CN115386588A

CN115386588A - Method for synthesizing insect sex pheromone by yeast

Info

Publication number: CN115386588A
Application number: CN202110545535.7A
Authority: CN
Inventors: 周志华; 蒋雨果; 严兴; 王平平; 黄勇平
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-11-25
Anticipated expiration: 2041-05-19
Also published as: CN115386588B

Abstract

The invention provides a yeast for synthesizing insect sex pheromone and a preparation method thereof. In particular, the invention provides a yeast cell having one, two, three or all four characteristics selected from: (1) expression of insect Δ 11-FAD, (2) expression of carboxylate reductase (CAR) and optionally its cofactors, (3) down-regulation of HFD1 expression, (4) down-regulation of OLE1 expression. The invention provides an artificial synthesis way of insect sex pheromone, breaks through the limitation that the synthesis way of the insect sex pheromone is not completely analyzed, and the steps of the synthesis way are shorter than those of the natural synthesis way.

Description

Method for synthesizing insect sex pheromone by yeast

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a method for synthesizing insect sex pheromone by using yeast.

Background

Helicoverpa armigera (Hubner) is a omnivorous pest that is a pest that damages multiple host plants. The insecticidal composition has the characteristics of strong adaptability, high survival rate, strong reproductive capacity and the like, is one of the most serious agricultural pests on crops such as cotton, corn, vegetables and the like in Asia regions, and causes great loss on the production of a large number of crops all over the world.

In order to reduce the damage of cotton bollworms to crops and increase the yield of crops, methods such as chemical control, physical control, biological control, and Bt transgenic crops have been used. But the problems of environmental pollution and pesticide residue caused by chemical pesticides are serious. Meanwhile, the cotton bollworm has serious drug resistance to chemical pesticides depending on the treatment mode of the pesticide. The physical prevention and control utilizes the light trapping and killing and other modes to kill the cotton bollworms, and the mode is environment-friendly but has low insecticidal efficiency. With the development of technology, people hope to Bt insect-resistant cotton. However, since the beginning of the use of Bt insect-resistant cotton in 1997, the resistance to cotton bollworms increased, and the difficulty in developing new insect-resistant targets increased. Under such circumstances, there is an urgent need to develop an efficient and environmentally friendly method for controlling cotton bollworms to cope with the damage of cotton bollworms to crops.

Insect pheromone (pheromone), also known as insect pheromone, is a trace chemical substance secreted by insect glands and capable of attracting intraspecific individuals to carry out information transmission and copulation to reproduce offspring, and is a species-specific communication system. Among them, sex pheromones play an important role in the reproduction and interspecies segregation of insects. The sex pheromone of the insects has the characteristics of high sensitivity, strong species specificity and the like, is convenient to use and low in cost, and is widely applied to prediction and prevention of pests in recent years.

The research of the bollworm, which is a target pest with serious influence on crop production, has attracted much attention, and the sex pheromone substance of the bollworm is extracted and identified in 1977, and the sex pheromone and the transmission mechanism of the sex pheromone are most understood since the first insect sex pheromone is found by the silkworm. The sex pheromone of cotton bollworm is mainly composed of cis-11-hexadecenal (Z11-16: 3. the two components are analyzed in the synthetic route of the cotton bollworm in vivo by an isotope labeling method. In the insect body, Z11-16; and Z9-16 is that Ald takes C18 saturated fatty acyl-CoA as a precursor, cis 11-octadecanoyl-CoA is firstly generated by dehydrogenation of delta 11 desaturase, then cis-9-hexadecanoyl-CoA is formed by shortening the carbon chain through beta-oxidation, and finally Z9-16 is synthesized under the action of fatty acyl reductase and primary alcohol oxidase. Since the primary alcohol oxidase in the insect body has not been identified, the synthetic pathways of Z11-16.

In addition to the noctuidae family to which the cotton bollworm belongs, Z11-16 and/or Z9-16 are sex pheromone components of many other moth-type insects, of which Z11-16 is Plutella xylostella (Plutella xylostella), plutella xylostella (chrysotichia tropicalis), phaedodes punctiferalis (chrysosporium), phaeophytyla armywormis (Tehama boniaella), pyraaphalocrocis nubilalis (cryptobla grandiella), southwestern corn borer (ascotaster reinticus), chilo suppressalis (Chilo supressalis), tryporyza incertulas (scirphagella incognita), nicotiana tabacia scopsis (mandshurica), and the like; ald, Z9-16, is the sex pheromone component of agricultural pests such as Plutella sera, ostrinia furnacalis (Ascogaster reticulatus), chilo supressalis (Chilo supressalis), scirpus suppressalis (Scirpophaga incertulas), plutella sacchari (Eldana saccharana), and Gekkonidae tobacco hornworm (Manduca texta).

By utilizing a synthetic biology method, a new way can be provided for synthesizing insect sex pheromone.

University of Lund in Sweden 2013

Task groupResearch uses saccharomyces cerevisiae as a basal disc cell, and introduces a Z11-16 OH synthesis pathway of the yellow cutworm into the basal disc cell, so that the synthesis of Z11-16 OH is realized, and the yield is 195 mu g/L. Since the primary alcohol oxidases required for the final synthesis of Z11-16 from Z11-16 oh to Z11-16 alds have not been identified, they further synthesize Z11-16 by chemical means (method of pyridinium chlorochromate (PCC) oxidation). The prior art methods still do not get rid of the use of chemical methods.

Disclosure of Invention

The inventor develops artificial synthesis routes of Z9-16 and Z11-16, only one insect-derived element is needed, Z9-16 and Z11-16 can be synthesized directly in yeast cells at the same time, fermentation yield is high, and yeast extract can be directly used for attracting insects such as male cotton bollworms, oriental tobacco budworms and chilo suppressalis, and therefore the artificial synthesis route has wide application prospect.

In a first aspect, the present invention provides an expression vector comprising:

(1) Insect delta 11-FAD coding sequence, and/or

(2) A carboxylate reductase (CAR), and optionally comprises the coding sequence of NpgA.

In one or more embodiments, the insect Δ 11-FAD is from a Spodoptera (Noctuidae) insect, preferably from the genera Heliothis (Helicoverpa), geotrichum (Agrotis), or Heliothis (Heliothis).

In one or more embodiments, the amino acid sequence of the insect Δ 11-FAD is selected from the amino acid sequences shown in the NCBI accession nos: AGP26038.1, AGR49312.1, ATJ44454.1, AAM28483.2, or variants having at least 90% identity thereto. In one or more embodiments, the insect Δ 11-FAD has a nucleotide sequence set forth in SEQ ID NOS: 95-98.

In one or more embodiments, the CAR is a carboxylic acid reductase of mycobacterium marinum (MmaCAR); a variant having an amino acid sequence as set forth in NCBI accession No. WP _012393886.1 or at least 90% identity thereto; the nucleotide sequence is shown as SEQ ID NO. 99.

In one or more embodiments, the cofactor is NpgA; a variant having an amino acid sequence as set forth in NCBI accession No. AAF12814.1 or at least 90% identity thereto; the nucleotide sequence is shown as SEQ ID NO. 100.

In one or more embodiments, the expression vector further comprises a promoter, a terminator. The promoter is selected from GAL1, TCCTDH and TEF1. The terminator is selected from: CYC1, RBL41B, PGK.

In one or more embodiments, the expression vector is a yeast expression vector. Yeast expression vectors include integrative vectors, episomal vectors, or yeast centromeric vectors.

In a second aspect the invention provides a yeast cell having one, two, three or all four characteristics selected from:

(1) The expression of the insect delta 11-FAD,

(2) Expressing a carboxylate reductase (CAR) and optionally expressing a cofactor therefor,

(3) The expression of HFD1 is down-regulated,

(4) OLE1 expression is down-regulated.

In one or more embodiments, the amino acid sequence of the insect Δ 11-FAD is selected from the amino acid sequences shown in the NCBI accession nos: AGP26038.1, AGR49312.1, ATJ44454.1, AAM28483.2, or variants having at least 90% identity thereto. In one or more embodiments, the insect Δ 11-FAD has a nucleotide sequence as set forth in SEQ ID NOS: 95-98.

In one or more embodiments, the cofactor is NpgA; a variant having an amino acid sequence as set forth in NCBI accession number AAF12814.1 or at least 90% identity thereto; the nucleotide sequence is shown as SEQ ID NO. 100.

In one or more embodiments, the yeast cell contains an expression vector as described herein in the first aspect.

In one or more embodiments, OLE1 expression is downregulated by replacing the promoter of OLE1 with a weakly expressing promoter. The weakly expressed promoter is CYC1.

In one or more embodiments, the HFD1 gene is knocked out using CRISPR technology.

In one or more embodiments, the yeast cell has down-regulated expression of one or more genes selected from the group consisting of: POX1, FAA4, GAL80. Preferably, the gene is knocked out using CRISPR technology.

In one or more embodiments, the yeast cell has down-regulated expression of a gene selected from any one of the group consisting of: (1) POX1, (2) POX1, FAA1 and FAA4, (3) POX1, FAA4 and GAL80.

In one or more embodiments, the yeast is saccharomyces cerevisiae.

In a third aspect, the present invention provides a method for producing yeast, or a method for synthesizing insect sex pheromones or precursors thereof by yeast, comprising in yeast:

(1) Express insect delta 11-FAD, and/or

(2) Downregulating OLE1 expression, and/or

(3) Expressing a carboxylate reductase (CAR) and optionally expressing a cofactor therefor, and/or

(4) Downregulation of HFD1 expression, and

optionally (5) downregulating expression of one or more genes selected from: POX1, FAA4, GAL80.

In one or more embodiments, the insect sex pheromone is Z11-16.

In one or more embodiments, the insect sex pheromone precursor is Z11-16 cooh and Z9-16 cooh.

In one or more embodiments, OLE1 expression is downregulated by replacing the promoter of OLE1 with a weakly expressing promoter. Preferably, the weakly expressed promoter is CYC1.

In one or more embodiments, the HFD1 gene is knocked out using CRISPR technology to down-regulate its expression.

In one or more embodiments, one or more genes selected from the group consisting of: POX1, FAA4, GAL80 to down-regulate its expression.

In one or more embodiments, (5) is downregulation of expression of a gene selected from any group of: (a) POX1, (b) POX1, FAA1 and FAA4, (c) POX1, FAA4 and GAL80.

In a further aspect herein is provided the use of a yeast cell as described in the third aspect herein for the production of Z11-16 Ald and Z9-16 Ald.

The fourth aspect of the invention also provides a yeast culture medium, which comprises 3g/L-7g/L monopotassium phosphate, 7g/L-10g/L ammonium sulfate, a carbon source, magnesium sulfate, uracil, metal ions and vitamins.

In one or more embodiments, the yeast culture medium comprises 6.3g/L potassium dihydrogen phosphate and 7.4g/L ammonium sulfate.

In one or more embodiments, the carbon source is glucose.

In one or more embodiments, the metal ion is selected from one or more of the following: zinc, cobalt, manganese, copper, calcium, iron, sodium and potassium. Preferably, the metal ion is selected from one or more of: zinc sulfate, cobalt chloride, manganese chloride, copper sulfate, calcium chloride, ferrous sulfate, sodium molybdate, boric acid, potassium iodide and ethylenediamine tetraacetic acid.

In one or more embodiments, the vitamin is selected from one or more of the following: biotin, calcium pantothenate, nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, and p-aminobenzoic acid.

In one or more embodiments, the pH of the medium is 4-8, preferably 6.

In one or more embodiments, the medium further contains n-dodecane; the concentration of n-dodecane is 5 to 20%, preferably 10% to 15%, more preferably 10%.

In one or more embodiments, the yeast culture medium is used to increase the yield of fatty acids and/or fatty aldehydes produced by yeast.

The present invention also provides a method for obtaining an insect sex pheromone or a precursor thereof, comprising culturing a yeast cell according to the second aspect of the present invention.

In one or more embodiments, the method comprises culturing the yeast cell using the medium of any of the embodiments of the fourth aspect herein.

The present invention also provides a method for obtaining fatty acids and/or fatty aldehydes, comprising culturing yeast cells with a medium according to any of the embodiments of the fourth aspect of the present invention.

The invention also provides the use of an insect sex pheromone or a precursor thereof obtained by a method as described herein for attracting insects. In one or more embodiments, the insect is a cotton bollworm.

In another aspect, the present invention provides a yeast cell with increased production of free fatty acids, said yeast cell having down-regulated expression of one or more genes selected from the group consisting of: POX1, FAA4, GAL80.

In one or more embodiments, the gene is knocked out using CRISPR technology to down-regulate expression.

The invention also provides a method of increasing the production of free fatty acids by a yeast cell comprising downregulating the expression of one or more genes of the yeast cell selected from the group consisting of: POX1, FAA4, GAL80. Preferably, the gene is knocked out using CRISPR technology to down regulate expression.

In one or more embodiments, the expression of a gene of the yeast cell selected from any one of the following groups is downregulated: (1) POX1, (2) POX1, FAA1 and FAA4, (3) POX1, FAA4 and GAL80.

The invention also provides gRNA for down-regulating the expression of HFD1, POX1, FAA4 and GAL80, and the sequence is shown in SEQ ID NO. 101-SEQ ID NO. 105.

The invention has the advantages that:

1. the artificial synthesis route of Z11-16 and Z9-16 is provided, and the following synthetic route of Z11-16 and Z9-16 in insects is broken through the limitation that the synthetic route of Z11-16 and Z9-16 is not completely resolved, and the steps of the synthetic route are shorter than those of the natural synthetic route;

2. optimizing a yeast fatty acid chassis;

3. screening a plurality of delta 11-fatty acyl desaturation elements with excellent efficiency;

4. the fermentation conditions for producing fatty alcohol and fatty aldehyde by yeast are optimized.

Drawings

FIG. 1 is a schematic diagram of the pathway for the synthesis of sex pheromones Z11-16 and Z9-16 in Heliothis armigera.

FIG. 2 is a diagram of the pathway for Ald metabolism of Saccharomyces cerevisiae Z11-16.

FIG. 3 is a graph showing the yield of free fatty acids produced by s.cerevisiae underpan cells.

FIG. 4 is a phylogenetic analysis of fatty acyl desaturases having cis 11 activity.

FIG. 5 is a graph of insect Δ 11-FAD gene production Z11-16 after expression in FA3 strain.

FIG. 6 is a graph showing Z11-16 COOH production by single-copy and multi-copy expression of the cotton bollworm FAD gene in FA3 strain.

FIG. 7 is a graph showing the production of Z11-16 COOH and Z9-16 by the FA5 strain OLE1 promoter after attenuation.

FIG. 8 is a graph of Ald yield for Z11-16 following expression of MmaCAR and NpgA genes in FA6 strain and knock-out of the hfd gene.

FIG. 9 is a graph of the Ald yield after condition optimization for Z11-16 and Z9-16.

FIG. 10 is a GC-EAD plot of detection of heterosynthesization Z11-16 Alds and Z9-16 Alds.

FIG. 11 is a Y-tube data analysis chart of insect behavior of fermentation products of a Helicoverpa armigera sex pheromone yeast cell factory.

Detailed Description

The invention provides a method for synthesizing insect sex pheromone Z11-16 and Z9-16 by using saccharomyces cerevisiae, aiming at solving the problem that the synthesis cost of Z11-16 and Z9-16 is expensive and the environmental pollution is large. Therefore, the invention provides a method for synthesizing the cotton bollworm sex pheromone Z11-16 and Z9-16 by using the saccharomyces cerevisiae, which comprises the following steps of optimizing a saccharomyces cerevisiae fatty acid chassis, optimizing insect fatty acyl desaturase elements, constructing a cotton bollworm sex pheromone yeast cell factory and a cotton bollworm sex pheromone yeast cell factory, performing fermentation optimization and functional analysis, and the like.

The term "insect sex pheromone" as used herein refers to primarily Z11-16 Ald and Z9-16 Ald, or mixtures thereof; "precursors of insect sex pheromones" are precursors of said sex pheromones present in yeast or mixtures thereof, including but not limited to Z11-16 COOH, Z9-16 COOH, Z11-16: acyl-CoA A, Z-16: acyl-CoA.

As used herein, a "Δ 11-FAD" or "Δ 11 desaturase" is an enzyme that dehydrogenates C16 and C18 fatty acyl-CoA in insects, producing cis-11-hexadecanoyl-CoA and cis 11-octadecanoyl-CoA for the synthesis of Z11-16 Ald and Z9-16 Ald in insects (as shown in FIG. 1). The inventors have found that, when expressed in yeast, the yeast can produce cis-11-palmitoyl-CoA and cis-9-palmitoyl-CoA directly (as shown in FIG. 2). Thus, by redesign, Δ 11-FAD can synthesize Z11-16 and Z9-16 in yeast in a different synthetic pathway than in insects. The insect Δ 11-FAD suitable for use herein may be from insects of the family Spodoptera (Noctuidae), preferably from the genus Trichoplusia (Helicoverpa), trichoplusia (Agrotis) or Trichoplusia (Heliothis). More preferably, the insect Δ 11-FAD is selected from the following insect Δ 11-FADs: yellow cutworm (Agrotis segetum, NCBI No.: AGP 26038.1), black cutworm (Agrotis ipsilon, NCBI No.: AGR 49312.1), helicoverpa armigera (NCBI No.: ATJ 44454.1), and Heliothis assulata (NCBI No.: AAM 28483.2). The nucleotide sequence of the insect delta 11-FAD is shown in SEQ ID NO 95-98. Preferably, the amino acid sequence of insect Δ 11-FAD is as NCBI No.: AAM 28483.2; the nucleotide sequence is shown as SEQ ID NO. 95.

As used herein, a "carboxylate reductase" or "CAR" is any enzyme that can reduce a carboxylic acid group to an aldehyde. An exemplary carboxylate reductase is a carboxylate reductase (e.g., mmaCAR) from mycobacterium (e.g., mycobacterium marinum). The amino acid sequence of MmaCAR is shown as NCBI accession number WP _ 012393886.1; the nucleotide sequence is shown as SEQ ID NO. 99. To increase the efficiency of MmaCAR, its cofactors, e.g., npgA, may be further expressed. Exemplary, the amino acid sequence of NpgA is shown as NCBI accession number AAF 12814.1; the nucleotide sequence is shown as SEQ ID NO. 100.

The yeast described herein is preferably Saccharomyces cerevisiae (e.g., IMX581, having the genotype MATa ura3-52 can 1. Delta.: cas9-natNT2 TRP1 LEU2 HIS 3), but may be any yeast having the same fatty acid metabolic pathway as Saccharomyces cerevisiae, and exemplary yeasts suitable for use herein are those having the ACC1, FAS1/2, OLE1, TE enzymes shown in FIG. 2.

In the present invention, the terms "Δ 11-FAD", "CAR", and "NpgA" also include variants of each of the indicated sequences that have the same function as each of the indicated proteins. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Amino acids with similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Also, for example, the addition of one or more amino acids at the amino-and/or carboxy-terminus will not generally alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically coded amino acid.

Variants of the polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. Any polypeptide having high homology (such as 70% or greater homology to the indicated sequence; preferably 80% or greater homology; more preferably 90% or greater homology, such as 95%,98% or 99% homology) to said Δ 11-FAD, CAR and NpgA, and having similar or identical functions, is also encompassed by the invention. The polypeptide fragment, derivative or analogue of the invention may also be: (i) A polypeptide formed by fusing a mature polypeptide to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol); or (ii) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

The invention also relates to polynucleotide sequences encoding Δ 11-FAD, CAR and NpgA or variants, analogs, derivatives thereof. The polynucleotide may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NOS: 95-100 or degenerate variants.

The present invention also relates to variants of the above polynucleotides encoding fragments, analogs and derivatives of the polypeptides having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby. As used herein, degenerate variants are referred to herein as encoding a polypeptide having NCBI No.: AGR49312.1, ATJ44454.1, AAM28483.2, AAM28483.2, WP _012393886.1, AAF12814.1, but with differences from the coding region sequences set forth in SEQ ID NO: 95-100. A "polynucleotide encoding a polypeptide" may be a polynucleotide comprising a sequence encoding the polypeptide, or may further comprise additional coding and/or non-coding sequences.

The full-length Δ 11-FAD, CAR, and NpgA nucleotide sequences of the invention or fragments thereof (e.g., primers or probes) can be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and amplified using commercially available DNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the sequences. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. Usually, the sequence is cloned into a vector, transferred into a cell, and then isolated from the propagated host cell by a conventional method. Methods known in the art for designing Primer and probe sequences are all useful herein, for example by software Primer Express.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also provides a recombinant vector, particularly a yeast expression vector, comprising the coding sequences for Δ 11-FAD, CAR and NpgA. Yeast expression vectors include integrative vectors, episomal vectors, or yeast centromeric vectors. An integration vector is used to integrate the coding sequence into the genome of the host for expression. In some embodiments, the integration vector integrates into the genome of the host for independent expression an expression cassette containing the coding sequence, the expression cassette containing (in the 5 'to 3' direction) a promoter, a gene of interest, and a terminator. Alternatively, the integration vector may also be one in which the coding sequence is integrated into a promoter in the host genome, and gene expression is achieved using the promoter. Integration may be single copy integration or multiple copy integration. Exemplary promoters GAL1, TCCTDH, TEF1; terminators such as CYC1, RBL41B, PGK. Episomal vectors are used to express the gene in a host (e.g., yeast) as a plasmid, and expression can be inducible (e.g., using an inducible promoter) or constitutive (e.g., using a constitutive promoter). In a preferred embodiment, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one cleavage site. When it is desired to express the target gene of the present invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operably linking the target gene with the promoter. If desired, the recombinant vector may further comprise an element selected from the group consisting of: a 3' polyadenylation signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; resistance selection markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, green fluorescent protein, etc.); an enhancer; or an operator. It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, terminators, enhancers.

One of ordinary skill in the art can construct expression vectors containing the genes described herein using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. When the gene of the invention is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide. The expression vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In general, any plasmid and vector may be used as long as it can replicate and is stable in the host. Exemplary yeast vectors include pMEL10, pMEL13, and pESC-Leu2d.

Vectors comprising the genes or expression cassettes of the invention may be used to transform appropriate host cells to allow the host to express the protein. The host cell may be a prokaryotic cell, such as E.coli, streptomyces, agrobacterium; or eukaryotic cells, such as yeast cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., escherichia coli), caCl may be used ₂ By treating, also withElectroporation was carried out. When the host is a eukaryote, the following DNA transfection methods may be used: liAC transformation, calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). When expressed in eukaryotic cells, the polynucleotide will provide enhanced transcription if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase gene transcription.

The invention also relates to inhibitors of OLE1, HFD1, POX1, FAA4, GAL80 and the like genes and application thereof. As used herein, "inhibit" and "down-regulate" are synonymous and refer to a partial or complete reduction in gene transcription, translation, activity of an encoded protein, interaction between a gene and an associated protein. Exemplary inhibitors include antibodies or ligands that specifically bind to the encoded protein of the gene; small molecule compounds that inhibit the activity of the gene or its encoded protein; a weak expression promoter used for replacing the original promoter (for example, the weak expression promoter is realized by transferring into a homologous recombination fragment, such as fusion of a target integration fragment and a nutrition screening label gene, and then genome integration is carried out by utilizing self homologous recombination of a host, or a plasmid containing sgRNA and the target integration fragment are introduced into a strain containing Cas9 protein to realize site-specific insertion based on a CRISPR/Cas9 system); inhibitory molecules that specifically interfere with the transcription and/or expression of the gene, such as interfering molecules that can form shRNA, ZFNs or TALENs containing specific DNA recognition domains, or grnas and gene knockout vectors for CRISPRs. Methods for making interfering molecules that interfere with the expression of a particular gene, once the target sequence is known, are well known to those skilled in the art. Herein, the promoter used to reduce the expression of OLE1 is CYC1; the gRNA for knocking out genes such as HFD1, POX1, FAA4, GAL80 and the like by CRISPR technology is shown as SEQ ID NO: 101-105. Methods of CRISPR knockouts and the reagents required therefor are well known in the art. Knockouts using CRISPR techniques also include the introduction of a CAS enzyme (e.g., CAS 9) in the cell.

The inventors found that expression of a CAR (e.g., mmaCAR) and its cofactor (NpgA) in yeast can be synthesized to give Z9-16; expression of single or multiple copies of the insect Δ 11-FAD in yeast can synthesize the precursor Z11-16 of Z11-16 Ald. Therefore, simultaneous expression of CAR and its cofactors and Δ 11-FAD allows yeast to produce Z11-16 and Z9-16. Moreover, the inventors found that downregulation of HFD1 expression can inhibit reoxidation of Z11-16 to Z11-16.

On the other hand, the inventors found that down-regulating the expression of POX1, FAA4, GAL80 genes in yeast can cause yeast to produce more free fatty acids. Since free fatty acids are the material for the synthesis of Z11-16 alds and Z9-16 alds, expression of Δ 11-FAD, CAR and its cofactors, down-regulated HFD1 expression in yeast down-regulated for these genes can synthesize more of Z11-16 alds and Z9-16 alds.

Furthermore, the inventors have found that down-regulating OLE1 expression in the yeast can increase the ratio of Z11-16 to Z9-16.

Thus, a host cell (e.g., a yeast cell) of the invention may have one, two, three, or all four characteristics selected from: characteristic (1) expression of insect Δ 11-FAD, characteristic (2) downregulation of OLE1 expression, characteristic (3) expression of carboxylate reductase (CAR) and optionally its cofactors, characteristic (4) downregulation of HFD1 expression, and optionally characteristic (5) downregulation of expression of one or more genes selected from the group consisting of: POX1, FAA4, GAL80; the yeast may synthesize insect sex pheromones or precursors thereof. The invention also provides a preparation method of the yeast, which comprises the following steps: (1) express insect Δ 11-FAD, and/or (2) down-regulate OLE1 expression, and/or (3) express carboxylate reductase (CAR) and optionally a cofactor thereof, and/or (4) down-regulate HFD1 expression, and optionally (5) down-regulate expression of one or more genes selected from: POX1, FAA4, GAL80. Any steps of the method can be performed simultaneously (e.g., simultaneously into an expression vector, a knock-out vector, or a promoter replacement vector) or sequentially and are not limited in order.

In specific examples, the genetic engineering corresponding to each yeast is as follows: FA1 is POX1 knockout; FA2 is POX1, FAA1 or FAA4 knock-out; FA3 is POX1, FAA4, GAL80 knockout; FA4 is FA3+ single copy characteristic (1); FA5 is FA3+ multicopy feature (1); FA6 is FA5+ feature (2); FAL1 is FA5+ feature (2) and feature (3); FAL2 is FA5+ feature (2), feature (3) and feature (4).

The expression or down-regulation of the above genes may be combined in any combination to obtain different synthetic products. For example, if it is desired to synthesize a low ratio of Z11-16 cooh and Z9-16 cooh, the insect Δ 11-FAD described herein can be expressed in yeast; insect Δ 11-FAD as described herein can be expressed in yeast and OLE1 expression down-regulated if it is desired to synthesize high proportions of Z11-16 COOH and Z9-16 COOH; if it is desired to synthesize Z9-16; insect Δ 11-FAD and CAR and its cofactors as described herein can be expressed in yeast and hfD1 expression optionally down-regulated if a low ratio of Z11-16 and Z9-16 Alds; if it is desired to synthesize high proportions of Z11-16 and Z9-16 alds, the insect Δ 11-FAD and CAR described herein and their cofactors can be expressed in yeast, down-regulating OLE1 expression, and optionally HFD1 expression; if it is desired to increase the synthetic yield, the expression of one or more of POX1, FAA4 and GAL80 may be further reduced on the basis of the above-mentioned modified yeast. Furthermore, if only an increase in the production of free fatty acids is desired, the expression of one or more of POX1, FAA4, GAL80 may be downregulated in yeast only.

In an exemplary embodiment, the genotype of the yeast is IMX581 pox1 Δ faa1 Δ faa4 Δ GAL80 Δ δ GAL1 GAL 11 GAL1p- Δ 11FAD-CYC1t OLE1 CYC1p-OLE1-OLE1t YMRW TCCTDHp-McSAR-RBL 41Bt-PGK1t-npgA-TEF1p hfd1 Δ.

Free fatty acids, Z11-16 Ald and/or Z9-16, or precursors thereof, can be obtained by culturing the yeast described herein. The conditions for culturing the yeast may be any conditions known in the art, for example, 30 ℃. The medium may be any medium used for culturing yeast, such as MM. It may also be an optimized yeast medium according to the invention.

Methods for extracting Z11-16 and Z9-16 from cultured yeast are known in the art, such as isopropanol-n-hexane extraction, and exemplary steps include: the cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in Tris-HCl (50mM pH = 7.5) buffer, the supernatant was discarded by centrifugation, and the cells were resuspended in 6.8% sodium sulfate solution, and 2mL of isopropanol was added: and (3) mixing the normal hexane (2:3) solution, carrying out shake reaction for at least 2h, centrifuging, concentrating the rotary-evaporated organic phase, and adding the normal hexane for dissolving. Further separation may be by chromatography.

Methods for extracting fatty acids from cultured yeast are well known in the art, and exemplary methods include the steps of: adding tetrabutylammonium hydroxide and dichloromethane mixed system (containing 50mg/L C: COOH and 200mM methyl iodide), shaking for at least 1h, centrifuging, concentrating rotary-evaporating organic phase, and adding n-hexane for dissolving. Further separation may be by chromatography.

The invention also provides an optimised yeast culture medium for increasing the yield of Z11-16 Ald and/or Z9-16. The fourth aspect of the present invention also provides a yeast culture medium comprising 3g/L to 7g/L (preferably 6.3 g/L) monopotassium phosphate, 7g/L to 10g/L (preferably 7.4 g/L) ammonium sulfate, a carbon source (e.g., glucose), magnesium sulfate, uracil, metal ions (including but not limited to 4.5mg/L zinc sulfate, 0.3mg/L cobalt chloride, 1mg/L manganese chloride, 0.3mg/L copper sulfate, 4.5mg/L calcium chloride, 3mg/L ferrous sulfate, 0.4mg/L sodium molybdate, 1mg/L boric acid, 0.1mg/L potassium iodide, 15mg/L ethylenediaminetetraacetic acid) and vitamins (including but not limited to 0.0006mg/L biotin, 0.012mg/L calcium pantothenate, 0.012mg/L nicotinic acid, 0.3mg/L inositol, 0.012mg/L thiamine hydrochloride, 0.012mg/L pyridoxine hydrochloride, 0.0024mg/L pyridoxine hydrochloride). To facilitate the extraction of Z11-16 and/or Z9-16 ald or precursors thereof, the culture medium also contains 5-20% (preferably 10% -15%, more preferably 10%) n-dodecane.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: conditions described in the laboratory Manual (New York: coldSpringHarbor laboratory Press, 2002), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Examples

The culture medium of the invention is as follows:

(1) YPD medium (1% yeast powder, 2% peptone, 2% glucose), solid medium added with 2% agar powder, and sterilized at 115 deg.C for 20min for activation and pre-culture of Saccharomyces cerevisiae.

(2) 2xYPAD medium (2% yeast powder, 4% peptone, 4% glucose, 80mg/L adenosine) was sterilized at 115 ℃ and used for the preparation of competent cells of Saccharomyces cerevisiae.

(3) YPD (G418) medium (1% yeast powder, 2% peptone, 2% glucose), solid medium added with 2% agar powder, sterilized at 115 ℃ for 20min, added with 200. Mu.g/mL G418 antibiotic, used for screening KanMX marker.

(4) SCO medium (0.67% yeast nitrogen source, 2% glucose) solid medium was added with 2% agar powder, sterilized at 115 ℃ for 20min, and the auxotrophic medium was used to screen for KlURA3 marker.

(5) LB culture medium (1% sodium chloride, 1% tryptone, 0.5% yeast powder), solid culture medium added with 2% agar powder, sterilized at 121 deg.C for 20min, for Escherichia coli activation and pre-culture.

(6) LB (Amp) medium (1% sodium chloride, 1% tryptone, 0.5% yeast powder, 100. Mu.g/mL ampicillin), 2% agar powder was added to the solid medium, sterilized at 121 ℃ and used for culturing plasmid-containing Escherichia coli.

(7) MM Synthesis Medium (pH 6.0,5g/L (NH) ₄ ) ₂ SO ₄ 、14.4g/L KH ₂ PO ₄ 、0.5g/L MgSO ₄ ·7H ₂ O、20g/L Glucose·H ₂ O, 200mg/L Uracil, metal ions and vitamins) for fatty acid and fatty aldehyde fermentation.

Example 1 Artificial design of the pathway for the Synthesis of Cotton bollworm sex pheromone in s.cerevisiae Chassis cells

The synthetic pathway in the cotton bollworm is shown in figure 1, in the insect body, Z11-16 Ald takes C16 saturated fatty acyl coenzyme A as a precursor, cis-11-hexadecanoyl coenzyme A is generated by dehydrogenation of delta 11 desaturase, and Z11-16 is synthesized under the action of fatty acyl reductase and oxidase; and Z9-16 Ald is prepared by taking C18 saturated fatty acyl-CoA as a precursor, dehydrogenating by delta 11 desaturase to generate cis 11-octadecanoyl-CoA, then carrying out beta-oxidation to shorten the carbon chain to form cis-9-hexadecanoyl-CoA, and finally synthesizing Z9-16 under the action of fatty acyl reductase and primary alcohol oxidase (Wang H L, et al, 2005). Although the intermediates in the sex pheromone synthesis pathway have been analyzed, the enzymes catalyzing the synthesis of sex pheromones have not been completely analyzed, and in order to synthesize sex pheromones of Helicoverpa armigera in Saccharomyces cerevisiae, the sex pheromone synthesis pathway of Helicoverpa armigera must be redesigned according to the characteristic of the fatty acid anabolism of Saccharomyces cerevisiae.

According to the characteristic of the synthetic metabolism of the fatty acid of the saccharomyces cerevisiae, a synthetic pathway of sex pheromone of the cotton bollworm is artificially designed in the saccharomyces cerevisiae and is shown in figure 2, the supply of precursor fatty acyl coenzyme A and the fatty acid is increased, the coding gene pox1 of fatty acyl CoA oxidase is knocked out, and the degradation of the fatty acid caused by beta oxidation is prevented; because the fatty acyl CoA synthetase encoding genes faa1 and faa4 exist, the synthesis of free fatty acid can be fed back and inhibited, and the faa1 and faa4 encoding genes are knocked out, so that a yeast chassis strain with high yield of free fatty acid can be constructed to provide a sufficient precursor for the synthesis of sex pheromone; synthesis of Z11-16 cooh and Z9-16 cooh is achieved by expression of exogenous Δ 11-FAD and yeast endogenous Δ 9-FAD (OLE 1), followed by direct reduction of Z11-16 cooh and Z9-16 cooh to Z11-16 ald by re-heterologous expression of the carboxylate reductase gene from mycobacterium marinum (mmcar) and the 4-phosphopentyltransferase gene whose coenzyme is from aspergillus nidulans (npga). Increasing the ratio of Z11-16 and Z9-16 by attenuating OLE1, inhibiting reoxidation of Z11-16 and Z9-16 to Z11-16 and Z9-16 by knocking out the hfd gene of yeast itself, increasing the ability to synthesize fatty aldehydes.

Example 2 construction of a Saccharomyces cerevisiae Chassis producing high free fatty acids

According to the synthetic route artificially designed in example 1 for the construction of sex pheromones of Helicoverpa armigera, a Saccharomyces cerevisiae chassis strain with high free fatty acid yield was first constructed to increase the supply of precursor fatty acyl-CoA and fatty acids. Fatty acyl CoA oxidase genes (pox 1) and two fatty acyl CoA synthetase genes (faa 1 and faa 4) in a yeast beta oxidation pathway are knocked out, so that degradation or activation of FFA can be reduced, and then the FFA enters other synthesis pathways, and the saccharomyces cerevisiae fatty acid chassis cell is constructed.

Synthesizing 24 primers, which respectively have the nucleotide sequences numbered SEQ ID NO. 24 in the sequence table, such as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and the like.

(1) The sgRNA-Cas9 plasmid for constructing the gene knockout strain comprises the following steps:

obtaining pMEL 10/13-gene-targettRNA linear fragment, using plasmid pMEL10 (KlURA 3) or pMEL13 (KanMX) as template, using TAKARA PrimeSTAR HS high fidelity enzyme to perform PCR amplification: amplifying by using a primer pair SEQ ID NO 1/2 to obtain a pMEL10/13-POX 1-targettRNA linear fragment; amplifying by using a primer pair SEQ ID NO of 3/4 to obtain a pMEL10/13-FAA 1-targettRNA linear fragment; amplifying by using a primer pair SEQ ID NO of 5/6 to obtain a pMEL10/13-FAA 4-targettRNA linear fragment; the pMEL10/13-GAL 80-targettRNA linear fragment is obtained by amplification with the primer pair SEQ ID NO: 7/8.

Hieff Using Shanghai assist of saint organisms Ltd

Pmu One Step Cloning Kit for pMEL 10/13-gene-self-circularizing recombination of a linear fragment of targettrna; after recombination, the E.coli TOP10 was competent transformed and plated on LB + Amp plates.

Selecting a single colony from the plate, culturing the single colony in an LB + Amp liquid culture medium for 2 hours, sending a bacterial liquid for sequencing, screening out positive clones, extracting plasmids, and respectively obtaining pMEL10/13-POX1-sgRNA-Cas9 plasmid, pMEL10/13-FAA4-sgRNA-Cas9 plasmid and pMEL10/13-GAL80-sgRNA-Cas9 plasmid.

(2) A knock-out cassette for use in constructing a strain, comprising the steps of:

PCR amplification was performed using the genome of yeast IMX581 (WT) as a template and PrimeSTAR HS Hi-Fi enzyme from TAKARA:

an upstream repair segment of the pox1 gene knockout segment is obtained by amplification of a primer SEQ ID NO. 9/10, and a downstream repair segment of the pox1 gene knockout segment is respectively amplified by a primer SEQ ID NO. 11/12. There is a homology arm of about 30bp between primers SEQ ID NO. 10 and SEQ ID NO. 11.

An upstream repair segment of the faa1 gene knockout segment is obtained by amplification of primers SEQ ID NO. 13/14, and downstream repair segments of the faa1 gene knockout segment are respectively amplified by primers SEQ ID NO. 15/16. There is a homology arm of about 30bp between primers SEQ ID NO. 14 and SEQ ID NO. 15.

An upstream repair segment of the faa4 gene knockout segment is obtained by amplification of primers SEQ ID NO. 17/18, and downstream repair segments of the faa4 gene knockout segment are respectively amplified by primers SEQ ID NO. 19/20. There is a homology arm of about 30bp between primers SEQ ID NO. 18 and SEQ ID NO. 19.

The upstream repair segment of the gal80 gene knockout segment is obtained by amplification of primers SEQ ID NO:21/22, and the downstream repair segment of the gal80 gene knockout segment is respectively amplified by primers SEQ ID NO: 23/24. There is a homology arm of about 30bp between primers SEQ ID NO. 22 and SEQ ID NO. 23.

Constructing a gene expression cassette, and forming a gene knockout cassette by a pMEL 10/13-gene-sgRNA-Cas 9 plasmid and an upstream and downstream repair fragment of a gene knockout fragment. Thus, a pox1 gene knockout cassette, a faa4 gene knockout cassette, and a gal80 gene knockout cassette were constructed.

(3) Preparation of Yeast competence

Strains IMX581, FA1, FA2 are made competent according to the Yeast competence preparation method (R Daniel Gietz, et al, 2007).

(4) Yeast transformation

Sequentially transforming the knockout expression cassettes in saccharomyces cerevisiae by using a LiAC transformation method (R Daniel Gietz, et al, 2007) to prepare corresponding yeast competence, for example, transforming the pox1 gene knockout cassette in IMX581 strain competence; transforming the faa1 knockout cassette and the faa4 knockout cassette in FA1 competence; the gal80 knock-out cassette was transformed in FA2 competence.

When the gene knockout box carries the KlURA3 label, coating the gene knockout box on an SCO plate; YPD + G418 plates were coated with the KanMX tag in the knock-out cassette.

(5) PCR validation

After the single clone grows out, carrying out PCR verification, selecting the single clone to the same plate for streak culture, and then treating the yeast single clone by DNA lysine buffer (TARAKA company) at 80 ℃ for 15min for PCR detection. Detecting the knock-out of the pox1 gene by using a primer pair SEQ ID NO 9/12; detecting the knockout of faa1 gene by using a primer pair SEQ ID NO. 13/16; detecting the knockout of faa4 gene by using a primer pair SEQ ID NO: 17/20; the primer pair SEQ ID NO:21/24 was used to detect the knockout of the gal80 gene.

(6) Marker gene recovered by removing sgRNA plasmid

Because the use of tags in yeast transformation is increased, the tags need to be recovered after each transformation is verified to be correct, and the procedure is performed with reference to the sgRNA plasmid tag recovery method (Robert Mans, et al, 2015).

(7) Fermentation of free fatty acid producing strains

Removing the sgRNA plasmid with correct PCR verification to construct a pox1 gene knockout strain FA1; constructing a faa1 gene knockout and faa4 gene knockout strain FA2 on the FA1 strain; a strain FA3 in which the gal80 gene was knocked out on the strain FA2 was constructed. Shake flask fermentations were performed on WT (IMX 581), FA1, FA2, FA3 strains, respectively, to produce free fatty acids.

The shake flask fermentation method is as follows: picking single colony into 3mL YPD tube, culturing at 30 deg.C and 250rpm for 24h, transferring into 10mL MM synthetic medium, and transferring to initial OD ₆₀₀ And (4) culturing at 30 ℃ and 250rpm for 72h, wherein the culture time is 0.1.

The fatty acid extraction and detection method comprises the following steps: sucking 200 μ L of fermented yeast liquid, adding 200 μ L of water for dilution, adding 20 μ L of tetrabutylammonium hydroxide and 400 μ L of dichloromethane mixed system (containing 50mg/L C: COOH and 200mM methyl iodide), placing in a water bottle shaker at 1500rpm, shaking for uniform mixing reaction for 1h, centrifuging at 15000g for 5min after the reaction is finished, sucking lower organic phase, adding 50 μ L of n-hexane for dissolution after rotary evaporation and concentration, taking out into a micro glass lining tube by using a pipetting gun, detecting by using a gas chromatograph GC-FID, performing chromatographic column TG-5MS (30 m × 0.25mM × 0.25 μm), maintaining nitrogen carrier gas flow at 1.25mL/min, sample loading 2 μ L, and flow split ratio of 1: free fatty acid production was analyzed 20. The procedure in which GC detects fatty acids is: the starting temperature is 120 ℃; at a rate of 30 c/min to 165 c, then at a rate of 2 c/min to 212 c, at an inlet temperature of 250 c and a detection temperature of 260 c.

The fatty acid detection result is shown in FIG. 3, the yield of free fatty acid of the yeast starting strain IMX581 is about 65mg/L, and the yield of free fatty acid of the FA1 strain with the pox1 gene knocked out in the IMX581 strain is slightly improved to about 74mg/L; the strain FA2 with the faa1 gene and the faa4 gene knocked out on the basis of FA1 has obviously improved production capacity of free fatty acid, and reaches 334mg/L; the deletion of the gal80 gene does not affect the production of fatty acid, and the yield of free fatty acid of the strain FA3 can reach 337mg/L, so that the FA3 strain is selected as a strain for producing free fatty acid by saccharomyces cerevisiae, and the research on the effectiveness of the method for synthesizing free fatty acid by saccharomyces cerevisiae is further explained.

Example 3 cloning and functional characterization of the Δ 11 fatty acyl desaturase elements

In the sex pheromone biosynthetic pathway of cotton bollworms, desaturases (FADs) are key proteins in the sex pheromone component biosynthetic pathway of moth insects. Has both position specificity and configuration specificity, can introduce Z-type or E-type double bonds at different positions in fatty acids with different chain lengths, and realizes the diversity of sex pheromone components. In the examples, four insects FAD of Lepidoptera were cloned and functionally characterized, and Δ 11 fatty acyl desaturase elements with better efficiency were selected.

Synthesizing 26 primers with SEQ ID NO 25, 26, 27 and the like in the sequence table to SEQ ID NO 50.

(1) Phylogenetic analysis of insect FAD

Several insect FADs, namely A (yellow cutworm Agrotis segetum), B (black cutworm Agrotis ipsilon), C (cotton bollworm Helicoverpa armigera) and D (tobacco budworm Heliothis assulata), which possibly have the activity of delta 11 (Z) are searched in a database of NCBI, the amino acid sequence of the insect FAD and the protein sequence reported by annotation are subjected to phylogenetic analysis, multi-sequence alignment is carried out through the MAFFT function in the Genious software, then the comparison result is introduced into MEGA5 software, the Neighbor-Joining method is adopted, bootstrand is set as 100, and the phylogenetic analysis is carried out by adopting a JTT model. According to the principle of the evolution analysis of biological systems, the closer the corresponding sequences are in the evolution distance, the stronger their homology and the greater the probability of containing the same function.

The Δ 11-FAD elements of the four insects were similar in homology to the annotated elements with Δ 11 activity, as shown in FIG. 4, based on phylogenetic analysis of fatty acyl desaturases with Δ 11 activity. Thus, the insect A, B, C, D fatty acyl-coa desaturase gene was functionally validated.

(2) Reverse extraction of insect RNA and cloning of fad Gene

Using a precooled cryopreservation tube and scissors, quickly cutting off information glands between the 8 th and the 9 th abdominal knots of the insects, enabling the information glands to fall into a centrifuge tube without RNase, adding 500 mu L of TRIzol (placed on ice), grinding the information glands into powder by using liquid nitrogen (a mortar needs DEPC treatment in advance, sterilization, drying and precooling), pouring the dry powder into the centrifuge tube filled with the TRIzol (each tube is about 100 mg), supplementing 500 mu L of LTRIZOL when the liquid nitrogen in the centrifuge tube is volatilized almost, and inverting and mixing the mixture for several times after the sample is slightly frozen; adding 200 mu L of chloroform, inverting and mixing for 15s; centrifuging at 12000rpm at 4 ℃ for 15min; taking supernatant liquid to a new centrifuge tube, adding isopropanol (about 500 mu L) with the same volume, inverting and uniformly mixing for several times; centrifuging at 12000rpm at 4 deg.C for 5min; the supernatant was decanted off, the precipitate was retained, and then 1mL of 75% ethanol was added for a little rinse; centrifuging at 12000rpm at 4 deg.C for 5min, pouring out ethanol, drying, and standing on ice for 5min; adding appropriate amount of enzyme-free water, and dissolving with flick; running glue to measure concentration, and performing reverse transcription by using a reverse First Strand cDNA Synthesis Kit to obtain insect cDNA;

amplifying corresponding insect cDNA by using a primer insect FAD-F/R to obtain an insect FAD gene, and amplifying the cotton bollworm cDNA by using a primer pair SEQ ID NO:25/26 to obtain a cotton bollworm FAD gene SEQ ID NO:95; for example, the primer pair SEQ ID NO. 27/28 amplifies the cotton bollworm cDNA to obtain the oriental tobacco budworm fad gene SEQ ID NO. 96; amplifying the agrotis ypsilon cDNA by the primer pair SEQ ID NO. 29/30 to obtain agrotis ypsilon fad gene SEQ ID NO. 97; the yellow cutworm fad gene is chemically synthesized by codon optimization, and a primer pair SEQ ID NO 31/32 is used for amplification to obtain the yellow cutworm fad gene SEQ ID NO 98.

Adding base A to the 5' end of the obtained insect fad gene by using Ex Taq enzyme of TAKARA company at 70 ℃ for 20min to respectively obtain a cotton bollworm fad gene + base A fragment, a oriental tobacco budworm fad gene + base A fragment, a black cutworm fad gene + base A fragment, a yellow cutworm fad gene + base A fragment.

A pMD18-T Vector kit of TAKARA company is adopted to connect a pMD18-T Vector linear Vector with an insect fad gene + base A fragment at 25 ℃ for 2h. Transformed in TOP10 competent cells of Escherichia coli, and the pMD18-T + insect fad gene plasmid is obtained after sequencing verification.

(2) Construction of gRNA-Cas9 expression plasmid

Similar to the step (1) in the example 2, the sgRNA plasmid of the cotton bollworm fad gene insertion site XI-3 is constructed, a primer pair SEQ ID NO:33/34 is used for amplifying pMEL10 or pMEL13 plasmid, after glue recovery, cyclization recombination is carried out, transformation is carried out in TOP10 competence, and sequencing verification is carried out to obtain the pMEL10/13-XI-3-sgRNA-Cas9 plasmid with the XI-3 site inserted.

(3) Construction of an insect fad Gene expression cassette

The fragments to be amplified when expressed at the XI-3 site of yeast are as follows:

amplifying DNA of IMX581 by using a primer pair SEQ ID NO. 35/36 to obtain an XI-3-up fragment at the upstream of the XI-3 site; amplifying pESC plasmid by using a primer pair SEQ ID NO:37/38 to obtain GAL1p promoter fragment with a homologous arm at the upstream of the X1-3 site; amplifying pESC plasmid by using a primer pair SEQ ID NO:39/40 to obtain CYC1t terminator fragment with a homologous arm at the downstream of the X1-3 locus; amplifying IMX581 DNA by using a primer pair SEQ ID NO. 41/42 to obtain an XI-3-down fragment at the downstream of an XI-3 locus;

pMD18-T + insect fad gene plasmid is used as a template, and primer pairs of SEQ ID NO 43/44, SEQ ID NO 45/46, SEQ ID NO 47/48 and SEQ ID NO 49/50 are respectively used for amplification to obtain insect fad gene fragments with homologous arms with GAL1 promoter and CYC1 terminator.

pMEL10/13-XI-3-sgRNA-Cas9 plasmid, XI-3-up fragment, GAL1p fragment, insect FAD gene fragment with homologous arms with GAL1 promoter and CYC1 terminator, CYC1t fragment and XI-3-down fragment form XI-3 expression cassette of GAL1 p-insect FAD-CYC1t gene. Therefore, XI-3 GAL1p-HarFAD-CYC1t gene expression box, XI-3 GAL1p-AseFAD-CYC1t gene expression box, XI-3 GAL1p-AipFAD-CYC1t gene expression box and XI-3 GAL1p-HasFAD-CYC1t gene expression box are constructed.

(4) Preparing FA3 yeast competence;

(5) Performing yeast transformation, and respectively transforming GAL1 p-insect FAD-CYC1t gene expression cassettes in FA3 competence, and correspondingly coating on SCO auxotroph plates or YPD +200 mug/mL G418 plates;

(6) Performing PCR detection, and detecting the DNA sequence of SEQ ID NO 35/42 by using a universal primer; detecting expressed Helicoverpa armigera FAD yeast monoclonals by using a primer pair SEQ ID NO. 25/26, detecting expressed Helicoverpa assulta FAD yeast monoclonals by using a primer pair SEQ ID NO. 27/28, detecting expressed Tilapia minutissima FAD yeast monoclonals by using a primer pair SEQ ID NO. 29/30, and detecting expressed Tilapia lutescens FAD yeast monoclonals by using a primer pair SEQ ID NO. 31/32, respectively.

(7) Removing sgRNA plasmids carrying marker genes, similar to (6) in example 2, removing sgRNA plasmids in strains of an insect fad gene expression cassette, which express XI-3 in FA3 competence, to obtain strains numbered FA3-HarFAD (FA 4), FA3-HasFAD, FA3-AipFAD, and FA 3-AseFAD;

(8) Fermentation of Saccharomyces cerevisiae heterosynthesis Z11-16 COOH

Removing the sgRNA, verifying the correctness by PCR, constructing correct strains FA3-AseFAD, FA3-AipFAD, FA3-HarFAD and FA3-HasFAD by gene expression cassettes, and performing shake flask fermentation on the strains to produce Z11-16 COOH. The fermentation and extraction assay was similar to (7) in example 2.

(9) Comparison of results for Saccharomyces cerevisiae heterosynthesis Z11-16 COOH

As can be seen from FIG. 5, the yield of Z11-16 COOH produced by the four insects Δ 11-FAD after expression in FA3 strain is higher than that of the other three insects Δ 11-FAD at Z11-16 COOH, and the yield is about 7.5mg/L. The cotton bollworm delta 11-FAD has higher expression activity in an FA3 strain, and a cotton bollworm delta 11-FAD element with higher expression activity in saccharomyces cerevisiae is screened out.

Example 4 multicopy expression of the Δ 11-FAD element from Helicoverpa armigera

After obtaining the cotton bollworm delta 11 fatty acyl desaturase element with better expression effect from the example 3, the multi-copy expression is carried out in the high fatty acid producing Chassis yeast strain FA3, and the yield of the sex pheromone precursor Z11-16 COOH is improved.

Synthesizing 8 primers respectively having the sequences from SEQ ID NO. 51, SEQ ID NO. 52, etc. to SEQ ID NO. 58 in the sequence table

(1) Construction of Cotton bollworm delta 11-fad Gene multicopy expression cassette

Firstly, amplifying DNA of IMX581 by using a primer pair SEQ ID NO:51/52 to obtain an upstream fragment of a Delta (Delta) site; 53/54 amplification FA4 strain DNA with primer pair SEQ ID NO, gain and mark the GAL1p-HarFAD-CYC1t gene fragment of gene homology arm with the upstream of (delta) site and KanMX; amplifying pMEL13 plasmid by using a primer pair SEQ ID NO:55/56 to obtain a KanMX marker gene; a primer pair SEQ ID NO:57/5 is used for amplifying DNA of IMX581, a downstream fragment of a Delta (Delta) site of a homologous arm of a KanMX marker gene promoter is obtained, and the Delta (Delta) is formed by the downstream fragment, GAL1p-HarFAD-CYC1t-KanMX gene expression box.

(2) Making FA3 yeast competent.

(3) Yeast transformation was performed by applying Delta (. Delta.) GAL1p-HarFAD-CYC1t-KanMX gene expression cassettes to FA3 strain and plating onto YPD + 200. Mu.g/mL G418 plates.

(4) And performing PCR detection, detecting by using a primer pair SEQ ID NO. 51/54, a primer pair SEQ ID NO. 25/26 and a primer pair SEQ ID NO. 56/58, and obtaining the strain FA5 which expresses the cotton bollworm delta 11-fad gene in multiple copies in the FA3 strain after the detection is correct.

(5) The FA3, FA4 and FA5 strains were subjected to fermentation extraction assay, similar to the procedure of example 2 (7).

(6) Analysis of fermentation results, as shown in FIG. 6, from the analysis of Z11-16 COOH synthesis yield, the synthesis yield of strain FA4 expressed in single copy of HarFAD in the cells of strain FA3 was about 7.5mg/L; the synthetic yield of the strain FA5 expressed by multiple copies of HarFAD is increased to 10.2mg/L. Preliminary evidence suggests that a method for multicopy expression of HarFAD is effective.

Example 5 promoter replacement of the OLE1 Gene

After multi-copy expression, the yield of Z11-16 COOH is improved, but the increase range is limited, and in order to improve the proportion of Z11-16 Ald synthesis, the operation is carried out by weakening the yeast self-fatty acyl desaturase gene ole1 (delta 9-fad) and replacing the ole1 gene self-promoter with a weak promoter CYC1 p.

Synthesizing 8 primers with SEQ ID NO 59, SEQ ID NO 60 and the like in the sequence table to SEQ ID NO 66 respectively.

(1) Constructing sgRNA plasmid replaced by the promoter of the OLE1 gene, amplifying pMEL10 plasmid by using a primer pair SEQ ID NO:59/60, recovering glue, performing cyclization recombination, transforming in TOP10 competence, and performing sequencing verification to obtain pMEL10-OLE1p-sgRNA-Cas9 plasmid replaced by the promoter of the OLE1 gene.

(2) Construction of ole1 Gene promoter attenuation substitution expression cassette

Amplifying DNA of IMX581 by using a primer pair SEQ ID NO 61/62 to obtain an upstream fragment after the ole1 promoter with the homologous arm of the CYC1p promoter is removed; amplifying DNA of IMX581 by using a primer SEQ ID NO:63/64 to obtain a CYC1p promoter gene segment; amplifying DNA of IMX581 according to SEQ ID NO 65/66 to obtain an ole1 gene upstream segment with a homologous arm of a CYC1 promoter; the sgRNA plasmid with the promoter of the OLE1 gene replaced and the several fragments form an OLE1: CYC1p-OLE1-OLE1t gene expression cassette.

(3) Prepare FA5 competence.

(4) And (3) yeast transformation, namely performing yeast transformation on the OLE1: CYC1p-OLE1-OLE1t gene expression cassette in FA5 competence, and coating the transformed yeast on an SCO auxotrophic culture medium plate.

(5) PCR verified that a yeast monoclonal of the expression cassette for the OLE 1:CYC1 p-OLE1-OLE1t gene expressed in FA5 competence was detected using primer pair SEQ ID NO 61/64. The sgRNA plasmid in the strain expressing the OLE 1:CYC1 p-OLE1-OLE1t gene expression cassette expressed in FA5 competence was removed to obtain the numbered FA6 strain.

(6) And performing fermentation extraction detection on the FA5 and FA6 strains.

(8) The results analyzed, as shown in FIG. 7, when the promoter replacement of the desaturase OLE1 gene for Z9-16 COOH acid production by yeast itself was performed, the productivity of Z11-16 COOH was increased by decreasing the production of Z9-16 COOH, and decreasing the production of Z9-16 COOH from 136mg/L to 87mg/L, so that the production of Z11-16 COOH was increased from 10mg/L to 47mg/L, which achieves the purpose of experimental design for increasing the synthesis ratio of Z11/Z9 to increase the precursors of Z11-16 Ald, and thus the FA6 strain was selected as the Z11-16 COOH acid-producing strain of Saccharomyces cerevisiae. Further illustrates the effectiveness of the method for researching the synthesis of Z11-16 COOH and Z9-16 COOH by the saccharomyces cerevisiae.

Example 6 heterologous expression of MmaCAR and NpgA genes

In the artificially designed yeast synthetic sex pheromone pathway, unsaturated fatty aldehydes are reduced from unsaturated fatty acids, so in order to shorten the synthetic route to improve the efficiency of mass conversion, the carboxylate reductase from mycobacterium marinum (MmaCAR) and its coenzyme factor 4-phosphate pentotransferase (NpgA, from aspergillus nidulans) are expressed heterologously in saccharomyces cerevisiae Z11-16 cooh acid strain FA6, whereby a further reduction of Z11-16 cooh and Z9-16 cooh to obtain the end products Z11-16 ald and Z9-16 ald.

Synthesizing 22 primers respectively having the sequences from SEQ ID NO 67, SEQ ID NO 68, etc. in the sequence table to SEQ ID NO 88

(1) Construction of mmacar and npga Gene expression cassettes

Using pUC57-MmaCAR plasmid synthesized by company and optimized by codon as template, adopting primer pair SEQ ID NO:67/68 to amplify carboxylesterase gene MmaCAR (SEQ ID NO: 99) of marine mycobacterium to obtain a segment with homologous arms with TCCTDHp promoter and RBL41Bt terminator; a codon-optimized pUC57-NpgA synthesized by a company is taken as a template, and an Aspergillus nidulans 4-phosphopentyltransferase gene NpgA (SEQ ID NO: 100) is amplified by adopting a primer SEQ ID NO:69/70 to obtain a fragment with homologous arms of a TEF1p promoter and a PGK1t terminator; using the DNA of yeast IMX581 as a template, amplifying by using a primer SEQ ID NO:71/72 to obtain an upstream fragment of the yeast ymrw15 insertion site, and amplifying by using a primer SEQ ID NO:73/74 to obtain a TCCTDHp promoter fragmentSegment, using primer SEQ ID NO 75/76 to amplify to obtain RBL41Bt segment, using primer SEQ ID NO 77/78 to amplify to obtain PGK1t segment, using primer SEQ ID NO 79/80 to amplify to obtain TEF1P segment, using primer SEQ ID NO 81/82 to amplify to obtain downstream segment of yeast ymrw15 insertion site, mixing the above-mentioned several segments with P ₂ The M ymrw15 plasmids are combined, and homologous arms are arranged among the fragments to form a ymrw15 gene expression cassette TCCTDHp-MmCAR-RBL41Bt-PGK1t-npgA-TEF1 p.

(2) FA6 yeast competence was prepared.

(3) Yeast transformation, the ymrw15 gene expression cassette TCCTDHp-MmCAR-RBL41Bt-PGK1t-npgA-TEF1p is transformed in the FA6 competence and spread on YPD + Hyg (hygromycin) plates.

(4) PCR verification, three pairs of primers SEQ ID NO 83/84, SEQ ID NO 85/86 and SEQ ID NO 87/88 are used for carrying out PCR detection on the single clone growing in the YMrw15: (TCCTDHp-MmCAR-RBL 41Bt-PGK1t-npgA-TEF1p gene expression cassette) transformed in FA6 competence, so as to construct the FAL1 strain.

Example 7 knock-out of the hfd gene

In order to improve the yield of the fatty aldehyde, a fatty aldehyde dehydrogenase encoding gene hfd1 in yeast needs to be knocked out to inhibit the oxidation of the fatty aldehyde, and the synthesis of Z11-16 Ald and Z9-16 Ald is finally completed.

Synthesizing 6 primers respectively having the numbers of SEQ ID NO 89, SEQ ID NO 90, etc. in the sequence table to SEQ ID NO 94

(1) Constructing a sgRNA plasmid with HFD gene knockout, amplifying a pMEL10 plasmid by using a primer SEQ ID NO:89/90, recovering glue, carrying out cyclization recombination, transforming in TOP10 competence, and carrying out sequencing verification to obtain a pMEL10-HFD1-sgRNA-Cas9 plasmid with HFD gene knockout.

(2) Construction of hfd Gene knockout cassette

Similar to example 2 (2), the genome of yeast IMX581 is used as a template, and the upstream and downstream homologous arms of HFD knockout fragments are respectively amplified by using a primer pair SEQ ID NO:91/92 and a primer pair SEQ ID NO:93/94, so that the upstream and downstream fragments of the HFD1 knockout fragments and the corresponding sgRNA expression plasmid form a HFD knockout cassette.

(3) FAL1 competence was prepared.

(4) Yeast transformation, hfd knock-out cassette was yeast transformed in FAL1 competence and plated on SCO plates. PCR detection of a single clone that transformed the growth of the hfd knockout cassette in FAL1 competence was performed using primer pair SEQ ID NO 91/94. The sgRNA plasmid with KlURA3 tag knocked out by hfd gene was removed to construct FAL2 strain.

(5) And (3) carrying out fermentation extraction detection on the FA6, FAL1 and FAL2 strains.

The fermentation method comprises the following steps: single colonies of FA6, FAL1 and FAL2 were picked up in 3mL YPD tubes, cultured at 30 ℃ and 250rpm for 24h, transferred to 10mL MM synthetic medium, and the initial OD ₆₀₀ And (4) =0.1, 30 ℃,250rpm, and culturing for 96h.

The extraction detection method comprises the following steps: collecting fermentation liquor, centrifugally collecting thalli, removing supernatant, adding 50mM Tris-HCl buffer solution with the same volume as the supernatant, pH7.5, for resuspension, centrifugally removing supernatant, adding 500 mu L6.8% sodium sulfate solution for resuspension, adding 2mL isopropanol: mixing a mixed solution of n-hexane (2:3), shaking, uniformly mixing, reacting for 2 hours, centrifuging to promote layering, absorbing an upper organic phase, performing rotary evaporation and concentration, adding 100 mu L of n-hexane for dissolution, detecting by using a gas chromatograph GC-FID (gas chromatograph GC-FID), using a chromatographic column TG-5MS (30 m multiplied by 0.25mm multiplied by 0.25 mu m), maintaining the nitrogen gas carrying capacity at 0.6mL/min, and the sample loading capacity of 2 mu L, wherein the split ratio is 1:10, the detection program is as follows: the starting temperature is 140 ℃; heating to 163.5 deg.C at a rate of 0.5 deg.C/min; then increased to 190 ℃ at a rate of 10 ℃/min, with both the inlet temperature and the detection temperature set at 300 ℃.

(6) Analysis of the results, as shown in fig. 8, after expressing mmaacr and coenzyme factor NpgA, and performing the knockout of hfd gene, constructing saccharomyces cerevisiae strain FAL2 capable of directly synthesizing Z11-16; the yield of Z9-16. Further illustrates the effectiveness of the method for researching the synthesis of Z11-16 and Z9-16 by the saccharomyces cerevisiae.

Example 8 optimization of fermentation Medium

After the saccharomyces cerevisiae is subjected to strain construction through gene editing, the strain has growth weakness to a certain extent, and due to the characteristics of rapid growth, easy culture, mass production and fermentation, adaptability to severe fermentation environment and the like of the saccharomyces cerevisiae, the conditions of a culture medium are optimized for obtaining a higher-yield fermentation product. Phosphorus and nitrogen sources are elements constituting important compounds such as nucleic acid lipids and are also essential elements required for cell growth, and appropriate concentrations are advantageous for production of target products. This patent improves the output of Z11-16 Ald and Z9-16 through carrying out condition optimization to potassium dihydrogen phosphate and ammonium sulfate in the culture medium.

(1) Production of Z11-16

Inoculating FAL2 strain to carry out shake flask fermentation, wherein the fermentation method is similar to that (2) in example 3, the concentration range of monopotassium phosphate in the synthetic medium components used for fermentation is controlled to be 3 g/L-15 g/L, the concentration range of ammonium sulfate is controlled to be 2 g/L-10g/L, and the components and the concentrations of the rest of the medium are unchanged.

(2) Production analysis of Z11-16 and Z9-16 by the strains after optimization of the culture medium conditions

As shown in FIG. 9, the medium condition-optimized medium was optMM (dipotassium hydrogen phosphate concentration was 6.3g/L, ammonium sulfate concentration was 7.4g/L, and the rest of the components were the same as MM medium), and during the same fermentation batch, the Ald yield of Z11-16 produced by fermentation was increased from 1.2mg/L to 1.8mg/L, and the Ald yield of Z9-16 was increased to a smaller extent than that of the non-optimized MM medium, and the yield was maintained at 3.0mg/L. Adding n-dodecane during fermentation is beneficial to extraction of fatty alcohol aldehydes (young quist J T, et al, 2013), setting that 10% of n-dodecane (optMM + Dode) is added to an optimized culture medium to produce Z11-16. And (3) using the optimized culture medium conditions to put a strain of the fermented FAL2 in a fermentation tank, wherein the final yields of Z11-16. Further illustrates the effectiveness of the method for researching the synthesis of Z11-16 and Z9-16 by the saccharomyces cerevisiae.

Example 9 electrophysiological function analysis of fermentation products of Cotton bollworm sex pheromone Yeast cell factory

Z11-16 and Z9-16 Alds obtained from the fermentation require a biological activity test in addition to GC-FID identification. Electrophysiological detection uses gas chromatography-antennal potential combination technology (GC-EAD), which is one of the most central techniques in insect behavior. Studying the identification of volatile chemicals by insects, a mixture can be screened effectively. Gas chromatography uses a capillary column that separates trace components from a complex mixture and detects the biological activity of the components by means of an antenna potential device.

(1) GC-EAD detection method

For GC-EAD detection, a GC2010pro gas chromatograph, HP-5MS (30 m.times.0.25 mm.times.0.25 μm) column was used, the sample loading was 2 μ L and the split ratio in the FID and EAD detectors was 1:1, the detection program is as follows: the initial temperature was 140 ℃ and was raised to 175 ℃ at a rate of 2 ℃/min and then to 300 ℃ at a rate of 40 ℃/min and held for 10min. The inlet temperature and the detection port temperature were both set at 300 ℃ and the nitrogen carrier gas flow was maintained at 2mL/min. The male cotton bollworm antenna 3-4 days after eclosion is used, a signal collector is connected between two electrodes, and EAD and FID signals are collected by using AutoSpike software.

(2) Analysis of GC-EAD detection results

As shown in FIG. 10, GC-EAD activity tests performed on Z11-16 and Z9-16 synthesized by Saccharomyces cerevisiae, the antenna of Helicoverpa armigera has signal reaction at the corresponding peak time and position of Z11-16 Ald and Z9-16 Ald in the sample, and can preliminarily judge that the constructed Saccharomyces cerevisiae cell synthesized Z11-16 and Z9-16 have physiological activity, further explaining the effectiveness of the method for researching the synthesis of Z11-16 and Z9-16 by Saccharomyces cerevisiae.

Example 10 insect behavioral analysis of fermentation products of Cotton bollworm sex pheromone Yeast cell factory

After the antenna electrophysiological detection of the biological activity of the yeast synthetic product, the behavioral response of the cotton bollworm to the synthetic product needs to be observed. The behavioral reaction of the cotton bollworm to the synthesized sex pheromone is observed by simulating through a Y-shaped tube olfactometer.

(1) Y-type olfactometer observation behavioural reaction method

And (3) observing the behavioral reaction by using a Y-shaped tube olfactometer, selecting the male bollworm moths within 2-5 days after eclosion, and starting a Y-shaped tube selection test after about 1 hour after entering a dark period. The whole device is arranged in a dark environment, and during test, the length of a trunk straight arm of the Y-shaped pipe model is about 60cm, and the inner diameter is about 6cm; the arms of the selection interval were about 45cm in length and about 6cm in internal diameter. Keeping the air speed of 0.6cm/s at the air inlet, placing a filter paper strip containing a sample reagent to be detected at the air inlet, enabling the sample application position to be located on the central axis of the bottle body as far as possible, enabling the air flow direction to be right opposite to the sample application position, releasing male cotton bollworm moths entering a dark period for 1 hour from a take-off point, standing for about 1 hour, observing and recording the cotton bollworm behavior reaction direction selection under red light (taking an entering side arm as a positive selection standard, and considering that the cotton bollworm moves randomly if the cotton bollworm does not enter).

In the Y-shaped tube test, positive control groups are respectively arranged: sample Vs n-hexane group; negative control group: WT fermentation extract Vs n-hexane group; experimental groups: FAL2 fermentation extract Vs n-hexane group, FAL2 crude pure Vs n-hexane group, FAL2 fermentation extract Vs WT fermentation extract group.

Positive control group: in the standard Vs n-hexane group, the standards were set at Z11-16: 3, spot amount Z11-16 on filter paper about 400ng Ald, Z9-16 about 12.5ng Ald, 10. Mu.l n-hexane was added.

Negative control group: in the WT fermentation extract Vs hexane group, since WT did not produce Z11-16 Ald and Z9-16.

Experimental groups: in FAL2 fermentation extract Vs n-hexane group, due to the mixed components in the fermentation extract, the fermentation extract is not purified, wherein the ratio of Z11-16: 2, the amount of Z11-16 applied to the filter paper was about 450ng Alds, about 900ng Z9-16 applied to the filter paper, and 10. Mu.l of n-hexane was added.

In the FAL2 fermentation extract Vs WT fermentation extract group, a system equivalent to the FAL2 crude extract is added on a filter paper sheet because WT does not produce Z11-16.

In the crude pure Vs n-hexane group of FAL2, crude FAL2 extract was subjected to crude purification (Ding B J, et al, 2014) to obtain Z11-16 and Z9-16 ald products, and Z11-16 and Z9-16. 10. Mu.l of n-hexane was added.

And (3) putting the male cotton bollworm moths of 2-4 days old from the flying point, observing cotton bollworm selection for about 1 hour, and counting the experimental results of effective selection.

(2) Analysis of observation behavior reaction result of Y-shaped tube olfactometer

As shown in fig. 11, the Y-tube data analysis of insect behavior for the fermentation product of the cotton bollworm sex pheromone yeast cell factory showed that in the positive control group, 64% of the 118 effective data were selected for the standard and 36% were selected for n-hexane, indicating that the ratio of 97:3, Z11-16 and Z9-16. In the negative control group, 27% of the 129 effective data selected WT yeast extract and 73% selected n-hexane, indicating that the fermentation extract of the unmodified WT strain was poor in cotton bollworm trapping effect.

In the experimental group, in the FAL2 fermentation extract Vs n-hexane group, in 156 effective data, 59% of FAL2 extract and 41% of n-hexane are selected.

In FAL2 crude product Vs n-hexane group, in 117 effective data groups, 62% of FAL2 crude product is selected, and 38% of n-hexane is selected.

In the FAL2 fermentation extract Vs WT fermentation extract group, in 93 effective data groups, 68% of FAL2 fermentation extracts were selected, and 32% of WT fermentation extracts were selected.

By referring to a positive control and a negative control, the experimental group shows that the cotton bollworm is trapped by using the cotton bollworm sex pheromone components Z11-16 and Z9-16 which are synthesized by saccharomyces cerevisiae and directly using yeast fermentation extracts or performing crude purification. Further, the effective method for synthesizing the cotton bollworm sex pheromone components Z11-16 and Z9-16 by using the saccharomyces cerevisiae is shown in the patent.

Sequence listing

<110> China academy of sciences molecular plant science remarkable innovation center

<120> method for synthesizing insect sex pheromone by yeast

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gattccattt tgaggattcc tatatccagt acggattaga agccgccgag c 51

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ctaacgccgc catccagtgt cgacttcgag cgtcccaaaa ccttctcaag 50

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cagcgacatg gaggcccaga at 22

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tcgacactgg atggcggcgt ta 22

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tgtcaaggag ggtattctgg gcctccatgt cgctgtcgag gagaacttct agtatattc 59

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atgaagcagg tgttgttgtc tgttgag 27

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taaatgatca tagccgtaac aatagggatg ttttagagct agaaatagca agttaaaat 59

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agctctaaaa catccctatt gttacggcta tgatcattta tctttcactg cggagaagt 59

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gttctgaggt attcgtatcg c 21

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<213> Artificial Sequence

<400> 62

accaaccaac gctcgccaaa tgaatagtca cggagaatct tgacgt 46

<210> 63

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 63

tcatttggcg agcgttggtt 20

<210> 64

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 64

atttagtgtg tgtatttgtg tttg 24

<210> 65

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 65

acacaaacac aaatacacac actaaatatg ccaacttctg gaactact 48

<210> 66

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 66

tctatggtaa ccggcagtaa t 21

<210> 67

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 67

gaacttagtt tcgaataaac acacataaac aaacaaaatg tccccaatta ccagagaag 59

<210> 68

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 68

tacttgacct gaacttaacg atttgctctc aatccgctta caacaagccc aacagcctc 59

<210> 69

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 69

caaagaagca ccaccaccag tagagacatg ggagatctca agacaagcag ttacataca 59

<210> 70

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 70

gaaagcatag caatctaatc taagttttaa ttacaaaatg gttcaagata cctcttctg 59

<210> 71

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 71

tcaatcaaag caacccacaa 20

<210> 72

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 72

agtaaaaaag gagtagaaac attttgaagc tatggcgcgt gcggtgtaag aaaatgaca 59

<210> 73

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 73

ctgtttctca aactttatgt cattttctta caccgcacgc gccatagctt caaaatgtt 59

<210> 74

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 74

ttctttccaa tctttcttct ctggtaattg gggacatttt gtttgtttat gtgtgttta 59

<210> 75

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 75

gtacgtttct gatttgaggc tgttgggctt gttgtaagcg gattgagagc aaatcgtta 59

<210> 76

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 76

gcgtatttta agtttaataa ctcgaaaatt ctgcgttagc cgaaaatctt tcaagcacg 59

<210> 77

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 77

aatgaatctt tctgtcgtgc ttgaaagatt ttcggctaac gcagaatttt cgagttatt 59

<210> 78

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 78

accatgtgct actggtgtat gtaactgctt gtcttgagat ctcccatgtc tctactggt 59

<210> 79

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 79

ttggagaggt agaagcagaa gaggtatctt gaaccatttt gtaattaaaa cttagatta 59

<210> 80

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 80

gaagatttat aatggtttat cggttgcatt ttccatgagt gatcccccac acaccatag 59

<210> 81

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 81

gtagaaacat tttgaagcta tggtgtgtgg gggatcactc atggaaaatg caaccgata 59

<210> 82

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 82

gccgtcctca tgatgtgtta 20

<210> 83

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 83

accgagggga gtcacttcat 20

<210> 84

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 84

ttgtggcaat ggcaaacctg 20

<210> 85

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 85

gctgttgctg ttccattgca 20

<210> 86

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 86

gcggcagctc tacatacagt 20

<210> 87

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 87

gtgcggttta ccagttgctg 20

<210> 88

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 88

gtgcggttta ccagttgctg 20

<210> 89

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 89

gcagtgaaag ataaatgatc agggtaaaat cattccaata gttttagagc tagaaatag 59

<210> 90

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 90

ctatttctag ctctaaaact attggaatga ttttaccctg atcatttatc tttcactgc 59

<210> 91

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 91

ttccagtaca ggcacaccag 20

<210> 92

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 92

aattaattaa ccttaaacat tacgtgttgg tgataaatta ctatggctat g 51

<210> 93

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 93

atagccatag taatttatca ccaacacgta atgtttaagg ttaattaatt atttgatg 58

<210> 94

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 94

gggtgatggt ttggcgaatg 20

<210> 95

<211> 1017

<212> DNA

<213> Artificial Sequence

<400> 95

atggcccaaa gctatcaatc aactacagtt ttgagtgagg agaaagaacc aacactggca 60

catttggtgc cccaagcatc gcccaggaag tatcaaatag tgtatccgaa ccttatcaca 120

tttggttact ggcacatcgc tggactttat ggcctttact tgtgcttcac ttccgcgaaa 180

tggtccacaa ttttattcag ctacatcctc ttcgtgttag cagaaatagg aatcacggct 240

ggcgctcaca gactctgggc ccacaaaact tacaaagcga aactaccatt agaaatactc 300

ttaatggtat tcaactccat cgcttttcaa aactcagcca ttgactgggt gagggaccat 360

cgtctccatc ataagtatag cgatacagat gctgatcccc acaatgccag ccgagggttc 420

ttctattccc atgtaggatg gctacttgtg agaaaacacc ctgaagtcaa aaagcgtggg 480

aaagaactca atatgtctga tatttacaac aatcctgttc tgcggtttca gaaaaaatac 540

gccattccct tcattggggc tgtttgtttc gccttaccta caatgatacc tgtttacttc 600

tggggagaaa cctggtccaa tgcttggcat atcaccatgc ttcgctacat catgaacctc 660

aatgtcacct tcttggtaaa cagcgccgct catatatggg gaaataaacc ttatgacgcg 720

aaaatattac ctgctcaaaa tgtagcagtg tcggtcgcca ctggtggaga aggtttccat 780

aattaccacc atgtcttccc ttgggattat cgagcagcgg aactcggtaa caatagcctc 840

aatctgacga ctaaattcat agatttattc gcagcaatcg gatgggcata tgacctgaag 900

acggtttcgg aggatatgat aaaacaaagg attaaacgca ctggagatgg aacggatctt 960

tggggacacg aacaaaaatg tgatgaagtg tgggatgtaa aagataaatc aagttaa 1017

<210> 96

<211> 1016

<212> DNA

<213> Artificial Sequence

<400> 96

atggcccaaa gctatcaatc aactacggtt ttgagtgagg agaaagaacc aacactgaca 60

catttggtgc cccaagcatc tcccaggaag taccaaatag tgtatccgaa ccttattacg 120

tttggttact ggcacatcgc tggactttat ggcctgtact tgtgcttcac ttctgcgaaa 180

tgggctacga ttttattcag ctacatcctc ttcgtgttag cagaaatagg catcacggct 240

ggcgctcaca gactctgggc ccacaaaact tacaaagcga aactaccatt agaattactc 300

ttaatggtgt tcaactccat cgctttccaa aactcggcta ttgactgggt gagggaccat 360

cgtctccatc ataagtatag cgatacagat gctgatcccc acaatgctag ccgagggttc 420

ttttattccc atgtgggatg gctactggtg agaaaacatc cagaagtcaa aaagcgtggg 480

aaagaactca atatgtctga tatttacaac aatcctgttc tgcggtttca gaaaaagtac 540

gccataccct tcattggggc tgtttgtttc gccttgccta caatgatacc tgtttacttc 600

tggggagaaa cctggtccaa tgcctggcat atcaccatgc ttcgctacat catgaacctt 660

aatgtcacct ttttggtaaa cagcgccgct catatatggg gaaataagcc ttatgacgcg 720

aaaatattac ctgcccaaaa tgtagcagtg tcagtagcca ctggtggaga aggttttcat 780

aattaccatc atgtctaccc ttgggattat cgagcagcgg aactcggtaa caatagcctt 840

aatctgacga ctaaatttat agatttcttc gcagcaatcg gatgggcata tgacctgaag 900

acggtttcgg aggatatgat aaaacagagg attaaacgca caggagatgg aacggatctt 960

tggggacacg aacaaaaatg tgataaagga tcgggtgtaa acgataaatt aagtta 1016

<210> 97

<211> 1047

<212> DNA

<213> Artificial Sequence

<400> 97

ggttggtcct ttaatgttac aattccgttt tggaaaatgg ctcaaggcgt ccaaacgact 60

acgatattca gggaagaaga gccagcattg actttcgtgg tacctcaaga gccgagaaag 120

tatcaaatcg tttatccaaa ccttatcaca tttgggtact ggcatatagc tggtctatac 180

ggtctatatt tgtacttcac ttcggcaaaa tggcaaacaa tgttattcag tttcatgctc 240

gtcgtgttag cagagttagg aataacagcc ggcgctcaca gattatgggc ccataaaaca 300

tataaagcga agcttccctt acaaattatc ctgatggtat tgaactccat tgccttccaa 360

aattccgcca ttgattgggt gagagaccac cgtctccatc ataagtacag tgacactgat 420

gcagaccctc acaatgctaa tcgtggtttc ttctattccc atgttggatg gttgctcgta 480

agaaaacatc ctgaagtcaa gagacgtgga aaggaacttg acatgtctga tatttacaac 540

aatccagtgc tgagatttca aaagaagtat gctataccct tcattggggc aatgtgcttt 600

gggctaccaa cttttatccc tgtctactgc tggggagaaa cctggactaa cgcttggcat 660

atcaccatgc ttcgctacat cgtgaacctc aacattactt tcctggtcaa cagtgctgct 720

catatctggg gaaacaagcc gtatgacagc aaaatattgc ctgcccaaaa tatagcagta 780

tccatagtaa ccggtggcga aggttttcat aactaccatc acgtttttcc ttgggattat 840

cgtgcagcag aattggggaa caattatctt aatttgacaa ctaagttcat agatttcttc 900

gcttggattg gatgggctta tgatctcaag acggtgtcta gtgatgttat aaaaagcagg 960

gcacaaagaa ctggtgatgg gacgaatctt tggggattag aagacaaagg tgaagaagaa 1020

attttgaaaa tctggaaaga caattaa 1047

<210> 98

<211> 1011

<212> DNA

<213> Artificial Sequence

<400> 98

atggctcaag gtgttcaaac tacaactatc ttgagagaag aagaaccatc tttgacattt 60

gttgttccac aagaaccaag aaagtaccaa atcgtttacc caaatttgat cactttcggt 120

tactggcata ttgctggttt gtacggtttg tacttatgtt tcacatctgc aaagtggcaa 180

actatcttgt tttcttttat gttggttgtt ttggcagaat tgggtattac agctggtgca 240

catagattgt gggctcataa aacttacaaa gcaaaattgc cattgcaaat catcttgatg 300

atcttgaact ctattgcttt tcaaaattca gcaattgatt gggttagaga tcatagattg 360

catcataagt actctgatac agatgctgat ccacataatg caactagagg tttcttttat 420

tcacatgttg gttggttgtt agttagaaag catccagaag ttaagagacg tggtaaagaa 480

ttggatatgt ctgatatcta taacaaccca gttttgagat tccaaaagaa atacgctatc 540

ccttttattg gtgcaatgtg ttttggtttg ccaactttta ttccagttta cttctggggt 600

gaaacatggt ctaacgcttg gcatatcact atgttgagat acatcttgaa tttgaacatc 660

acatttttgg ttaactcagc tgcacatatt tggggttaca agccatacga tatcaagatc 720

ttgccagctc aaaacatcgc agtttctatt gttacaggtg gtgaagtttc aatcacaact 780

acaactttct ttccatggga ttacagagct gcagaattgg gtaacaacta cttgaatttg 840

acaactaagt ttattgattt ctttgcttgg attggttggg catatgattt gaagactgtt 900

tcttcagatg ttattaaatc aaaagctgaa agaacaggtg acggtactaa tttgtggggt 960

ttggaagata agggtgaaga agatttcttg aagatctgga aagataatta a 1011

<210> 99

<211> 3525

<212> DNA

<213> Artificial Sequence

<400> 99

atgtccccaa ttaccagaga agaaagattg gaaagaagaa tccaagactt gtacgctaac 60

gatccacaat ttgctgctgc taaaccagct actgctatta ctgctgctat tgaaagacca 120

ggtttgccat tgccacaaat tattgaaact gttatgaccg gttacgctga tagaccagct 180

ttggctcaaa gatctgttga atttgttact gatgctggta ctggtcatac caccttgaga 240

ttattgccac atttcgaaac catctcttac ggtgaattgt gggatagaat ttctgctttg 300

gctgatgttt tgtctactga acaaactgtt aagccaggtg atagagtttg tttgttgggt 360

ttcaactctg ttgattacgc cactattgat atgaccttgg ctagattggg tgctgttgct 420

gttccattgc aaacttcagc tgcaattact caattgcaac ctatcgttgc tgaaactcag 480

ccaactatga ttgctgcttc tgttgatgct ttagcagatg ctactgaatt ggctttgtct 540

ggtcaaactg ctactagagt tttggttttc gatcaccaca gacaagttga tgctcataga 600

gctgctgttg aatctgctag agaaagatta gctggttctg ctgttgttga aactttggcc 660

gaagctattg ctagaggtga tgttccaaga ggtgcttctg ctggttcagc tccaggtact 720

gatgtttctg atgattcttt ggccttgttg atctacactt ctggttctac tggtgctcca 780

aaaggtgcta tgtatccaag aagaaacgtt gccacttttt ggagaaagag aacttggttt 840

gaaggtggtt acgaaccatc tattaccttg aatttcatgc caatgtctca cgtcatgggt 900

agacaaatct tgtatggtac tttgtgtaat ggtggtactg cttactttgt tgccaagtct 960

gatttgtcca ctttgttcga agatttggct ttggttagac caactgaatt gacattcgtt 1020

cctagagttt gggatatggt gttcgatgaa ttccaatccg aagtcgatag aagattggtt 1080

gatggtgcag atagagttgc tttggaagct caagttaagg ccgaaattag aaacgatgtt 1140

ctaggtggta gatacacctc tgctttgaca ggttctgctc caatttctga cgaaatgaag 1200

gcttgggttg aagagttgtt ggatatgcat ttggttgaag gttatggttc aactgaagcc 1260

ggtatgattt tgatcgatgg tgctattaga aggccagctg ttttggatta taagttggtt 1320

gacgttccag acttgggtta ctttttgaca gatagaccac atccaagggg tgaattattg 1380

gttaagaccg attctttgtt cccaggttat taccaaagag ccgaagttac tgcagatgtt 1440

tttgatgctg atggtttcta cagaaccggt gatattatgg ctgaagttgg tccagaacaa 1500

ttcgtctatt tggacagaag aaacaacgtc ttgaagttgt cccaaggtga attcgttacc 1560

gtttctaaat tggaagctgt tttcggtgat tccccattgg ttaggcaaat ctatatctac 1620

ggtaattccg ctagagctta cttgttggct gttatagttc caactcaaga agctttggac 1680

gctgttccag ttgaagaatt gaaagctaga ttaggcgact ccttgcaaga agttgctaaa 1740

gctgctggtt tacagtctta cgaaattcca agagacttca tcattgaaac tactccatgg 1800

actttggaga acggtttgtt gactggtatt agaaaattgg ccagaccaca gttgaaaaaa 1860

cattatggtg aactgctgga acagatctat actgatttgg ctcatggtca agctgacgaa 1920

ttgagatctt taagacaatc tggtgctgat gctcctgttt tggttactgt atgtagagct 1980

gccgctgctt tgttaggtgg ttcagcatct gatgttcaac cagatgctca ttttactgac 2040

ttaggtggtg attcattgtc cgctttgtct ttcactaact tgttgcacga aattttcgac 2100

atcgaagttc cagtaggtgt tatcgtttct ccagctaatg acttacaagc tttggcagat 2160

tatgttgaag ctgcaagaaa accaggttct tctagaccta cttttgcttc agttcatggt 2220

gcttctaatg gtcaagttac tgaagttcat gctggtgatt tgtctttgga taagttcatt 2280

gatgctgcta cattggctga agctccaaga ttgccagcag ctaatactca agttagaact 2340

gttttgttga caggtgctac tggttttttg ggtagatact tggctttaga atggttggaa 2400

aggatggatt tggtcgacgg taaattgatt tgcttggtca gagctaagtc tgatactgaa 2460

gctagagcca gattggataa gacttttgat tctggtgacc cagaactgtt ggctcattat 2520

agagctttag ctggtgatca cttggaagtt ttggctggtg ataagggtga agctgatttg 2580

ggtttagata gacaaacttg gcaaagattg gctgataccg ttgatttgat agttgatcca 2640

gctgccttgg ttaatcatgt tttgccatac tctcaactgt ttggtccaaa tgctttgggt 2700

actgcagaat tattgagatt ggcattgacc tctaagatca agccatattc ttacacctct 2760

accattggtg ttgctgatca aattccacca tctgctttta ctgaagatgc cgatattaga 2820

gttatttccg ctacaagagc tgttgatgac tcttatgcta atggctactc taattctaaa 2880

tgggccggtg aagttttgtt aagagaagct catgatttgt gcggtttacc agttgctgtt 2940

ttcagatgtg atatgatatt ggctgacact acttgggctg gtcaattgaa tgttccagat 3000

atgttcacca ggatgatttt gtctttagct gctacaggta ttgctcctgg ttctttttac 3060

gaattggcag ctgatggtgc cagacaacgt gctcattatg atggtttgcc agttgaattc 3120

attgccgaag caatttctac tttaggtgct caatcacaag acggtttcca tacttaccat 3180

gttatgaacc catacgatga tggtatcggt ttggatgaat ttgtcgactg gttgaatgaa 3240

tctggttgcc caattcaaag aattgccgat tacggtgatt ggttgcaaag atttgaaact 3300

gctttgagag ctttgccaga cagacaaaga cattcttcat tattgcctct gttgcacaac 3360

tacagacaac cagaaagacc tgttagaggt tctattgctc caactgatag attcagagct 3420

gcagttcaag aagcaaaaat tggtcctgat aaggatatcc cacatgttgg tgctcctatt 3480

atcgttaagt acgtttctga tttgaggctg ttgggcttgt tgtaa 3525

<210> 100

<211> 1035

<212> DNA

<213> Artificial Sequence

<400> 100

atggttcaag atacctcttc tgcttctacc tctccaattt tgactagatg gtacattgat 60

accagaccat tgacagcttc tactgctgct ttgccattat tggaaacttt acaaccagcc 120

gatcaaatct ccgttcaaaa gtactatcac ttgaaggaca agcacatgtc tttggcttct 180

aacttgttga agtacttgtt cgttcacaga aactgcagaa ttccatggtc ctctatcgtt 240

atttctagaa ctccagatcc acatagaagg ccatgttata ttccaccatc tggttctcaa 300

gaggattctt ttaaagatgg ttacaccggt atcaacgtcg agtttaatgt ttctcatcaa 360

gcttccatgg ttgctattgc tggtactgct tttactccaa attctggtgg tgattctaag 420

ttgaaaccag aagttggtat cgatattacc tgcgtcaacg aaagacaagg tagaaatggt 480

gaagaaaggt ccttggaatc tttgagacag tacatcgata tcttctccga agttttttct 540

accgctgaaa tggccaacat tagaagattg gatggtgtct cttcttcctc attgtctgct 600

gatagattgg ttgattatgg ctacaggttg ttctatactt actgggcttt gaaagaagcc 660

tacattaaga tgactggtga agctttgttg gctccatggt taagagaatt ggaattttcc 720

aatgttgttg ctccagctgc tgttgctgaa tctggtgatt cagctggtga ttttggtgaa 780

ccatatactg gtgttagaac gacgttgtac aagaacttgg ttgaagatgt tagaattgaa 840

gttgctgcct taggtggtga ctatttgttt gctactgcag ctagaggtgg tggtattggt 900

gcttcttcta gacctggtgg tggtccagat ggttctggta ttagatctca agatccttgg 960

aggccattca agaagttgga tattgaaagg gatattcaac catgtgctac tggtgtatgt 1020

aactgcttgt cttga 1035

<210> 101

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 101

tttgacctaa tattctacac 20

<210> 102

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 102

tgattccttt gtaatttcat 20

<210> 103

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 103

tcctcattct tataatattc 20

<210> 104

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 104

aataaacctt aagtttccac 20

<210> 105

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 105

gtttataaaa gtaacatgat 20

Claims

1. An expression vector comprising:

(1) Insect delta 11-FAD coding sequence, and/or

(2) A coding sequence for a carboxylate reductase, and optionally a coding sequence for NpgA,

preferably, the expression vector has one or more characteristics selected from the group consisting of:

the insect delta 11-FAD is from an insect of the family Spodoptera (Noctuidae), preferably from the genus Trichoplusia (Helicoverpa), trichoplusia (Agrotis) or Trichoplusia (Heliothis); more preferably, the amino acid sequence of the insect Δ 11-FAD is selected from the amino acid sequences shown in the NCBI accession nos: AGP26038.1, AGR49312.1, ATJ44454.1, AAM28483.2, or variants having at least 90% identity thereto,

the carboxylate reductase is a carboxylate reductase of a marine mycobacterium; preferably, the carboxylate reductase amino acid sequence as NCBI accession number WP _012393886.1 or with at least 90% identity variant thereof,

the amino acid sequence of said NpgA is as shown in NCBI accession number AAF12814.1 or a variant having at least 90% identity thereto,

the expression vector also comprises a promoter and a terminator; preferably, the promoter is selected from one or more of GAL1, TCCTDH, TEF1, and/or the terminator is selected from one or more of CYC1, RBL41B, PGK1,

the expression vector is a yeast expression vector.

2. A yeast cell having one, two, three, or all four characteristics selected from:

(1) The expression of the insect delta 11-FAD,

(3) The expression of HFD1 is down-regulated,

(4) The expression of OLE1 is down-regulated,

preferably, the yeast cell has one or more characteristics selected from the group consisting of:

the amino acid sequence of said NpgA is as shown in NCBI accession No. AAF12814.1 or a variant having at least 90% identity thereto,

the yeast cell containing the expression vector of claim 1,

downregulating expression of one or more genes of the yeast cell selected from the group consisting of: POX1, FAA4, GAL80,

the yeast is saccharomyces cerevisiae.

3. A method for producing yeast, or a method for synthesizing an insect sex pheromone or a precursor thereof from yeast, comprising:

(1) Express insect delta 11-FAD, and/or

(2) Downregulating OLE1 expression, and/or

(4) Downregulation of HFD1 expression, and

optionally (5) downregulating expression of one or more genes selected from the group consisting of: POX1, FAA4, GAL80,

preferably, the method has one or more characteristics selected from the group consisting of:

the insect sex pheromone is Z11-16 Ald and Z9-16,

the insect sex pheromone precursor is Z11-16 COOH and Z9-16 COOH,

the amino acid sequence of NpgA is as shown in NCBI accession number AAF12814.1 or a variant having at least 90% identity thereto.

4. Use of the yeast cell of claim 2 for the production of an insect sex pheromone or a precursor thereof,

preferably, the insect sex pheromone is Z11-16 and Z9-16.

5. A yeast culture medium comprises 3g/L-7g/L potassium dihydrogen phosphate, 7g/L-10g/L ammonium sulfate, carbon source, magnesium sulfate, uracil, metal ions and vitamins,

preferably, the yeast culture medium has one or more characteristics selected from the group consisting of:

the yeast culture medium comprises 6.3g/L potassium dihydrogen phosphate and 7.4g/L ammonium sulfate,

the carbon source is glucose, and the carbon source is glucose,

the metal ion is selected from one or more of the following: zinc, cobalt, manganese, copper, calcium, iron, sodium and potassium,

the vitamins are selected from one or more of the following: biotin, calcium pantothenate, nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, and p-aminobenzoic acid,

the pH of the medium is 4 to 8, preferably 6,

the culture medium also contains n-dodecane, preferably in a concentration of 5-20%,

the yeast culture medium is used for improving the yield of fatty acid and/or fatty aldehyde produced by yeast.

6. A method for obtaining an insect sex pheromone or a precursor thereof, comprising culturing the yeast cell of claim 2,

preferably, the method comprises culturing the yeast cell using the yeast medium of claim 5.

7. Use of an insect sex pheromone or a precursor thereof obtained by the method of claim 3 for attracting insects, preferably the insects are Heliothis armigera.

8. A yeast cell with increased production of free fatty acids, said yeast cell having down-regulated expression of one or more genes selected from the group consisting of: POX1, FAA4, GAL80,

preferably, the expression of a gene selected from any one of the groups of genes of the yeast cell is down-regulated: (1) POX1, (2) POX1, FAA1 and FAA4, (3) POX1, FAA4 and GAL80.

9. A method of increasing free fatty acid production of a yeast cell, comprising down-regulating expression of one or more genes of the yeast cell selected from the group consisting of: POX1, FAA4, GAL80,

preferably, the method comprises down-regulating expression of a gene of the yeast cell selected from any one of the group consisting of: (1) POX1, (2) POX1, FAA1 and FAA4, (3) POX1, FAA4 and GAL80.

10. The sequence of gRNA for down-regulating the expression of HFD1, POX1, FAA4 and GAL80 is shown in SEQ ID NO. 101-SEQ ID NO. 105.