr Ile Pro Asn Asn Ala Thr Asn

35 40 45

Leu Lys Leu Val Tyr Thr Gln Asn Asn Pro Leu Tyr Met Ser Val Leu

50 55 60

Asn Ser Thr Ile His Asn Leu Arg Phe Thr Ser Asp Thr Thr Pro Lys

65 70 75 80

Pro Leu Val Ile Val Thr Pro Ser His Val Ser His Ile Gln Gly Thr

85 90 95

Ile Leu Cys Ser Lys Lys Val Gly Leu Gln Ile Arg Thr Arg Ser Gly

100 105 110

Gly His Asp Ser Glu Gly Met Ser Tyr Ile Ser Gln Val Pro Phe Val

115 120 125

Ile Val Asp Leu Arg Asn Met Arg Ser Ile Lys Ile Asp Val His Ser

130 135 140

Gln Thr Ala Trp Val Glu Ala Gly Ala Thr Leu Gly Glu Val Tyr Tyr

145 150 155 160

Trp Val Asn Glu Lys Asn Glu Asn Leu Ser Leu Ala Ala Gly Tyr Cys

165 170 175

Pro Thr Val Cys Ala Gly Gly His Phe Gly Gly Gly Gly Tyr Gly Pro

180 185 190

Leu Met Arg Asn Tyr Gly Leu Ala Ala Asp Asn Ile Ile Asp Ala His

195 200 205

Leu Val Asn Val His Gly Lys Val Leu Asp Arg Lys Ser Met Gly Glu

210 215 220

Asp Leu Phe Trp Ala Leu Arg Gly Gly Gly Ala Glu Ser Phe Gly Ile

225 230 235 240

Ile Val Ala Trp Lys Ile Arg Leu Val Ala Val Pro Lys Ser Thr Met

245 250 255

Phe Ser Val Lys Lys Ile Met Glu Ile His Glu Leu Val Lys Leu Val

260 265 270

Asn Lys Trp Gln Asn Ile Ala Tyr Lys Tyr Asp Lys Asp Leu Leu Leu

275 280 285

Met Thr His Phe Ile Thr Arg Asn Ile Thr Asp Asn Gln Gly Lys Asn

290 295 300

Lys Thr Ala Ile His Thr Tyr Phe Ser Ser Val Phe Leu Gly Gly Val

305 310 315 320

Asp Ser Leu Val Asp Leu Met Asn Lys Ser Phe Pro Glu Leu Gly Ile

325 330 335

Lys Lys Thr Asp Cys Arg Gln Leu Ser Trp Ile Asp Thr Ile Ile Phe

340 345 350

Tyr Ser Gly Val Val Asn Tyr Asp Thr Asp Asn Phe Asn Lys Glu Ile

355 360 365

Leu Leu Asp Arg Ser Ala Gly Gln Asn Gly Ala Phe Lys Ile Lys Leu

370 375 380

Asp Tyr Val Lys Lys Pro Ile Pro Glu Ser Val Phe Val Gln Ile Leu

385 390 395 400

Glu Lys Leu Tyr Glu Glu Asp Ile Gly Ala Gly Met Tyr Ala Leu Tyr

405 410 415

Pro Tyr Gly Gly Ile Met Asp Glu Ile Ser Glu Ser Ala Ile Pro Phe

420 425 430

Pro His Arg Ala Gly Ile Leu Tyr Glu Leu Trp Tyr Ile Cys Ser Trp

435 440 445

Glu Lys Gln Glu Asp Asn Glu Lys His Leu Asn Trp Ile Arg Asn Ile

450 455 460

Tyr Asn Phe Met Thr Pro Tyr Val Ser Lys Asn Pro Arg Leu Ala Tyr

465 470 475 480

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

485 490 495

Asn Asn Tyr Thr Gln Ala Arg Ile Trp Gly Glu Lys Tyr Phe Gly Lys

500 505 510

Asn Phe Asp Arg Leu Val Lys Val Lys Thr Leu Val Asp Pro Asn Asn

515 520 525

Phe Phe Arg Asn Glu Gln Ser Ile Pro Pro Leu Pro Arg His Arg His

530 535 540

<210> 11

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctaaaatcca gatcccccga tgattacgcc aaccttcca 39

<210> 12

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aattggcaat gcatcggagc cgggggatct ggattttag 39

<210> 13

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gctccgatgc attgccaatt gatcatgagc ggagaattaa 40

<210> 14

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acgagctccc caaaaccctc cccgatctag taacatagat 40

<210> 15

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gagggttttg gggagctcgt gatcatgagc ggagaattaa 40

<210> 16

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

taaaacgacg gccagtgaat tccccgatct agtaacatag at 42

<210> 17

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tacccgggga tcctctagag tcgacatggg taagaactac aagtccttgg attccgttg 59

<210> 18

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

actcgagctt gcatgcctgc aggtcgactc attcgaagtg ggagaattgt tgtctcaag 59

<210> 19

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tgtaaaacga cggccagtgc caagcttgac gtcgttgtgg ttggtgcttt ccttacattc 60

<210> 20

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aatccaagga cttgtagttc ttacccataa gatctgcatc tgttaatcag aaaaactcag 60

<210> 21

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aacaattctc ccacttcgaa tgaagccatt ctctcgcaga tgatgttcac tttctg 56

<210> 22

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gagtgtcgtg ctccaccatg ttggaagctt cgagtcacta cggttagtcc gtttgcattc 60

Claims

1. An expression vector for the synthesis of cannabidiol comprising enzymes required for the expression of cannabidiol, wherein the enzymes are AAE1, TKS, OAC, CsPT4 and CBDAS.

2. A tobacco cell comprising the vector of claim 1.

3. Use of the tobacco cell of claim 2 for the production of cannabidiol.

4. A method for constructing engineering bacteria for synthesizing cannabidiol is characterized in that recombinant vectors of coding genes AAE1, TKS, OAC, CsPT4 and CBDAS are constructed in a mode of combining gene expression cassettes, and the recombinant vectors are transformed into agrobacterium to obtain the engineering bacteria.

5. The method of claim 4, wherein the recombinant vector is obtained by adding the genes encoding OAC, CsPT4 and CBDAS of interest to pUC19-TKS to obtain intermediate vectors, and then ligating pC2300S vector to obtain the intermediate vectors, and adding the gene AAE1 by homologous recombination.

6. The method of claim 5, wherein the intermediate vector is constructed by adding 15-20 degenerate bases N to the genes OAC and CsPT4, respectively.

7. A method for producing cannabidiol by heterologous expression, characterized in that tobacco leaves are infected with an engineering bacterium comprising any one of claims 4 to 6 to obtain an expression product.

8. The method for producing cannabidiol as claimed in claim 7, wherein the tobacco is infected after resuspension by culturing the engineered bacteria using LB medium.

9. The method of producing cannabidiol as claimed in claim 8, wherein the content of cannabidiol in the tobacco lamina is 0.69054 mg/mL.