CN114262695B - Saccharomyces cerevisiae engineering bacterium for producing CBGA precursor and construction method and application thereof - Google Patents

Abstract

The invention relates to a Saccharomyces cerevisiae engineering bacterium for producing a CBGA precursor, a construction method and application thereof, wherein the Saccharomyces cerevisiae engineering bacterium for producing the CBGA precursor is obtained by constructing a recombinant expression vector capable of simultaneously expressing tetrone synthase, olive acid cyclase and acyl activating enzyme and converting the recombinant expression vector into a Saccharomyces cerevisiae competent cell with thioester hydrolase genes knocked out. The saccharomyces cerevisiae engineering bacteria can effectively improve the OA yield of the strain by optimizing the expression of the acyl activating enzyme and the expression of the thioesterase through down-regulation, can predict the yield of the hexanoyl coenzyme A and lays a foundation for further constructing a new high-efficiency synthetic CBGA precursor OA strain.

Description

Saccharomyces cerevisiae engineering bacterium for producing CBGA precursor and construction method and application thereof

Technical Field

The invention relates to the technical field of metabolic engineering, in particular to a saccharomyces cerevisiae engineering bacterium for producing a CBGA precursor and a construction method and application thereof.

Background

Fatty acyl-CoA is essential for the life activities of plants, for example acetyl-CoA and malonyl-CoA are both essential substrates for the synthesis of fatty acids. Long-Chain Fatty Acyl-CoA plays a key role in many vital activities such as Plant lignin, phospholipid, triacylglycerol, jasmonic Acid generation and Fatty Acid beta-oxidation [ J.M. Shockey, M.S. Fulda, J.A. browse, arabidopsis thaliana Long-Chain amino-enzyme A synthetic Genes at particulate Acid in Fatty Acid and Glycerolide, plant Physiology,129 (2002) 1710-1722; M.Stout, Z.Boubakir, S.J.Ambrose, R.W.Purves, J.E.Page, the hexanoyl-CoA presensor for housing biosyntheses is formed by acyl-activating enzymes in housing hydrates, the Plant Journal,71 (2012) 353-365 ]. Short-chain fatty acyl-coenzyme A in plants can be used as a substrate to participate in the generation of various important secondary metabolites, and is closely related to human health. The source of the particular flavor in beer-picric Acid synthesis precursors are isovaleryl-coa, butyryl-coa [ h.xu, f.zhang, b.liu, d.v.huhman, l.w.sumner, r.a.dixon, g.wang, charaterization of the formatting of Branched Short-Chain Fatty Acid: coAs for Bitter Acid biosyntheses in Hop Glandular Trichomes, mol. Plant,6 (2013) 1301-1317.]; hexanoyl-coa is a substrate for cannabigerolic acid (CBGA). CBGA is being pursued by more and more medical and scientific researchers as a new drug because of its pharmacological effects of stimulating lipid metabolism, alleviating colitis, inducing apoptosis of colon cancer cells and reducing inflammation. Variants of various secondary metabolites in nature tend to have higher medicinal activity than endogenous compounds. Variant heptanoyl branches such as CBGA hexanoyl branches are more active than CBGA produced by hexanoyl branches in pharmacological efficacy.

The biosynthetic pathway of CBGA in cannabis is well understood. The synthesis method mainly comprises three steps: the generation of acyl group, the generation of phloroglucinol compound (OA) and isopentenyl modification. The content of upstream acyl groups directly affects the downstream OA and CBGA yields.

The production of plant fatty Acyl-CoA is catalyzed by Acyl-activating enzyme (AAE), for example, H1CCL2 catalyzes the production of isovaleryl-CoA and H1CCL4 catalyzes the production of isobutyryl-CoA and butyryl-CoA in hops. CsAAE1 in cannabis catalyzes the production of hexanoyl-CoA. To date, no other AAE genes than CsAAE1 have been reported to catalyze hexanoyl-coa production in yeast and further produce OA and CBGA. Therefore, the search for acyl-activating enzymes capable of catalyzing the production of hexanoyl-coa, and thus further CBGA, plays an important role in increasing CBGA production in yeast.

Acyl-coa can be produced catalytically by acyl-activating enzymes, but is also hydrolyzed by thioesterase enzymes in the cell. In yeast, three thioesterase enzymes, eht, eeb and Mgl, are involved in the hydrolysis of short-chain acyl-CoA. Wherein Eeb, eht catalyze mainly The hydrolysis of C2, C4, C6, C8 acyl-CoA [ m.j.knight, i.d.fill, p.cumow, the yeast enzyme Eht1 is an octanoyl-CoA: ethanol acyl transferase, sodium alcohol function as a thioesterase, yeast,31 (2014) 463-474; s.m.g.saerens, k.j.verstrepen, s.d.m.van Laere, a.r.d.voet, p.van Dijck, f.r.delbaux, j.m.thevelein, the Saccharomyces cerevisiae EHT1 and EEB1 Genes enzyme Enzymes with Medium-chain Fatty Acid Ethyl esters Synthesis and hydrolysics Capacity, journal of Biological Chemistry 281 (2006) 4446-4456; gajewski, r.pavlovic, m.fischer, e.balls, m.gringer, engineering fungal de novo facial acid synthesis for short chain facial acid production, nat commu, 8 (2017) 14650-14650 ]. Does it inhibit the hydrolysis of hexanoyl-coa and promote more efficient transfer of the product to OA after the three thioester hydrolases in yeast are knocked out? These problems have not been reported in research to date.

Disclosure of Invention

Based on this, it is an object of the present invention to provide an acyl-activating enzyme for use in the production of a CBGA precursor.

The specific technical scheme is as follows:

an acyl-activating enzyme for producing a CBGA precursor, which comprises At4g05160, atAAE1, atAAE12 or CsAAE3.

The sequences of the acyl activating enzyme expression genes At4g05160, atAAE1, atAAE12, csAAE3 and CsAAE1 are optimized by selecting yeast preference codons by a Gene Optimizer. The gene or protein sequence from which the signal peptide is not removed is located as Full Length (FL), and the gene or protein sequence from which the signal peptide is removed is defined as a Truncated Polypeptide (TP), respectively.

The gene sequence of At4g05160 is shown in SEQ ID NO.6, or is a sequence which is completely complementary and paired with SEQ ID NO.6, or is a nucleotide sequence which is shown in SEQ ID NO.6 and can encode the same functional protein by replacing, deleting and/or adding one or more nucleotides, or is a sequence which has At least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown in SEQ ID NO. 6; or the gene sequence of the At4g05160 is shown in SEQ ID No.10, or is a sequence which is completely complementary and paired with SEQ ID No.10, or is a nucleotide sequence which is shown in SEQ ID No.10 and can encode the same functional protein by replacing, deleting and/or adding one or more nucleotides, or is a sequence which has At least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown in SEQ ID No. 10;

the gene sequence of the AtAAE1 is shown as SEQ ID NO.7, or is a sequence which is completely complementary and matched with the SEQ ID NO.7, or is a nucleotide sequence which is shown as the SEQ ID NO.7 and can encode the same functional protein by replacing, deleting and/or adding one or more nucleotides, or is a sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as the SEQ ID NO. 7; or, the gene sequence of the AtAAE1 is shown as SEQ ID NO.11, or is a sequence which is completely complementary and matched with the SEQ ID NO.11, or is a nucleotide sequence which is shown as SEQ ID NO.11, is subjected to substitution, deletion and/or addition of one or more nucleotides and can encode the same functional protein, or is a sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as SEQ ID NO. 11;

the gene sequence of the AtAAE12 is shown as SEQ ID NO.8, or is a sequence which is completely complementary and matched with the SEQ ID NO.8, or is a nucleotide sequence which is shown as the SEQ ID NO.8 and can encode the same functional protein by replacing, deleting and/or adding one or more nucleotides, or is a sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as the SEQ ID NO. 8; or, the gene sequence of the AtAAE12 is shown as SEQ ID NO.12, or is a sequence which is completely complementary and matched with the SEQ ID NO.12, or is a nucleotide sequence which is shown as SEQ ID NO.12 and can encode the same functional protein by replacing, deleting and/or adding one or more nucleotides, or is a sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as SEQ ID NO. 12;

the gene sequence of the CsAAE3 is shown as SEQ ID NO.9, or is a sequence which is completely complementary and matched with the SEQ ID NO.9, or is a nucleotide sequence which is shown as the SEQ ID NO.9 and can code the same functional protein by replacing, deleting and/or adding one or more nucleotides, or is a sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as the SEQ ID NO. 9; or the gene sequence of the CsAAE3 is shown as SEQ ID NO.13, or is a sequence which is completely complementary and matched with the SEQ ID NO.13, or is a nucleotide sequence which is shown as the SEQ ID NO.13, is subjected to substitution, deletion and/or addition of one or more nucleotides and can encode the same functional protein, or is a sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as the SEQ ID NO. 13.

The invention also aims to provide a construction method of the saccharomyces cerevisiae engineering bacteria for producing the CBGA precursor.

The technical scheme for realizing the purpose is as follows:

a construction method of Saccharomyces cerevisiae engineering bacteria for producing CBGA precursors comprises the following steps:

(1) Constructing a recombinant expression vector 2 capable of simultaneously expressing a tetrone synthase, an olive acid cyclase and the acyl activating enzyme;

(2) Preparing a Saccharomyces cerevisiae competent cell 1 with the thioester hydrolase gene knocked out;

(3) Transforming the recombinant expression vector 2 obtained in the step 1 into a saccharomyces cerevisiae competent cell 1; obtaining the saccharomyces cerevisiae engineering bacteria for producing the CBGA precursor.

In some embodiments, the thioesterase gene knock-out comprises the steps of:

(3.1) constructing a guide RNA expression vector for targeted removal of thioesterase hydrolase;

(3.2) transforming the guide RNA expression vector obtained in the step 3.1 into a saccharomyces cerevisiae competent cell 1 to obtain a mutant strain competent cell;

(3.3) transforming the recombinant expression vector 2 to the mutant strain competent cell obtained in the step 3.2 to obtain the Saccharomyces cerevisiae engineering bacteria for producing the CBGA precursor.

In some embodiments, the acyl activating enzyme gene is AtAAE1, the nucleic acid sequence of which is the full-length sequence shown in SEQ ID No.8 without the signal peptide removed, or the sequence perfectly complementary and paired with SEQ ID No.8, or the nucleotide sequence shown in SEQ ID No.8 with one or more nucleotides substituted, deleted and/or added and can encode the same functional protein, or the sequence at least having a similarity of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more to SEQ ID No. 6; or the gene sequence of the AtAAE1 is a truncated sequence which is shown as SEQ ID NO.12 and is removed from the signal peptide, or is a sequence which is completely complementary and matched with the SEQ ID NO.12, or is a nucleotide sequence which is shown as the SEQ ID NO.12 and has one or more nucleotides substituted, deleted and/or added and can encode the same functional protein, or is a nucleotide sequence which has at least the similarity of more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99% with the sequence shown as the SEQ ID NO. 6.

In some embodiments, the thioester hydrolase gene is at least one of Eht1, eeb and Mgl.

In some embodiments, the mutant strain competent cells have three genes Eht, eeb and Mgl knocked out simultaneously.

The invention also aims to provide the saccharomyces cerevisiae engineering bacteria for producing the CBGA precursor.

The technical scheme for realizing the purpose is as follows:

the saccharomyces cerevisiae engineering bacteria for producing the CBGA precursor are obtained by the construction method.

It is also an object of the present invention to provide a method for producing a CBGA precursor.

The technical scheme for realizing the purpose is as follows:

a method of producing a CBGA precursor, comprising the steps of:

(1) Constructing engineered saccharomyces cerevisiae producing CBGA precursors as described in claim 7;

(2) Fermenting and culturing the saccharomyces cerevisiae engineering bacteria for producing the CBGA precursor in the step (1) to obtain a fermentation culture product;

(3) And (3) extracting and purifying the fermentation culture product obtained in the step (2) to obtain a CBGA precursor.

In some of these embodiments, the fermentation culture described above may be carried out continuously, or in a batch process (batch culture) or discontinuously in a fed-batch or repeated fed-batch process. A summary of the general properties of the known cultivation methods is available in the textbook of Chmiel (BioprozeBtechnik.1: einfiihung in die Bioverfahrentechnik (Gustav Fischer Verlag, stuttgart, 1991)) or Storhas (Bioreaktren and periphere Einrichtungen (Vieweg Verlag, braunschweig/Wiesbaden, 1994)).

In some of these embodiments, the above fermentation culture product extraction purification includes, but is not limited to, one or more measures selected from the group consisting of: a) Partial (> 0% to < 80%) to complete (100%) or substantially complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98% or > 99%) water removal; b) Partial (> 0% to < 80%) to complete (100%) or substantially complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98% or > 99%) removal of biomass, the latter optionally being inactivated prior to removal; c) Partial (> 0% to < 80%) to complete (100%) or essentially complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3% or > 99.7%) removal of organic by-products formed during the cultivation; and d) partial (> 0%) to complete (> 100%) or essentially complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3% or > 99.7%) removal of components of the used fermentation medium or starting material which are not consumed in the culture from the spent medium, thereby achieving concentration or purification of the desired target metabolite. The CBGA precursor, further preferably a phloroglucinol compound, having a desired content is isolated in this way.

It is another object of the present invention to provide a recombinant expression vector simultaneously expressing a tetrone synthase, an olive acid cyclase and an acyl activating enzyme.

The invention also aims to provide an application of the recombinant expression vector in the production of CBGA, CBG, THCA, CBDA and CBD.

Another object of the present invention is to provide a method for preparing CBGA, comprising the steps of:

(1) Constructing saccharomyces cerevisiae engineering bacteria for expressing tetrone synthase, olive acid cyclase and acyl activating enzyme, wherein the acyl activating enzyme can catalyze the generation of hexanoyl coenzyme A in saccharomyces cerevisiae;

(2) In the saccharomyces cerevisiae engineering bacteria obtained in the step (1), hexanoic acid is catalyzed by the acyl activating enzyme to generate hexanoyl coenzyme A, and then phloroglucinol compounds are generated under the catalysis of tetraketone synthase and olive acid cyclase;

(3) Preparing CBGA by using the phloroglucinol compound generated in the step (2).

In some embodiments, the acyl activating enzyme gene is AtAAE1.

In some embodiments, the gene sequence of AtAAE1 is shown in SEQ ID NO.7 or SEQ ID NO. 11.

In some embodiments, the construction method of the saccharomyces cerevisiae engineering bacteria in the step (1) comprises the following steps:

(1-1) constructing a saccharomyces cerevisiae engineering bacterium integrating tetraketone synthase and olive acid cyclase genes on a genome;

(1-2) constructing an expression vector of the gene of the acyl activating enzyme;

(1-3) constructing and obtaining the saccharomyces cerevisiae engineering bacteria simultaneously expressing the tetrone synthase, the olive acid cyclase and the acyl activating enzyme.

In some embodiments, step (1) further comprises knocking out the thioester hydrolase gene in the engineered saccharomyces cerevisiae.

In some embodiments thereof, the knockout thioester hydrolase gene is one or both of Eht and Eeb.

In some embodiments, the knock-out thioester hydrolase gene is Eht, eeb, and Mgl.

An object of the present invention is also to provide an application of an acyl-activating enzyme in the preparation of CBGA.

In some embodiments, the acyl activating enzyme is AtAAE1, and the gene sequence of AtAAE1 is shown in SEQ ID NO.7 or SEQ ID NO. 11.

In some embodiments, the acyl-activating enzyme catalyzes the production of hexanoyl-coa from hexanoic acid in saccharomyces cerevisiae.

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

the inventor of the invention finds an acyl-activating enzyme expression gene AtAAE1 which can effectively catalyze the generation of hexanoyl coenzyme A in a yeast body by constructing a saccharomyces cerevisiae engineering strain (chassis strain 1) with a tetraketone synthase and an olive acid cyclase gene integrated on a genome, and successfully constructs a saccharomyces cerevisiae strain (chassis strain 2 for expressing a recombinant expression vector) which can simultaneously express the tetraketone synthase, the olive acid cyclase and the acyl-activating enzyme; on the basis, a guide RNA expression vector for targeted removal of thioesterase is constructed and transformed into a chassis strain 2, so that the saccharomyces cerevisiae engineering bacteria capable of producing the CBGA precursor in a large scale are obtained.

The influence of each thioesterase hydrolase mutation on the production of OA is verified by measuring the OA content, and the results show that: when Eht and Eeb are knocked out in the chassis strain 2 respectively, the content of OA in the yeast can be increased by 1.5 times and 1.2 times. The OA content of the mutant strain with two genes of Eht1 and Eeb knocked out simultaneously is improved by 1.6 times, and the OA content of the mutant strain with three genes of Eht1, eeb1 and Mgl knocked out simultaneously is improved by 1.81 times.

The saccharomyces cerevisiae engineering bacteria can effectively improve the OA yield of the strain through optimizing the expression of the acyl activating enzyme and the expression of the thioesterase through down-regulation, can predict the yield of the hexanoyl coenzyme A and lays a foundation for further constructing a new high-efficiency synthetic CBGA precursor OA strain.

Drawings

FIG. 1 is a schematic diagram of the acyl-activating enzyme yeast recombinant expression vector pYES2-Gal1p-AtAAE1FL-CYT1t in example 3.

FIG. 2 is a statistical chart comparing the yields of partially expressed acyl-activating enzyme OA in the engineered Saccharomyces cerevisiae of example 4, wherein CK group is a negative control group.

FIG. 3 is a statistical chart comparing the yields of partially expressed acyl-activating enzyme OA in the engineered Saccharomyces cerevisiae of example 4, wherein CK group is a negative control group.

FIG. 4 is a schematic diagram of the guide RNA expression vector targeting the knockdown of thioesterase in example 6.

FIG. 5 is a statistical chart comparing the growth and OA yield of engineered Saccharomyces cerevisiae after the thioesterase removal in example 6, wherein A is: growth comparison statistical chart, and B is OA yield comparison statistical chart, wherein the host of the experimental group is a thioester hydrolase gene non-knock-out control group.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following more detailed description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present disclosure does not necessarily refer to the same embodiment, although it may. Moreover, the phrase "in another embodiment" as used in this disclosure does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification the meaning of "a", "an" and "the" include plural referents. The meaning of "in.

The present invention will be described in further detail with reference to specific examples.

Example 1 construction of Chassis Strain 1

The sequence CsS-CsOAC is inserted between the pGOAC 1 and the pGOAC 2 at the site of the vector by a method described in reference [ X.Luo, M.A.Reiter, L.d' Espaux, J.Wong, C.M.Denby, A.Lechner, Y.Zhang, A.T.Grzybowski, S.Harth, W.Lin, H.Lee, C.Yu, J.Shin, K.Deng, V.T.Bennites, G.Wang, E.E.K.Baidoo, Y.Chen, I.Dev, C.J.Petzold, J.D.Keasling, complex biosyntheses of natures and the unknown Nature,567 (2019) 123-126 ]. The expression cassette pYES2-CsTKS-CsOAC was obtained, wherein the synthetic (universal biosynthesis) sequences of CsTKS-CsOAC were as follows:

＞CsTKS-CsOAC(SEQ ID NO.1)

the expression cassette pYES2-CsTKS-CsOAC was amplified with primers 1114-GAL1p-F-49 and 1114-CYC1t-R-50 in tables 1-3, and the size was 2623bp. The following amplification procedure was used:

TABLE 1-1

Components	Volume of
		2xPhusion master mix with HF buffer	25ul
Stencil (20 ng/ul)	2ul
		Primer 1 (10 uM)	1ul
Primer 2 (10 uM)	1ul
		Adding water	Make up to 50ul

The amplification conditions were as follows:

tables 1 to 2

TABLE 1-3 primers used for the construction of Bacillus bifidus 1

Detecting the specificity of the target band by electrophoresis, and cutting and recovering the gel. The expression cassette pYES2-CsTKS-CsOAC was inserted into ARS1114a site of Saccharomyces cerevisiae strain (CEN. PK2-1C) genome, CEN. PK2-1C single clone was picked up and inoculated to 5ml YPD, cultured overnight at 30 ℃ with shaking table at 230 rpm. Transfer to fresh YPD medium (approximately 10ml of bacteria per transformation) at 1: 200 starting OD600 around 0.1. When OD600=0.4, the cells were centrifuged, washed once with 0.1M LiAC, pH5.7, centrifuged to collect the cells, and reagents were added to each transformed sample as shown in the following table:

tables 1 to 4

Reagent	Volume of
		PEG 3500 50％w/v	240μl
LiAc 1.0M	36μl
		Boiled SS-carrier DNA(10mg/mL)	10μl
DNA mix plus Water	74μl
		Total volume	360μl

100ng of pCas-ARS1114a sgRNA plasmid (the construction method of pCas-ARS1114a sgRNA plasmid refers to the construction method of the thiolylase targeting site vector in example 6) and 500ng of corresponding expression cassette fragment were added to each sample. After 30 minutes of standing, the plate was heat-shocked at 42 degrees for 15 minutes. Centrifugation at 4000g for 5 minutes removed the supernatant. The cells were incubated overnight at 30 ℃ with 500ul YPD resuspended. The next day, 100ul of cells were blotted onto YPD +300ng/ml G418 resistant plates, blow-dried, and then cultured at 30 ℃ for 2-3 days. The grown clones were identified by colony PCR using primers V1114-F-71 and Gal1R in tables 1-3 to confirm correct insertion into ARS1114a of the CEN. PK2-1C genome of the strain. Correctly cloning YPD shake bacteria and then storing the strain as a basidiomycete 1 for free expression and integrated expression of acyl-activating enzyme candidate genes in the next step.

EXAMPLE 2 optimization of acyl-activating enzyme Gene sequences

Exogenous genes expressed in yeast often fail to achieve the desired effect due to codon inapplicability. The inventors optimized the At4g05160, atAAE1, atAAE12, csAAE3 and CsAAE1 sequences using Gene Optimizer to select yeast-preferred codons. The gene or protein sequence without the signal peptide removed is located as Full Length (FL), the gene or protein sequence without the signal peptide removed is defined as Truncated Polypeptide (TP), and the peroxisome signal peptide is generally C-terminal SKL of the protein. FL and TP sequences are shown below, and the gene sequences are determined and then transferred to general-purpose biosynthesis.

The optimized nucleotide sequences are respectively as follows:

＞At4g05160 FL(SEQ ID NO.6)

＞AtAAE1 FL(SEQ ID NO.7)

＞AtAE12 FL(SEQ ID NO.8)

＞CsAAE3 FL(SEQ ID NO.9)

＞At4g05160 TP(SEQ ID NO.10)

＞AtAE1 TP(SEQ ID NO.11)

＞AtAE12 TP(SEQ ID NO.12)

＞CsAAE3 TP(SEQ ID NO.13)

EXAMPLE 3 construction of acyl-activating enzyme Yeast expression vectors

Based on the sequences synthesized in example 2, primers in the tables were designed (as shown in tables 3-3) and cloned into the 3' end of GAL1 promoter of pYES2 vector to drive the expression of each sequence correctly. Insert acquisition and linearization of pYES2 vector was amplified from a 2 × Phusion master mix with HF buffer (NEB) in the following specific amplification system:

TABLE 3-1

Components	Volume of
		2×Phusion master mix with HF buffer	25ul
Stencil (20 ng/ul)	2ul
		Primer F (10 uM)	1ul
Primer R (10 uM)	1ul
		Adding water	Make up to 50ul

The amplification conditions were as follows:

TABLE 3-2

TABLE 3-3 primers for construction of candidate Gene FL and TP expression vectors

Note: in the base sequence, the bold bases represent homologous sequences, and the non-bold portions are amplification primers.

And (5) detecting whether the size of the target band is correct and single through electrophoresis, and cutting and recovering the correct band. The gene vector was constructed according to the NEB HIFI DNA assembly instructions. The reaction system is as follows: 0.2pmol of linearized pYES2 vector, the ratio of the number of moles of fragment to the number of moles of vector was 3: 1,5ul 2 XHIFI master mix, supplemented to 10ul with water. The reaction was carried out at 50 ℃ for 30 minutes. All reactions were transformed to DH5a competence and positive clones were selected on LB/Amp medium. And (3) sequencing after colony PCR to verify the correctness, extracting plasmids of the clones with the correct sequencing, and determining the concentration and the purity of the clones by using Nanodrop. Through verification, FL and TP vectors of each candidate acyl-activating enzyme gene are successfully constructed, wherein the construction schematic diagram of the expression vector of the gene AtAAE1FL is shown in figure 1.

Example 4 expression of acyl-activating enzyme Gene in Yeast and functional verification

Verification of the function of each protein when the acyl activating enzyme is expressed freely: 100ng of each acyl-activating enzyme expression vector is respectively transferred into the bacillus chassis 1, and positive transformants are screened on an SD-Ura screening plate. Positive transformants were picked, 400ul SD-Ura was added to a 96-well deep-well plate, and cultured for 24 hours at 30 ℃ on a high-speed shaker at 800 rpm. Yeast cells were inoculated into 400ul SG-Ura medium at a ratio of 1:40, 1mM hexanoic acid (100 mM hexanoic acid stock solution was made up with 0.1M NaOH) was added, and cultured on a 30 ℃ high speed shaker at 800rpm for 72-96 hours.

Adding acetonitrile in the same volume, shaking in a high-speed shaking table at 30 ℃ for 30min to break the yeast cells, and centrifuging at 3000rpm for 10min. The supernatant was transferred to a UPLC loading vial and UPLC tested for the production of OA and related compounds in each sample.

Verification of the function of each protein when the acyl activating enzyme is expressed integrally: the constructed yeast expression vector is used as a template, and the primers in the table 4-1 are used for respectively amplifying expression cassettes of different acyl activating enzymes. Electrophoresis is used for detecting the specificity of the target band, and the gel is cut to recover the target band, and the fragments are used for integrating expression cassettes expressing different acyl activating enzymes.

Chassis 1 competence was prepared, 100ng of sgRNA vector at X2 locus and 500ng of expression cassette fragment were added, and positive clones were screened on YPD +300ng/ml G418 plates. Colony PCR was performed with primers X2-up-260/GAL1R to confirm the insertion of the cassette into the genome. And (3) culturing the positive clone by using 400ul YPD culture medium for 24 hours, inoculating the positive clone into 400ul YPG culture medium according to the ratio of 1:40 to induce and express target gene expression, cracking cells after 72 hours, and determining the total OA content.

TABLE 4-1 primers for integration and expression of acyl-activating enzyme genes

The statistical results are shown in fig. 2 and 3, and experiments show that in free expression, atAAE1 can catalyze hexanoic acid to generate hexanoyl-coenzyme a in yeast regardless of FL or TP, so that OA is further generated under catalysis of CsTKS and CsOAC. This trend was not observed for the other proteins At4g05160, atAAE12 and CsAAE3. After the AtAAE1 is integrated on the yeast genome, the OA content in a negative control (the basidiomycetes 1 without the transformed AAE gene) is compared, so that the AtAAE1 can be further determined to be capable of effectively catalyzing hexanoic acid to generate hexanoyl coenzyme A, and then OA is generated under the catalysis of CsTKS and CsOAC, so that the method is applied to the heterologous production of CBGA.

Example 5 construction of Chassis Strain 2

Method 1

The procedure described in reference example 1 was used to insert the sequence CsTKS-CsOAC-CsAAE1 between the sites pGAL1 and CYC1t, respectively, in the vector pYES 2. Wherein the synthetic (universal biosynthesis) sequences of the CsTKS-CsOAC-CsAAE1 are respectively as follows:

＞CsTKS-CsOAC-CsAAE1(SEQ ID NO.38)

with reference to the same method and reagents as in example 1 above, a strain in which the expression cassette pYES2-CsTKS-CsOAC-CsAAE1 was correctly inserted into the ARS1114a site of the CEN. PK2-1C genome was verified and obtained as the strain of Bacillus licheniformis 2.

Method 2

The yeast expression vector for acyl-activating enzyme described in example 3 was inserted into Chassis bacterium 1 constructed in example 1, and Chassis bacterium 2 was constructed in the same manner as described above, with reference to example 4.

Example 6 thioester hydrolase Gene knock-out and validation

Although hexanoyl-coa can be produced smoothly in yeast, the thioesterase present in yeast can hydrolyze hexanoyl-coa to hexanoic acid, reducing the flux of hexanoic acid to the OA and CBGA metabolic pathways. In order to investigate the process of reducing the expression of thioesterase and improving the expression of thioesterase, the inventor designs an experiment for knocking out three thioesterase in a yeast genome, constructs single mutant strains, double mutant strains and triple mutant yeast strains of three genes of ScEb 1, scEht1 and ScMgl2 respectively, determines the OA content and analyzes the function of the thioesterase. The specific process is as follows:

firstly, sgRNA expression vectors of genes Eeb, eht and Mgl are constructed, corresponding genes are targeted, and mutations are introduced. The sequences of the sgRNAs are shown in tables 6-1, and schematic maps of the sgRNAs expression vectors are shown in FIG. 4. The construction process is as follows: the F primer of each sgRNA is matched with Cas9R-0001 to amplify a first segment expressed by the sgRNA, and the R primer of each sgRNA is matched with the Cas9F-0001 primer to amplify a second segment expressed by the sgRNA. The pCas plasmid is cut by XmaI/BglII enzyme, and the target band is recovered by cutting the gel. The gene vector was constructed according to the NEB HIFI DNAsystem instructions.

The reaction system is as follows: 0.2pmol of linearized pCAS vector with a ratio of moles fragment to moles vector of 3: 1,5ul 2 XHIFI master mix (NEB) supplemented to 10ul with water. The reaction was carried out at 50 ℃ for 30 minutes. All reactions were transformed to DH5a competence and positive clones were selected on LB/Amp medium. The correctness of the clone is verified by sequencing after colony PCR, plasmid extraction is carried out on the clone with the correct sequencing, and the concentration and the purity of the clone are determined by using Nanodrop.

TABLE 6-1 thioesterase hydrolase genes Eeb, eht and Mgl sgRNA and mutant sequences

Preparation of mutated donor DNA for each gene: the samples were mixed uniformly according to the following system:

TABLE 6-2

The program set in the PCR instrument was as follows:

tables 6 to 3

Preparation of mutants: the preparation of the competent cells of Bacillus bifidus 2 was performed as described in example 1, and 100ng of pCAS-Eht sgRNA, pCAS-Eeb sgRNA, pCAS-Mgl2 sgRNA were co-transformed with 5ul of the prepared donor DNA into Bacillus bifidus 2 (wherein the acyl activating enzyme was CsAAE 1) and screened on YPD +300ng/ul G418 resistant plates. The obtained positive clones were subjected to colony PCR amplification, and the PCR products were subjected to Sanger sequencing to confirm the correctness of the corresponding gene mutation. The same double mutant and triple mutant preparation process is the same as that of single mutant, and the difference is different in competence.

Eht1, eeb and Mgl single mutant, double mutant and triple mutant were cultured by shaking 400ul YPD for 24 hours at 800rpm in a high speed shaker, and the ratio of 1:40 were inoculated into YPD +1mM hexanoic acid medium and OA content was measured after 72 hours of culture, and the results are shown in FIG. 5. The results show that the yeast growth is not obviously affected when Eht and Eeb1 are knocked out, but the OA yield in the yeast is respectively improved by 1.5 times and 1.2 times. While the knockout of Mgl2 did not result in a significant increase in OA production. The OA yield in the double mutant eeb1/eht1 is improved to be 1.6 times of that in the control, and the OA yield in the triple mutant eeb1/eht1/mgl2 is improved to be 1.81 times of that in the control. The three thioester hydrolases knocked out in yeast can effectively inhibit the hydrolysis of hexanoyl-CoA and promote the generation of OA. The discovery can complement the function of the acyl activating enzyme AtAAE1 mutually, and lays a foundation for constructing a new high-efficiency synthetic CBGA precursor OA strain.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Hangzhou En and Biotechnology Co., ltd

<120> Saccharomyces cerevisiae engineering bacterium for producing CBGA precursor, and construction method and application thereof

<160> 54

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1794

<212> DNA

<213> Artificial Sequence

<400> 1

atgaatcatt taagagctga aggtccagcc tccgttttgg ccatcggtac cgctaaccct 60

gaaaacattt tgttgcaaga cgaattccca gactactact tcagagtcac taagtccgaa 120

cacatgaccc aattgaagga gaagttcaga aagatttgtg acaagtccat gattagaaag 180

agaaactgtt tcttgaacga agaacacttg aagcaaaacc caagattggt tgaacatgaa 240

atgcaaactt tggacgctag acaagacatg ttggttgttg aagtccctaa gttgggtaag 300

gatgcctgtg ctaaggccat taaagaatgg ggtcaaccta agtccaagat tacccacttg 360

attttcacct ctgcctccac cactgacatg cctggtgctg attaccactg cgctaagtta 420

ttgggtttgt ctccatccgt taagagagtt atgatgtacc aattgggttg ctacggtggt 480

ggtactgttt taagaattgc taaggatatt gctgaaaaca acaagggtgc cagagtctta 540

gctgtctgct gtgacattat ggcttgttta ttcagaggtc catctgaatc cgacttggaa 600

ttgttggttg gtcaagctat cttcggtgac ggtgctgctg ccgttattgt tggtgctgaa 660

ccagacgaat ccgttggtga aagaccaatt tttgaattgg tttccaccgg tcaaactatt 720

ttgccaaatt ccgaaggtac catcggtggt catatcagag aagccggttt gatcttcgac 780

ttacataagg atgtcccaat gttgatctct aacaacattg aaaagtgttt gatcgaagct 840

tttaccccaa ttggtatttc tgactggaac tctatcttct ggattaccca tcctggtggt 900

aaggctattt tggataaggt cgaggaaaaa ttgcacttga agtctgacaa gttcgttgac 960

tctagacacg tcttgtccga acatggtaat atgtcctctt ccaccgtttt attcgttatg 1020

gatgagttga gaaagagatc cttagaagaa ggtaagtcca ccaccggtga tggttttgag 1080

tggggtgttt tgttcggttt cggtccaggt ttgaccgtcg aaagagttgt tgttagatct 1140

gtcccaatta agtacgcagc cacaagcggt tctacgggct ccacgggctc taccggcagt 1200

gggaggagca ctgggtcaac gggatcaaca ggtagtggaa gatcacacat ggttgccgtc 1260

aagcacttga tcgttttgaa gttcaaggat gaaatcactg aagctcaaaa ggaagaattc 1320

ttcaaaacct acgtcaactt agtcaatatt attccagcca tgaaggacgt ctattggggt 1380

aaggacgtta ctcaaaagaa taaggaggaa ggttatactc atatcgttga ggtcactttc 1440

gaatctgttg agactattca agactacatc atccacccag cccacgttgg tttcggtgat 1500

gtttatcgtt ccttctggga aaaattgttg atcttcgact acacccctag aaagggatcc 1560

taactcgaga gcttttgatt aagccttcta gtccaaaaaa cacgtttttt tgtcatttat 1620

ttcattttct tagaatagtt tagtttattc attttatagt cacgaatgtt ttatgattct 1680

atatagggtt gcaaacaagc atttttcatt ttatgttaaa acaatttcag gtttaccttt 1740

tattctgctt gtggtgacgc gtgtatccgc ccgctctttt ggtcacccat gtat 1794

<210> 2

<211> 68

<212> DNA

<213> Artificial Sequence

<400> 2

aaagacgaag cctatttact atcaacgcta tgtaagtttg tactattatc acggattaga 60

agccgccg 68

<210> 3

<211> 71

<212> DNA

<213> Artificial Sequence

<400> 3

cctttcaatc actgattcgt ttcacgatgt tagatgatgc tattgactgt gcaaattaaa 60

gccttcgagc g 71

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 4

gcaaagggga agagaagatt gc 22

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 5

gggttttttc tccttgacgt taaagt 26

<210> 6

<211> 1635

<212> DNA

<213> Artificial Sequence

<400> 6

atggaaaagt ctggttatgg tagagatggt atctacagat ctttaagacc aaccttggtt 60

ttgccaaagg atccaaatac ttccttggtg tctttcctgt tcagaaactc ttcttcatac 120

ccatccaaat tggctattgc tgattctgat actggtgact ctttgacctt ctcacaattg 180

aaatctgctg ttgctagatt ggctcatggt tttcatagat tgggtatcag aaagaacgat 240

gtcgttttga ttttcgcccc aaactcttac caatttcctt tgtgtttttt ggccgttact 300

gctattggtg gtgtttttac tactgctaac ccactgtaca ctgttaacga agtttctaag 360

caaatcaagg acagcaaccc aaagatcatc atttctgtta atcagctgtt cgacaagatc 420

aagggtttcg atttgccagt tgttttgttg ggttctaagg ataccgttga aattccacca 480

ggttccaact ctaagatctt gtctttcgat aacgtcatgg aattgtccga acctgtttct 540

gaatatccat tcgtcgaaat caagcaatct gatacagctg ctttgttgta ctcttctggt 600

actactggta cttctaaggg tgttgaattg actcacggta actttattgc tgcctctttg 660

atggttacca tggatcaaga tttgatgggt gaataccatg gtgttttctt gtgtttcttg 720

ccaatgttcc atgttttcgg tttggctgtt attacctact ctcaattgca aagaggtaac 780

gccttggttt ctatggctag atttgaattg gaactggtct tgaagaacat cgaaaagttc 840

agagttaccc acttgtgggt tgttccacca gtttttttgg ctttgtctaa gcaatccatc 900

gtcaagaagt tcgacttgtc atccttgaag tatattggtt ctggtgctgc tccattgggt 960

aaagacttga tggaagaatg tggtagaaac atcccaaacg tcttgttgat gcaaggttac 1020

ggtatgactg aaacttgtgg tatcgtttct gttgaagatc caagattggg caagagaaat 1080

tctggttctg ctggtatgtt ggctccaggt gttgaagctc aaattgtctc tgttgaaact 1140

ggtaaatccc aaccaccaaa tcaacaaggt gaaatttggg ttagaggtcc caatatgatg 1200

aagggttatt tgaacaatcc acaggctacc aaagaaacga tcgataagaa atcttgggtt 1260

cacactggtg atttgggcta ctttaatgaa gatggtaact tgtacgttgt cgacaggatc 1320

aaagaactga ttaagtacaa gggttttcaa gttgctccag cagaattgga aggtttgttg 1380

gtttctcatc cagatatttt ggatgccgtt gttatcccat ttccagatga agaggctggt 1440

gaagttccta ttgcttttgt tgttagatcc cctaactcct ccattaccga acaagatatc 1500

caaaagttca ttgccaaaca agttgcccca tacaagagat tgagaagagt ctcttttatc 1560

agcttggtcc caaaatcagc tgctggtaag attctaagaa gagaattggt tcagcaagtc 1620

aggtctaaga tgtga 1635

<210> 7

<211> 1671

<212> DNA

<213> Artificial Sequence

<400> 7

atgaagatgg aaggcactat taagtctcca gctaattacg ttccactgac tccaatttca 60

ttcttggata gatctgctgt tgtttacgcc gatagagttt ctatcgttta cggttctgtt 120

aagtacactt ggagacaaac cagagataga tgcgttagaa ttgcttctgc tttgtctcaa 180

ttgggtattt ctactggtga tgttgtttct gttttggctc caaatgttcc agctatggtt 240

gaattgcatt ttggtgttcc aatggctggt gctttgttgt gtactttgaa catcagacac 300

gattcctcat tggttgctgt tttgttgaga cattctggta ctaaggttat cttcgccgat 360

catcaattct tgcaaattgc tgaaggtgcc tgcgaaattt tgtctaacaa aggtgataag 420

gtccccatct tggttttgat tccagaacca ttgactcagt ctgtctccag aaaaaaaagg 480

tccgaagaaa tgatggaata cgaagatgtt gttgctatgg gcaagtctga tttcgaagtt 540

attagaccaa ccgatgaatg tgatgccatc tccgttaact atacttctgg tacaacttct 600

tcaccaaagg gtgttgttta ttctcataga ggtgcttact tgaactcttt ggctgcagtt 660

ttattgaacg agatgcattc ttctccaact tacttgtgga ctaatccaat gtttcactgt 720

aacggttggt gtttgttatg gggtgttact gctattggtg gtacaaacat ttgcttgaga 780

aatgttaccg ctaaggccat ctttgacaat atctctcaac ataaggttac ccacatgggt 840

ggtgctccaa ctattttgaa catgattatt aacgccccag agtccgaaca aaaaccattg 900

ccaggtaagg tttctttcat tactggtgct gctccaccac cagctcatgt tattttcaaa 960

atggaagagt tgggcttctc catgttccat tcttatggtt tgactgaaac ttacggtcca 1020

ggtactatat gtacttggaa accagaatgg gattccttgc caagagaaga acaagctaaa 1080

atgaaggcta gacaaggtgt taaccacttg ggtttagaag aaatccaagt taaggatcca 1140

gttaccatga gaactttgcc agctgatggt gttactatgg gtgaagttgt ttttagaggt 1200

aacaccgtta tgaacggcta cttgaaaaat ccagaagcta ccaaagaagc ttttaaaggc 1260

ggttggtttt ggtctggtga tttgggtgta aaacatccag atggttacat cgaattgaag 1320

gacagatcca aggatatcat tatttccggt ggtgagaaca tctcctccat tgaagttgaa 1380

tctactttgt tcacccatcc atgtgttttg gaagctgcag ttgttgcaag accagatgaa 1440

tattggggtg aaactgcttg tgctttcgtt aagttgaaag atggttctaa ggcttccgct 1500

gaagagttga tttcttactg tagagacaga ttgccacatt acatggctcc aagatctatc 1560

gtctttgaag atttgccaaa aacctccact ggtaaggttc aaaagttcgt cttgagaact 1620

aaggctaagg ctttggtttc tttgtctaag aagggtcgtt ctaagctgtg a 1671

<210> 8

<211> 1737

<212> DNA

<213> Artificial Sequence

<400> 8

atggataact tggctttgtg cgaagctaac aatgttccat tgactccaat cacttttttg 60

aagagggctt ctgaatgtta cccaaacaga acctctatta tctacggtaa gactagattc 120

acttggccac aaacttacga tagatgttgt agattggctg cctccttgat ttctttgaac 180

attggtaaga acgacgttgt ttctgttgtt gctccaaata ctccagctat gtacgaaatg 240

cactttgctg ttccaatggc tggtgctgtt ttgaatccta ttaacactag attggatgcc 300

acctccattg ctgctatttt gagacatgct aagccaaaga tcttgttcat ctacagatct 360

ttcgaaccat tggccagaga aatcttgcaa ttgctatctt ccgaagattc caacttgaac 420

ttgccagtta ttttcatcca cgaaatcgac ttcccaaaga gagtttcttc tgaagaatct 480

gactacgaat gcttgattca aagaggtgaa cctactcctt tgttgttggc tagaatgttc 540

tgcatccaag atgaacatga cccaatctca ttgaattaca cctctggtac tactgctgat 600

ccaaagggtg ttgttatttc tcatagaggt gcttacttgt ctaccttgtc tgctattatt 660

ggttgggaaa tgggtacttg tccagtttat ttgtggactt tgccaatgtt tcactgtaac 720

ggttggactt ttacttgggg tactgctgct agaggtggta cttctgtatg tatgagacat 780

gttactgctc cagagatcta caagaacatc gaaatgcata acgttaccca tatgtgttgc 840

gttccaactg ttttcaacat cttgttgaag ggtaactcct tggatttgtc tcatagatct 900

ggtccagttc atgttttgac tggtggttct ccaccaccag ctgctttggt taagaaagtt 960

caaagattgg gtttccaagt tatgcatgct tacggtttga ctgaagctac aggtcctgtt 1020

ttgttttgtg aatggcaaga tgaatggaac aggttgccag aaaatcaaca gatggaattg 1080

aaagctagac agggcttgtc tattttgggt ttgaccgaag ttgatgtgag gaacaaagaa 1140

acccaagaat ctgttccaag agatggtaaa actatgggtg aaatcgttat gaagggctcc 1200

tctataatga agggttactt gaaaaatcca aaggctacct acgaagcttt caaacatggt 1260

tggttgaatt ctggtgatgt tggtgttatt catccagatg gtcatgttga gatcaaggat 1320

agatccaagg atatcattat ttccggtggt gagaacatct cctctgttga agttgagaac 1380

attatctaca aataccccaa ggttttggaa actgccgttg ttgctatgcc acatccaact 1440

tggggtgaaa ctccatgtgc ttttgttgtt ttagaaaagg gcgaaactaa caacgaagat 1500

agagaagata agctggtcac caaagaaaga gacttgattg aatactgcag ggaaaacttg 1560

ccacatttca tgtgtccaag aaaggtcgtt tttttggacg aattgccaaa gaatggtaac 1620

ggcaagattt tgaagccaaa gttgagagat atcgccaaag gtttggttgc tgaagatgaa 1680

gttaatgtca gatctaaggt tcaaagacca gttgaacatt tcacctccag attgtga 1737

<210> 9

<211> 1632

<212> DNA

<213> Artificial Sequence

<400> 9

atggaaaagt ctggttatgg tagagatggt atctacagat ctttaagacc accattgcat 60

ctgccaaaca acaacaattt gtccatggtg tctttcttgt tccgtaactc ttcttcatac 120

ccacaaaagc cagctttgat cgactctgaa actaaccaga ttttgtcctt ctcacacttc 180

aagtccaccg ttattaaggt ttctcatggc tttttgaacc tgggcatcaa aaagaacgat 240

gtcgttttga tctacgcccc aaactctatt catttcccag tttgtttctt gggcattatt 300

gcttcaggtg ctattgctac tacttctaac ccattataca ccgtgtctga attgtccaag 360

caagttaagg attccaatcc aaagttgatc attaccgttc cacagttgtt ggaaaaggtt 420

aagggtttta acttgcccac cattttgatt ggtccagact ctgaacaaga atcctcttca 480

gataaggtta tgaccttcaa cgacttggtt aacttaggtg gttcttctgg ttcagaattc 540

ccaatcgttg atgacttcaa gcaatctgat actgctgctt tgttgtactc ttctggtact 600

actggtatgt ctaagggtgt tgttttgacc cacaagaact ttattgcctc ttctttgatg 660

gtcaccatgg aacaagattt ggttggtgaa atggacaacg ttttcttgtg tttcttgcca 720

atgttccacg ttttcggttt ggctattatt acctacgctc aattgcaaag aggtaacacc 780

gttatatcta tggccagatt cgatttggag aagatgttga aggatgtcga aaagtacaag 840

gttacccatt tgtgggttgt tccaccagtt attttggctt tgtctaagaa ctccatggtc 900

aagaagttca acctgtcctc cattaagtat attggttctg gtgctgctcc attgggtaaa 960

gatttgatgg aagaatgctc taaggttgtc ccatatggta tagttgctca aggttacggt 1020

atgactgaaa cttgtggtat cgttagcatg gaagatatta gaggtggtaa gagaaattct 1080

ggttctgcag gtatgttagc ttctggtgtt gaagctcaaa tcgtttctgt tgatactttg 1140

aaacccttgc caccaaatca attgggtgaa atttgggtca aaggtccaaa tatgatgcag 1200

ggttacttca acaatccaca agctactaag ttgaccatcg ataagaaagg ttgggttcat 1260

actggtgact tgggttactt tgatgaagat ggtcacttgt acgttgtcga cagaatcaaa 1320

gaactgatca agtacaaagg tttccaagtt gctccagctg aattggaagg tttgttagtt 1380

tctcatccag aaatcttgga tgccgttgtt attccatttc cagatgctga agctggtgaa 1440

gttccagttg cttatgttgt tagatctccc aactcttcct tgactgaaaa cgatgtgaaa 1500

aagttcattg ctggtcaagt tgcctctttc aagagattga gaaaggttac cttcatcaac 1560

tccgttccaa aatctgcttc tggtaagatc ttgagaagag agttgattca aaaggtcagg 1620

tccaacatgt ga 1632

<210> 10

<211> 1626

<212> DNA

<213> Artificial Sequence

<400> 10

atggaaaagt ctggttatgg tagagatggt atctacagat ctttaagacc aaccttggtt 60

ttgccaaagg atccaaatac ttccttggtg tctttcctgt tcagaaactc ttcttcatac 120

ccatccaaat tggctattgc tgattctgat actggtgact ctttgacctt ctcacaattg 180

aaatctgctg ttgctagatt ggctcatggt tttcatagat tgggtatcag aaagaacgat 240

gtcgttttga ttttcgcccc aaactcttac caatttcctt tgtgtttttt ggccgttact 300

gctattggtg gtgtttttac tactgctaac ccactgtaca ctgttaacga agtttctaag 360

caaatcaagg acagcaaccc aaagatcatc atttctgtta atcagctgtt cgacaagatc 420

aagggtttcg atttgccagt tgttttgttg ggttctaagg ataccgttga aattccacca 480

ggttccaact ctaagatctt gtctttcgat aacgtcatgg aattgtccga acctgtttct 540

gaatatccat tcgtcgaaat caagcaatct gatacagctg ctttgttgta ctcttctggt 600

actactggta cttctaaggg tgttgaattg actcacggta actttattgc tgcctctttg 660

atggttacca tggatcaaga tttgatgggt gaataccatg gtgttttctt gtgtttcttg 720

ccaatgttcc atgttttcgg tttggctgtt attacctact ctcaattgca aagaggtaac 780

gccttggttt ctatggctag atttgaattg gaactggtct tgaagaacat cgaaaagttc 840

agagttaccc acttgtgggt tgttccacca gtttttttgg ctttgtctaa gcaatccatc 900

gtcaagaagt tcgacttgtc atccttgaag tatattggtt ctggtgctgc tccattgggt 960

aaagacttga tggaagaatg tggtagaaac atcccaaacg tcttgttgat gcaaggttac 1020

ggtatgactg aaacttgtgg tatcgtttct gttgaagatc caagattggg caagagaaat 1080

tctggttctg ctggtatgtt ggctccaggt gttgaagctc aaattgtctc tgttgaaact 1140

ggtaaatccc aaccaccaaa tcaacaaggt gaaatttggg ttagaggtcc caatatgatg 1200

aagggttatt tgaacaatcc acaggctacc aaagaaacga tcgataagaa atcttgggtt 1260

cacactggtg atttgggcta ctttaatgaa gatggtaact tgtacgttgt cgacaggatc 1320

aaagaactga ttaagtacaa gggttttcaa gttgctccag cagaattgga aggtttgttg 1380

gtttctcatc cagatatttt ggatgccgtt gttatcccat ttccagatga agaggctggt 1440

gaagttccta ttgcttttgt tgttagatcc cctaactcct ccattaccga acaagatatc 1500

caaaagttca ttgccaaaca agttgcccca tacaagagat tgagaagagt ctcttttatc 1560

agcttggtcc caaaatcagc tgctggtaag attctaagaa gagaattggt tcagcaagtc 1620

aggtga 1626

<210> 11

<211> 1662

<212> DNA

<213> Artificial Sequence

<400> 11

atgaagatgg aaggcactat taagtctcca gctaattacg ttccactgac tccaatttca 60

ttcttggata gatctgctgt tgtttacgcc gatagagttt ctatcgttta cggttctgtt 120

aagtacactt ggagacaaac cagagataga tgcgttagaa ttgcttctgc tttgtctcaa 180

ttgggtattt ctactggtga tgttgtttct gttttggctc caaatgttcc agctatggtt 240

gaattgcatt ttggtgttcc aatggctggt gctttgttgt gtactttgaa catcagacac 300

gattcctcat tggttgctgt tttgttgaga cattctggta ctaaggttat cttcgccgat 360

catcaattct tgcaaattgc tgaaggtgcc tgcgaaattt tgtctaacaa aggtgataag 420

gtccccatct tggttttgat tccagaacca ttgactcagt ctgtctccag aaaaaaaagg 480

tccgaagaaa tgatggaata cgaagatgtt gttgctatgg gcaagtctga tttcgaagtt 540

attagaccaa ccgatgaatg tgatgccatc tccgttaact atacttctgg tacaacttct 600

tcaccaaagg gtgttgttta ttctcataga ggtgcttact tgaactcttt ggctgcagtt 660

ttattgaacg agatgcattc ttctccaact tacttgtgga ctaatccaat gtttcactgt 720

aacggttggt gtttgttatg gggtgttact gctattggtg gtacaaacat ttgcttgaga 780

aatgttaccg ctaaggccat ctttgacaat atctctcaac ataaggttac ccacatgggt 840

ggtgctccaa ctattttgaa catgattatt aacgccccag agtccgaaca aaaaccattg 900

ccaggtaagg tttctttcat tactggtgct gctccaccac cagctcatgt tattttcaaa 960

atggaagagt tgggcttctc catgttccat tcttatggtt tgactgaaac ttacggtcca 1020

ggtactatat gtacttggaa accagaatgg gattccttgc caagagaaga acaagctaaa 1080

atgaaggcta gacaaggtgt taaccacttg ggtttagaag aaatccaagt taaggatcca 1140

gttaccatga gaactttgcc agctgatggt gttactatgg gtgaagttgt ttttagaggt 1200

aacaccgtta tgaacggcta cttgaaaaat ccagaagcta ccaaagaagc ttttaaaggc 1260

ggttggtttt ggtctggtga tttgggtgta aaacatccag atggttacat cgaattgaag 1320

gacagatcca aggatatcat tatttccggt ggtgagaaca tctcctccat tgaagttgaa 1380

tctactttgt tcacccatcc atgtgttttg gaagctgcag ttgttgcaag accagatgaa 1440

tattggggtg aaactgcttg tgctttcgtt aagttgaaag atggttctaa ggcttccgct 1500

gaagagttga tttcttactg tagagacaga ttgccacatt acatggctcc aagatctatc 1560

gtctttgaag atttgccaaa aacctccact ggtaaggttc aaaagttcgt cttgagaact 1620

aaggctaagg ctttggtttc tttgtctaag aagggtcgtt ga 1662

<210> 12

<211> 1728

<212> DNA

<213> Artificial Sequence

<400> 12

atggataact tggctttgtg cgaagctaac aatgttccat tgactccaat cacttttttg 60

aagagggctt ctgaatgtta cccaaacaga acctctatta tctacggtaa gactagattc 120

acttggccac aaacttacga tagatgttgt agattggctg cctccttgat ttctttgaac 180

attggtaaga acgacgttgt ttctgttgtt gctccaaata ctccagctat gtacgaaatg 240

cactttgctg ttccaatggc tggtgctgtt ttgaatccta ttaacactag attggatgcc 300

acctccattg ctgctatttt gagacatgct aagccaaaga tcttgttcat ctacagatct 360

ttcgaaccat tggccagaga aatcttgcaa ttgctatctt ccgaagattc caacttgaac 420

ttgccagtta ttttcatcca cgaaatcgac ttcccaaaga gagtttcttc tgaagaatct 480

gactacgaat gcttgattca aagaggtgaa cctactcctt tgttgttggc tagaatgttc 540

tgcatccaag atgaacatga cccaatctca ttgaattaca cctctggtac tactgctgat 600

ccaaagggtg ttgttatttc tcatagaggt gcttacttgt ctaccttgtc tgctattatt 660

ggttgggaaa tgggtacttg tccagtttat ttgtggactt tgccaatgtt tcactgtaac 720

ggttggactt ttacttgggg tactgctgct agaggtggta cttctgtatg tatgagacat 780

gttactgctc cagagatcta caagaacatc gaaatgcata acgttaccca tatgtgttgc 840

gttccaactg ttttcaacat cttgttgaag ggtaactcct tggatttgtc tcatagatct 900

ggtccagttc atgttttgac tggtggttct ccaccaccag ctgctttggt taagaaagtt 960

caaagattgg gtttccaagt tatgcatgct tacggtttga ctgaagctac aggtcctgtt 1020

ttgttttgtg aatggcaaga tgaatggaac aggttgccag aaaatcaaca gatggaattg 1080

aaagctagac agggcttgtc tattttgggt ttgaccgaag ttgatgtgag gaacaaagaa 1140

acccaagaat ctgttccaag agatggtaaa actatgggtg aaatcgttat gaagggctcc 1200

tctataatga agggttactt gaaaaatcca aaggctacct acgaagcttt caaacatggt 1260

tggttgaatt ctggtgatgt tggtgttatt catccagatg gtcatgttga gatcaaggat 1320

agatccaagg atatcattat ttccggtggt gagaacatct cctctgttga agttgagaac 1380

attatctaca aataccccaa ggttttggaa actgccgttg ttgctatgcc acatccaact 1440

tggggtgaaa ctccatgtgc ttttgttgtt ttagaaaagg gcgaaactaa caacgaagat 1500

agagaagata agctggtcac caaagaaaga gacttgattg aatactgcag ggaaaacttg 1560

ccacatttca tgtgtccaag aaaggtcgtt tttttggacg aattgccaaa gaatggtaac 1620

ggcaagattt tgaagccaaa gttgagagat atcgccaaag gtttggttgc tgaagatgaa 1680

gttaatgtca gatctaaggt tcaaagacca gttgaacatt tcacctga 1728

<210> 13

<211> 1620

<212> DNA

<213> Artificial Sequence

<400> 13

atggaaaagt ctggttatgg tagagatggt atctacagat ctttaagacc accattgcat 60

ctgccaaaca acaacaattt gtccatggtg tctttcttgt tccgtaactc ttcttcatac 120

ccacaaaagc cagctttgat cgactctgaa actaaccaga ttttgtcctt ctcacacttc 180

aagtccaccg ttattaaggt ttctcatggc tttttgaacc tgggcatcaa aaagaacgat 240

gtcgttttga tctacgcccc aaactctatt catttcccag tttgtttctt gggcattatt 300

gcttcaggtg ctattgctac tacttctaac ccattataca ccgtgtctga attgtccaag 360

caagttaagg attccaatcc aaagttgatc attaccgttc cacagttgtt ggaaaaggtt 420

aagggtttta acttgcccac cattttgatt ggtccagact ctgaacaaga atcctcttca 480

gataaggtta tgaccttcaa cgacttggtt aacttaggtg gttcttctgg ttcagaattc 540

ccaatcgttg atgacttcaa gcaatctgat actgctgctt tgttgtactc ttctggtact 600

actggtatgt ctaagggtgt tgttttgacc cacaagaact ttattgcctc ttctttgatg 660

gtcaccatgg aacaagattt ggttggtgaa atggacaacg ttttcttgtg tttcttgcca 720

atgttccacg ttttcggttt ggctattatt acctacgctc aattgcaaag aggtaacacc 780

gttatatcta tggccagatt cgatttggag aagatgttga aggatgtcga aaagtacaag 840

gttacccatt tgtgggttgt tccaccagtt attttggctt tgtctaagaa ctccatggtc 900

aagaagttca acctgtcctc cattaagtat attggttctg gtgctgctcc attgggtaaa 960

gatttgatgg aagaatgctc taaggttgtc ccatatggta tagttgctca aggttacggt 1020

atgactgaaa cttgtggtat cgttagcatg gaagatatta gaggtggtaa gagaaattct 1080

ggttctgcag gtatgttagc ttctggtgtt gaagctcaaa tcgtttctgt tgatactttg 1140

aaacccttgc caccaaatca attgggtgaa atttgggtca aaggtccaaa tatgatgcag 1200

ggttacttca acaatccaca agctactaag ttgaccatcg ataagaaagg ttgggttcat 1260

actggtgact tgggttactt tgatgaagat ggtcacttgt acgttgtcga cagaatcaaa 1320

gaactgatca agtacaaagg tttccaagtt gctccagctg aattggaagg tttgttagtt 1380

tctcatccag aaatcttgga tgccgttgtt attccatttc cagatgctga agctggtgaa 1440

gttccagttg cttatgttgt tagatctccc aactcttcct tgactgaaaa cgatgtgaaa 1500

aagttcattg ctggtcaagt tgcctctttc aagagattga gaaaggttac cttcatcaac 1560

tccgttccaa aatctgcttc tggtaagatc ttgagaagag agttgattca aaaggtctga 1620

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 14

agggccgcat catgtaatta gttat 25

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 15

gggttttttc tccttgacgt taaagt 26

<210> 16

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 16

cgtcaaggag aaaaaaccca tgggtaagaa ttacaaatcc ttgga 45

<210> 17

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 17

attacatgat gcggcccttt aggatccctc gaaatgagaa aat 43

<210> 18

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 18

cgtcaaggag aaaaaaccca tggaaaagtc tggttatggt agagat 46

<210> 19

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 19

attacatgat gcggcccttc acatgttgga cctgaccttt t 41

<210> 20

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 20

cgtcaaggag aaaaaaccca tgaagatgga aggcactatt aagtct 46

<210> 21

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 21

attacatgat gcggcccttc acagcttaga acgacccttc tta 43

<210> 22

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 22

cgtcaaggag aaaaaaccca tggataactt ggctttgtgc g 41

<210> 23

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 23

attacatgat gcggcccttc acaatctgga ggtgaaatgt tc 42

<210> 24

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 24

cgtcaaggag aaaaaaccca tggaaaagtc tggttatggt agagat 46

<210> 25

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 25

attacatgat gcggcccttc acatcttaga cctgacttgc tgaac 45

<210> 26

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 26

cgtcaaggag aaaaaaccca tggaaaagtc tggttatggt agagat 46

<210> 27

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 27

attacatgat gcggcccttc agaccttttg aatcaactct cttct 45

<210> 28

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 28

cgtcaaggag aaaaaaccca tgaagatgga aggcactatt aagtct 46

<210> 29

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 29

attacatgat gcggcccttc aacgaccctt cttagacaaa gaa 43

<210> 30

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 30

cgtcaaggag aaaaaaccca tggataactt ggctttgtgc g 41

<210> 31

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 31

attacatgat gcggcccttc aggtgaaatg ttcaactggt ctt 43

<210> 32

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 32

cgtcaaggag aaaaaaccca tggaaaagtc tggttatggt agagat 46

<210> 33

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 33

attacatgat gcggcccttc acctgacttg ctgaaccaat t 41

<210> 34

<211> 68

<212> DNA

<213> Artificial Sequence

<400> 34

agtaaattgc ctccatttct ttttcctcgg gcagagaaac tcgcaggcaa agggcgcgtg 60

gggatgat 68

<210> 35

<211> 74

<212> DNA

<213> Artificial Sequence

<400> 35

ctacacggaa accccaataa aggaaacgaa gaagtgacct tagccttcgt gcaaattaaa 60

gccttcgagc gtcc 74

<210> 36

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 36

ctatcctctt taggttaatt gtcgctg 27

<210> 37

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 37

gggttttttc tccttgacgt taaagt 26

<210> 38

<211> 4900

<212> DNA

<213> Artificial Sequence

<400> 38

atgaatcatt taagagctga aggtccagcc tccgttttgg ccatcggtac cgctaaccct 60

gaaaacattt tgttgcaaga cgaattccca gactactact tcagagtcac taagtccgaa 120

cacatgaccc aattgaagga gaagttcaga aagatttgtg acaagtccat gattagaaag 180

agaaactgtt tcttgaacga agaacacttg aagcaaaacc caagattggt tgaacatgaa 240

atgcaaactt tggacgctag acaagacatg ttggttgttg aagtccctaa gttgggtaag 300

gatgcctgtg ctaaggccat taaagaatgg ggtcaaccta agtccaagat tacccacttg 360

attttcacct ctgcctccac cactgacatg cctggtgctg attaccactg cgctaagtta 420

ttgggtttgt ctccatccgt taagagagtt atgatgtacc aattgggttg ctacggtggt 480

ggtactgttt taagaattgc taaggatatt gctgaaaaca acaagggtgc cagagtctta 540

gctgtctgct gtgacattat ggcttgttta ttcagaggtc catctgaatc cgacttggaa 600

ttgttggttg gtcaagctat cttcggtgac ggtgctgctg ccgttattgt tggtgctgaa 660

ccagacgaat ccgttggtga aagaccaatt tttgaattgg tttccaccgg tcaaactatt 720

ttgccaaatt ccgaaggtac catcggtggt catatcagag aagccggttt gatcttcgac 780

ttacataagg atgtcccaat gttgatctct aacaacattg aaaagtgttt gatcgaagct 840

tttaccccaa ttggtatttc tgactggaac tctatcttct ggattaccca tcctggtggt 900

aaggctattt tggataaggt cgaggaaaaa ttgcacttga agtctgacaa gttcgttgac 960

tctagacacg tcttgtccga acatggtaat atgtcctctt ccaccgtttt attcgttatg 1020

gatgagttga gaaagagatc cttagaagaa ggtaagtcca ccaccggtga tggttttgag 1080

tggggtgttt tgttcggttt cggtccaggt ttgaccgtcg aaagagttgt tgttagatct 1140

gtcccaatta agtacgcagc cacaagcggt tctacgggct ccacgggctc taccggcagt 1200

gggaggagca ctgggtcaac gggatcaaca ggtagtggaa gatcacacat ggttgccgtc 1260

aagcacttga tcgttttgaa gttcaaggat gaaatcactg aagctcaaaa ggaagaattc 1320

ttcaaaacct acgtcaactt agtcaatatt attccagcca tgaaggacgt ctattggggt 1380

aaggacgtta ctcaaaagaa taaggaggaa ggttatactc atatcgttga ggtcactttc 1440

gaatctgttg agactattca agactacatc atccacccag cccacgttgg tttcggtgat 1500

gtttatcgtt ccttctggga aaaattgttg atcttcgact acacccctag aaagggatcc 1560

taactcgaga gcttttgatt aagccttcta gtccaaaaaa cacgtttttt tgtcatttat 1620

ttcattttct tagaatagtt tagtttattc attttatagt cacgaatgtt ttatgattct 1680

atatagggtt gcaaacaagc atttttcatt ttatgttaaa acaatttcag gtttaccttt 1740

tattctgctt gtggtgacgc gtgtatccgc ccgctctttt ggtcacccat gtattcttgg 1800

ggccttacca ccagtggact ttcttgctgt ttgctttgtt ctggccattg tttgcgttta 1860

tatatttatg ttagatgttt ttcttattaa ctagaaagaa agaatataaa aggttgagga 1920

aagagatgta tcccgaagaa tacacagtct tttatatatg tatttcaaca aggagccgtg 1980

gagggtacta aaaagaaaaa tcgcccgggc atttcgttat cttccacgct aaaagtcaag 2040

gagagatatt acggccagga tcgcaaaggt gcagagcaag gaaatgtgag aaattgtgag 2100

aacgataatg tatgggacaa tgcgaaaatg tgagaacgag agcaaaaatc ttttttgtat 2160

ctccccgccg aatttggaaa ccgcgttctg aaaacttcgc atcttcacat agtaaaactg 2220

ttccgagcgc ttctccccat aatggttagt ggtaaaaacc gaagttgttt actttagcaa 2280

atgcccgcga atacggtggt aaattgccac ccccccttcc ccattcattg ggtaaagacc 2340

aatttgatgg ataaattggt tgtggaaaag gtctaattct ttttcctata aataccgaga 2400

tattttttct atatgatggt ttccgtcgca ttattgtact ctatagtact aaagcaacaa 2460

acaaaaacaa gcaacaaata taatatagta aaatagatct atgggtaaga attacaaatc 2520

cttggattct gttgttgctt ctgacttcat cgctttgggt atcacttccg aggtcgctga 2580

aaccttacac ggtcgtttgg ctgaaattgt ttgtaactac ggtgctgcta ccccacaaac 2640

ctggattaac atcgctaatc atattttgtc tccagatttg ccattttctt tgcatcaaat 2700

gttgttctac ggttgttata aggatttcgg tccagctcct ccagcttgga ttccagatcc 2760

agaaaaggtt aagtccacta acttgggtgc cttattggaa aaaagaggta aggaattctt 2820

aggtgttaaa tacaaagacc caatctcttc tttctctcac ttccaagaat tctctgttag 2880

aaacccagaa gtttactgga gaaccgtttt aatggacgag atgaagatct ccttttccaa 2940

ggatccagaa tgtatcttaa gacgtgatga tattaataac ccaggtggtt ccgaatggtt 3000

gccaggtggt tacttgaact ccgctaagaa ctgcttgaac gttaattcca acaagaagtt 3060

aaacgacact atgatcgttt ggagggacga aggtaacgat gacttgcctt tgaacaaatt 3120

aactttggac caattaagaa agagagtctg gttggttggt tacgctttgg aagaaatggg 3180

tttggaaaaa ggttgtgcca ttgctatcga catgccaatg cacgtcgacg ctgtcgttat 3240

ttacttggct attgtcttgg ctggttacgt tgttgtttct atcgccgact ccttctccgc 3300

cccagaaatt tccactagat tgagattgtc taaggctaag gccattttta cccaagatca 3360

tatcattcgt ggtaagaagc gtattccatt atactctaga gtcgttgaag ctaagtctcc 3420

aatggccatt gttattccat gctctggttc caatatcggt gccgaattga gggacggtga 3480

tatctcttgg gactattttt tggaaagagc taaagaattt aagaactgcg aattcaccgc 3540

cagagaacaa ccagttgacg cttacactaa catcttattc tcttctggta ccaccggtga 3600

accaaaagct attccatgga cccaagctac tcctttgaaa gccgctgctg atggttggtc 3660

ccacttagat attagaaagg gtgacgttat tgtttggcca accaacttgg gttggatgat 3720

gggtccatgg ttggtttatg cttccttgtt gaatggtgcc tccatcgctt tgtacaacgg 3780

ttctccattg gtttccggtt ttgctaagtt tgttcaagat gctaaggtca ctatgttagg 3840

tgttgttcct tctatcgtca gatcctggaa atctactaac tgtgtttctg gttacgattg 3900

gtctactatc cgttgcttct cctcttccgg tgaagcttct aacgttgacg aatatttatg 3960

gttgatgggt agagccaatt ataagcctgt cattgaaatg tgtggtggta ctgagattgg 4020

tggtgctttc tccgctggtt ccttcttgca agctcaatct ttgtcctctt tttcttctca 4080

atgtatgggt tgcactttgt acatcttgga taagaatggt tacccaatgc caaagaataa 4140

accaggtatt ggtgaattgg ccttgggtcc agttatgttc ggtgcttcca agactttatt 4200

gaacggtaac caccatgatg tttactttaa gggtatgcct actttgaacg gtgaagtttt 4260

gagaagacac ggtgacattt tcgaattaac ttccaacggt tactaccatg ctcacggtag 4320

agctgatgat accatgaaca tcggtggtat caagatctct tccattgaaa tcgagcgtgt 4380

ttgtaacgaa gttgacgaca gagttttcga aactactgcc atcggtgtcc cacctttggg 4440

tggtggtcct gaacaattgg tcattttctt cgtcttgaag gattctaacg ataccaccat 4500

cgacttgaac caattgagat tgtctttcaa cttgggtttg caaaagaagt tgaacccatt 4560

gttcaaagtc accagagttg ttccattgtc ctccttgcca cgtaccgcca ctaacaagat 4620

tatgagaaga gtcttgagac aacaattttc tcatttcgag ggatcctaac tcgaggcgaa 4680

tttcttatga tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa 4740

attttaaagt gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct 4800

gtaggtcagg ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac 4860

cggcatgccg agcaaatgcc tgcaaatcgc tccccatttc 4900

<210> 39

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 39

tccccccggg agagatggcc ggcatggt 28

<210> 40

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 40

ggaagatctc tagacctata tccactagac agaagtttg 39

<210> 41

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 41

tcgcagtggg agatttcaag gttttagagc tagaaatagc aagttaaaat aag 53

<210> 42

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 42

cttgaaatct cccactgcga aaagtcccat tcgccaccc 39

<210> 43

<211> 79

<212> DNA

<213> Artificial Sequence

<400> 43

gacgactcat taaaatcacc cactgcgtta caggttttca gcaagagatc ttataatcgg 60

ctcatgacta ccaccagca 79

<210> 44

<211> 79

<212> DNA

<213> Artificial Sequence

<400> 44

aacgcagtgg gtgattttaa tgagtcgtcc taaataccag aggttgtgca cgttccaaaa 60

ttaccaccag aaatttatt 79

<210> 45

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 45

acgccgctat tatacaacgg gttttagagc tagaaatagc aagttaaaat 50

<210> 46

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 46

ccgttgtata atagcggcgt aaagtcccat tcgccacccg 40

<210> 47

<211> 75

<212> DNA

<213> Artificial Sequence

<400> 47

aacaccgttt tattatagcg gcgttgtaat tgcagaataa caacatcctc tagcattaaa 60

cacgcatgct tcaaa 75

<210> 48

<211> 79

<212> DNA

<213> Artificial Sequence

<400> 48

attacaacgc cgctataata aaacggtgtt ggaccaatga tataagatat tgtgttaatg 60

acttgaggaa aagatttcc 79

<210> 49

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 49

actttaagtg tgtacgaaat cccatg 26

<210> 50

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 50

aaaacatggg atttcgtaca cactta 26

<210> 51

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 51

actacacttg gaattaaccg tcaccgactt atgatagatc ttcagataat gccctgatga 60

gaggctcatg gcttcccc 78

<210> 52

<211> 79

<212> DNA

<213> Artificial Sequence

<400> 52

tcataagtcg gtgacggtta attccaagtg tagtccttaa tgctagaggt tgctcaagat 60

ctaaagtcac cacccgtag 79

<210> 53

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 53

cttgtgaaac aaataattgg gttttagagc tagaaatagc aagttaaaat aag 53

<210> 54

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 54

ccaattattt gtttcacaag aaagtcccat tcgccaccc 39

Claims (7)

1. A construction method of a saccharomyces cerevisiae engineering bacterium for producing olivinic acid is characterized by comprising the following steps:

(1) Constructing a recombinant expression vector 2 capable of simultaneously expressing a tetrone synthase, an olive acid cyclase and an acyl activating enzyme;

(2) Preparing a Saccharomyces cerevisiae competent cell 1 with the thioester hydrolase gene knocked out;

(3) Transforming the recombinant expression vector 2 obtained in the step 1 into a saccharomyces cerevisiae competent cell 1; obtaining saccharomyces cerevisiae engineering bacteria for producing the olive acid;

the expression gene of the acyl activating enzyme is AtAAE1, and the gene sequence of the AtAAE1 is shown in SEQ ID NO.7 or SEQ ID NO. 11;

the thioester hydrolase genes are Eht1, eeb and Mgl.

2. The construction method according to claim 1, wherein the thioester hydrolase gene knock-out comprises the steps of:

(3.1) constructing a guide RNA expression vector for targeted removal of thioesterase hydrolase;

(3.2) transforming the guide RNA expression vector obtained in the step 3.1 into a saccharomyces cerevisiae competent cell to obtain a mutant strain competent cell;

(3.3) transforming the recombinant expression vector 2 to the mutant strain competent cell obtained in the step 3.2 to obtain the saccharomyces cerevisiae engineering bacteria for producing the olive acid.

3. An engineering strain of saccharomyces cerevisiae for producing olivinic acid, which is constructed by the construction method of any one of claims 1 to 2.

4. A method of producing olive acid comprising the steps of:

(1) Constructing the engineering bacteria of saccharomyces cerevisiae for producing the olivinic acid according to the claim 3;

(2) Fermenting and culturing the saccharomyces cerevisiae engineering bacteria for producing the olive acid in the step (1) to obtain a fermentation culture product;

(3) And (3) extracting and purifying the fermentation culture product obtained in the step (2) to obtain the olive acid.

5. The method for producing olive acid according to claim 4, wherein the thioester hydrolase gene knock-out comprises the steps of:

(3.1) constructing a guide RNA expression vector for targeted knocking-out of thioesterase;

and (3.2) transforming the guide RNA expression vector obtained in the step 3.1 into a saccharomyces cerevisiae competent cell 1 to obtain a mutant strain competent cell.

6. A preparation method of CBGA is characterized by comprising the following steps:

(1) Constructing saccharomyces cerevisiae engineering bacteria for expressing tetrone synthase, olive acid cyclase and acyl activating enzyme, wherein the acyl activating enzyme can catalyze the generation of hexanoyl coenzyme A in saccharomyces cerevisiae; the acyl activating enzyme gene is AtAAE1;

(2) In the saccharomyces cerevisiae engineering bacteria obtained in the step (1), hexanoic acid is catalyzed by the acyl activating enzyme to generate hexanoyl coenzyme A, and further, the olive acid is generated under the catalysis of tetrone synthase and olive acid cyclase;

(3) Preparing CBGA through the olive acid generated in the step (2);

the construction method of the saccharomyces cerevisiae engineering bacteria in the step (1) comprises the following steps:

(1) Constructing a recombinant expression vector capable of simultaneously expressing a tetrone synthase, an olive acid cyclase and an acyl activating enzyme;

(2) Preparing a Saccharomyces cerevisiae competent cell with the thioester hydrolase gene knocked out;

(3) Transforming the recombinant expression vector obtained in the step (1) into a saccharomyces cerevisiae competent cell; obtaining the saccharomyces cerevisiae engineering bacteria;

the thioester hydrolase gene knock-out is Eht, eeb and Mgl;

the gene sequence of the AtAAE1 is shown as SEQ ID NO.7 or SEQ ID NO. 11.

7. The CBGA production method according to claim 6, wherein said thioester hydrolase gene knock-out comprises the steps of:

constructing a guide RNA expression vector for targeted removal of thioesterase;

and transforming the obtained guide RNA expression vector to a saccharomyces cerevisiae competent cell to obtain a mutant strain competent cell.

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