CN111118052A

CN111118052A - Recombinant saccharomyces cerevisiae, construction method thereof and application of recombinant saccharomyces cerevisiae in production of hydroxy fatty acid

Info

Publication number: CN111118052A
Application number: CN202010060712.8A
Authority: CN
Inventors: 卢文玉; 李冰; 刘静静; 张传波
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2020-05-08

Abstract

The invention discloses a recombinant saccharomyces cerevisiae, a construction method thereof and application thereof in production of hydroxy fatty acid.A key gene acyl-CoA oxidase POX1 in β oxidation pathway related to fatty acid catabolism in saccharomyces cerevisiae is knocked out by a homologous recombination method, acyl-CoA activators FAA1 and FAA4 are knocked out to obtain a recombinant bacterium (I), cytochrome P450 oxidase CYP52M1 and cytochrome P450 reductase SbCPR are introduced into the recombinant bacterium (I) to obtain a recombinant strain (II), or cytochrome P450 oxidase CYP52M1 and cytochrome P450 reductase AtCPR1 are introduced into the recombinant bacterium (I) to obtain a recombinant bacterium (III), or cytochrome P450 oxidase and cytochrome P450 reductase fusion protein gene CYP52M1-AtCPR1 are introduced into the recombinant bacterium (I) to obtain a recombinant bacterium (IV).

Description

Recombinant saccharomyces cerevisiae, construction method thereof and application of recombinant saccharomyces cerevisiae in production of hydroxy fatty acid

Technical Field

The invention relates to the technical field of biology, in particular to recombinant saccharomyces cerevisiae, a construction method thereof and application thereof in producing hydroxy fatty acid.

Background

Long chain hydroxy fatty acids are widely used in various fields such as food, chemical, cosmetic industries, and as synthetic intermediates for ceramides, polyesters, and cyclic lactones due to their unique properties, the pharmaceutical properties of hydroxy fatty acids, including anti-biotic, anti-inflammatory, and anti-diabetic effects are also disclosed^[9]. Omega-hydroxy fatty acid is fatty acid with hydroxyl at the end, is a multifunctional compound with wide application, such as adhesive, lubricant and potential anticancer agent, and is used as a material for synthesizing polymers such as bioplastic and the like, and the produced bioplastic has better water resistance, nontoxicity, biocompatibility and chemical versatility.

In view of the wide commercial use of hydroxy fatty acids, researchers have made extensive efforts to produce hydroxy fatty acids by chemical or biological methods. Direct synthesis of the desired specific hydroxy fatty acid molecule by chemical catalysis is difficult to achieve due to the limitations of selective carbon atoms and harsh reaction conditions. In biological systems, the synthesis of hydroxy fatty acids can be achieved by selective enzymatic reactions using yeast or E.coli to achieve specific hydroxylation of fatty acid molecules. For example, bacillus megaterium can hydroxylate oleic acid molecules at 1,2, and 3 carbon atom positions, producing different hydroxyoleic acids. Candida tropicalis can also produce hydroxy fatty acids as by-products when alkanes or fatty acids are cultured as the main carbon source.

Saccharomyces cerevisiae is a traditional and well-studied host strain with mature genetic tools for the manipulation of metabolic pathways and has been successfully manipulated to produce many valuable platform chemicals and biofuels. Furthermore, saccharomyces cerevisiae is easy to culture in chemically defined media, and can utilize inexpensive renewable raw materials and exhibit a rapid growth rate, which is advantageous for a wider industrial application. The endogenous fatty acid synthesis pathway of saccharomyces cerevisiae can provide a precursor for the synthesis of hydroxy fatty acids, and the subsequent hydroxylation reaction is completed by the subsequently introduced cytochrome P450 oxidase CYP52M1 and reductase CPR. Based on the discovery of the saccharomyces cerevisiae endogenous pathway and the hydroxyl fatty acid exogenous synthetic gene, the method and the technology of synthetic biology are utilized to construct and optimize the biosynthesis pathway of the hydroxyl fatty acid in the saccharomyces cerevisiae to produce the hydroxyl fatty acid, so that the method has great potential.

At present, the heterologous synthesis of omega (omega-1) -hydroxy fatty acids of a specific structure (C16-C18) in Saccharomyces cerevisiae by synthetic biological methods has not been reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a recombinant saccharomyces cerevisiae, a construction method thereof and application thereof in producing hydroxy fatty acid.

The second purpose of the invention is to provide a construction method of the recombinant saccharomyces cerevisiae (I) for producing the hydroxy fatty acid.

The third purpose of the invention is to provide a recombinant saccharomyces cerevisiae (II) for producing the hydroxy fatty acid.

The fourth purpose of the invention is to provide a construction method of the recombinant saccharomyces cerevisiae (II) for producing the hydroxy fatty acid.

The fifth purpose of the invention is to provide a recombinant saccharomyces cerevisiae for producing hydroxy fatty acid (III).

The sixth purpose of the invention is to provide a construction method of the recombinant saccharomyces cerevisiae (III) for producing the hydroxy fatty acid.

The seventh object of the present invention is to provide a recombinant saccharomyces cerevisiae producing hydroxy fatty acid (IV).

The eighth purpose of the invention is to provide a construction method of the recombinant saccharomyces cerevisiae (IV) for producing the hydroxy fatty acid.

The ninth purpose of the invention is to provide the use of the recombinant saccharomyces cerevisiae (I), (II), (III) and (IV) for producing the hydroxy fatty acid.

The technical scheme of the invention is summarized as follows:

a construction method of a recombinant saccharomyces cerevisiae (I) for producing hydroxyfatty acid comprises the following steps of knocking out an acyl-CoA oxidase encoding gene expression box which is a key gene in β oxidation pathway related to fatty acid catabolism on a saccharomyces cerevisiae genome through a homologous recombination method, and knocking out an acyl-CoA activating enzyme encoding gene expression box, so as to obtain the recombinant saccharomyces cerevisiae (I) for producing hydroxyfatty acid, wherein the acyl-CoA oxidase encoding gene expression box is composed of an acyl-CoA oxidase encoding gene POX1, the acyl-CoA oxidase encoding gene expression box is composed of an acyl-CoA activating enzyme encoding gene FAA1, the acyl-CoA activating enzyme encoding gene expression box is composed of an acyl-CoA activating enzyme encoding gene FAA4, the acyl-CoA 1 encoding gene nucleotide sequence is shown by SEQ ID NO.1, the acyl-CoA synthetase encoding gene FAA1 nucleotide sequence is shown by SEQ ID NO.2, and the acyl-CoA synthetase encoding nucleotide sequence is shown by SEQ ID NO. 4.

The recombinant saccharomyces cerevisiae (I) for producing the hydroxy fatty acid constructed by the method.

A construction method of recombinant saccharomyces cerevisiae (II) for producing hydroxy fatty acid comprises the following steps: and (2) introducing a cytochrome P450 oxidase gene expression cassette and a cytochrome P450 reductase gene expression cassette into the HO site of the recombinant saccharomyces cerevisiae (I) by a homologous recombination method to obtain the recombinant saccharomyces cerevisiae (II) for producing the hydroxy fatty acid. The cytochrome P450 oxidase gene expression box is composed of a promoter P_PGK1Cytochrome P450 oxidase-encoding gene CYP52M1 and terminator T_ADH1Composition is carried out; the cytochrome P450 reductase gene expression box is composed of a promoter P_TDH3Cytochrome P450 reductase-encoding gene SbCPR and terminator T_TDH2Composition is carried out; the nucleotide sequence of the cytochrome P450 oxidase coding gene CYP52M1 is shown in SEQ ID NO. 4; the promoter P_PGK1The nucleotide sequence of (A) is shown in SEQ ID NO.8, and a terminator T_ADH1The nucleotide sequence of (A) is shown in SEQ ID NO.9, and the promoter P_TDH3The nucleotide sequence of (A) is shown in SEQ ID NO.10, and a terminator T_TDH2The nucleotide sequence of SEQ ID NO. 11.

The recombinant saccharomyces cerevisiae for producing the hydroxy fatty acid constructed by the method is II.

A construction method of recombinant saccharomyces cerevisiae (III) for producing hydroxy fatty acid comprises the following steps: introducing a cytochrome P450 oxidase gene CYP52M1 expression cassette and a cytochrome P450 reductase gene AtCPR1 expression cassette into HO sites of the recombinant saccharomyces cerevisiae (I) by a homologous recombination method to obtain the recombinant saccharomyces cerevisiae (III) for producing hydroxy fatty acid; the cytochrome P450 reductase gene expression box is composed of a promoter P_TDH3Cytochrome P450 reductase-encoding gene AtCPR1 and terminator T_TDH2Composition is carried out; the promoter P_TDH3The nucleotide sequence of (A) is shown in SEQ ID NO.9, terminator T_TDH2The nucleotide sequence of SEQ ID NO. 10.

The recombinant saccharomyces cerevisiae for producing the hydroxy fatty acid constructed by the method is (III).

A construction method of recombinant saccharomyces cerevisiae (IV) for producing hydroxy fatty acid comprises the following steps: introducing a cytochrome P450 oxidase gene expression cassette and a cytochrome P450 reductase gene fusion protein gene CYP52M1-AtCPR1 expression cassette into a recombinant saccharomyces cerevisiae strain (I) by a homologous recombination method to obtain recombinant saccharomyces cerevisiae (IV) for producing hydroxy fatty acid; the cytochrome P450 oxidase and cytochrome P450 reductase fusion protein gene expression cassette is composed of a promoter P_PGK1Cytochrome P450 oxidase and cytochrome P450 reductase fusion protein-encoding gene CYP52M1-AtCPR1 and terminator T_ADH1And (4) forming. Nucleotide sequence of cytochrome P450 oxidase and cytochrome P450 reductase fusion protein gene CYP52M1-AtCPR1Shown as SEQ ID NO. 7.

The recombinant saccharomyces cerevisiae (IV) for producing the hydroxy fatty acid constructed by the method.

The application of the recombinant saccharomyces cerevisiae (I), (II), (III) and (IV) for producing the hydroxy fatty acid by fermentation. The recombinant saccharomyces cerevisiae utilizes glucose to synthesize the hydroxy fatty acid from the head, and the obtained hydroxy fatty acid types comprise omega-hydroxy-hexadecanoic acid, omega-1-hydroxy-hexadecanoic acid, omega-hydroxy-oleic acid and omega-1-hydroxy-oleic acid.

Experiments prove that the recombinant strain 2 can produce 37.8mg/L of hydroxy fatty acid through shake flask fermentation; the strain 3 can produce 83.2mg/L of hydroxy fatty acid; strain 4 produced 148.7mg/L of hydroxy fatty acid. And (3) performing a 5L fermentation tank feeding fermentation experiment on the recombinant bacterium 4 with the highest yield of the hydroxy fatty acid, wherein the recombinant bacterium can produce 347mg/L of hydroxy fatty acid after 144 h. The invention lays a foundation for artificially synthesizing the hydroxy fatty acid.

Drawings

FIG. 1 is a diagram of the metabolic pathway for producing hydroxy fatty acid by Saccharomyces cerevisiae.

FIG. 2 shows the results of gas-mass spectrometry analysis of hydroxy fatty acids with different structures:

a, detecting and analyzing results of gas chromatography of hydroxy fatty acids with different structures;

b is GC-MS detection analysis of hydroxy fatty acid with the structure and a comparison graph thereof (A is an omega-1-hydroxy-hexadecanoic acid mass spectrum comparison graph, B is an omega-hydroxy-hexadecanoic acid mass spectrum comparison graph, C is an omega-1-hydroxy-oleic acid mass spectrum comparison graph, and D is an omega-hydroxy-oleic acid mass spectrum comparison graph).

FIG. 3 analysis of the production of hydroxyfatty acids by recombinant strains (A is a comparison of the production of hydroxyfatty acids by recombinant strains B04 and B05, and B is a comparison of the production of hydroxyfatty acids by recombinant strains B05 and B06).

FIG. 4 shows the growth curve and yield analysis of the recombinant strain 4 in a 5L fermentor (FIG. A shows batch fermentation, and FIG. B shows fed-batch fermentation).

Detailed Description

The present invention will be further illustrated by the following specific examples.

The test methods used in the following examples are all conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Saccharomyces cerevisiae strain is Saccharomyces cerevisiae BY4741 (American ATCC 4002569), and is hereinafter referred to as Saccharomyces cerevisiae.

Example 1 construction method of recombinant Saccharomyces cerevisiae (I) for producing hydroxy fatty acid

Module construction

Homology arms POX1A-up, POX1B-down, FAA1A-up, FAA1B-down, FAA4A-up, FAA4B-down were designed based on the DNA sequences of the acyl-CoA oxidase-encoding gene POX1, the acyl-CoA activator-encoding gene FAA1 and the acyl-CoA 4 provided on NCBI. The templates are all from the genome of Saccharomyces cerevisiae BY 4741; selectable marker gene his3 was derived from plasmid Pxp320 (available from addge, Inc.WWW.addgene.org)。

Taking a saccharomyces cerevisiae BY4741 genome as a template,

POX1A-F (SEQ ID NO.12),

HIS3-POX1A-R (SEQ ID NO.13) as primer

Amplification homology arm POX1A-up (SEQ ID NO.14)

HIS3-POX1B-F (SEQ ID NO.15),

POX1B-R (SEQ ID NO.16) is used as a primer,

amplification homology arm POX1B-down (SEQ ID NO.17)

With FAA1A-F (SEQ ID NO.18),

LEU2-FAA1A-R (SEQ ID NO.19) as primer

Amplification homology arm FAA1A-up (SEQ ID NO.20)

LEU2-FAA1B-F (SEQ ID NO.21),

FAA1B-R (SEQ ID NO.22) as a primer,

amplification homology arm FAA1B-down (SEQ ID NO.23)

With FAA4A-F (SEQ ID NO.24),

LEU2-FAA4A-R (SEQ ID NO.25) as primer

Amplification homology arm FAA4A-up (SEQ ID NO.26)

LEU2-FAA4B-F (SEQ ID NO.27),

FAA4B-R (SEQ ID NO.28) as a primer,

amplification homology arm FAA4B-down (SEQ ID NO.29)

Using PRS403 (ATCC in USA) as template,

POX1A-HIS3-F(SEQ ID NO.30)，

POX1B-HIS3-R (SEQ ID NO.31) is used as a primer,

amplification marker gene his3(SEQ ID NO.32)

The genome of the saccharomyces cerevisiae S288C is taken as a template,

FAA1A-LEU2-F(SEQ ID NO.33)，

FAA1B-LEU2-R (SEQ ID NO.34) is used as a primer,

amplification marker gene leu2(SEQ ID NO.35)

The genome of the saccharomyces cerevisiae S288C is taken as a template,

FAA4A-LEU2-F(SEQ ID NO.36)，

FAA4B-LEU2-R (SEQ ID NO.37) is used as a primer,

amplification marker gene leu2(SEQ ID NO.35)

The PCR enzyme used in the present invention is Phanta Super-Fidelity DNA Polymerase from Biotech, Inc. of Nanjing Novowed. A50. mu.L PCR amplification system is shown in Table 1:

TABLE 1

An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 55 ℃ for 15sec, and extension at 72 ℃ for 1min (35 cycles); extension at 72 ℃ for 5min (1 cycle).

The fusion PCR system used in the present invention is shown in Table 2:

TABLE 2

The fusion program was set up on a PCR instrument. The fusion conditions were pre-denaturation at 95 ℃ for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 15sec, and extension at 72 ℃ for 1min (11 cycles); extension at 72 ℃ for 5min (1 cycle).

And purifying and recovering the fragment obtained by PCR amplification by using an agarose gel DNA recovery kit (Tiangen Biochemical technology Beijing, Ltd.) to obtain a DNA fragment used for transforming the saccharomyces cerevisiae, then carrying out fusion PCR on the DNA fragment, and amplifying a gene expression cassette used for transforming the saccharomyces cerevisiae by using a fusion PCR product as a template to finally obtain the DNA fragments used for transforming the saccharomyces cerevisiae, wherein the DNA fragments are POX1A-HIS3-POX1B, FAA1A-LEU2-FAA1B and FAA4A-LEU2-FAA4B respectively.

Yeast transformation

Initial Saccharomyces cerevisiae BY4741 was cultured in YPD medium for 12h, and 300. mu.L of the initial Saccharomyces cerevisiae BY4741 was added to 3mL of fresh YPD medium and cultured for 5 h. Centrifuging at 3000rpm for 5min at room temperature, collecting thallus, discarding supernatant, and sterilizing with ddH₂O washing the cells, centrifuging at 3000rpm for 5min to collect the cells, and discarding the supernatant. Then 1mL of 100mM lithium acetate is added into the thalli, the cells are evenly suspended by gentle blowing and beating, the thalli are placed at room temperature for 5min and then centrifuged at 3000rpm for 5min at normal temperature, and yeast competent cells are prepared. The transformation mix consisted of 240. mu.L PEG (50% W/V), 36. mu.L 1.0M lithium acetate, 10. mu.L ss-DNA, 300ng each of the transformation fragments POX1A, HIS3 and POX 1B. Finally using sterilized ddH₂The content of O is filled to 360 mu L. Sequentially adding the above components into the freshly prepared Saccharomyces cerevisiae competent cells, uniformly blowing by using a pipette gun, standing in a water bath at 42 ℃ for 30min, centrifuging at 4000rpm at normal temperature for 2min, removing the supernatant, adding 1mLYPD liquid culture medium, and culturing at 30 ℃ and 220rpm for 2 h. Then centrifuging at 4000rpm for 5min at normal temperature, discarding the supernatant, washing with sterile water for 2 times, finally resuspending the cells with 100 μ L of sterile water, and screening by coating a histidine-deficient plate. The screening culture condition is 30 ℃, and the culture lasts more than 48 h.

Thus, by a homologous recombination method, the coding gene of acyl-CoA oxidase in a saccharomyces cerevisiae metabolic pathway is knocked out, colony PCR verification is carried out, single colonies which are successfully transformed are selected, and fragments FAA1A-LEU2-FAA1B and FAA4A-LEU2-FAA4B are respectively transformed by the same method to obtain the recombinant strain 1.

Example 2 construction method of recombinant Saccharomyces cerevisiae (II) for producing hydroxy fatty acid

Respectively amplifying homologous arms HOup (SEQ ID NO.42) and HOdown (SEQ ID NO.43) BY taking a Saccharomyces cerevisiae BY4741 genome as a template and HOA-F (SEQ ID NO.38), PGK1p-HOA-R (SEQ ID NO.39), HIS3-HOB-F (SEQ ID NO.40) and HOB-R (SEQ ID NO.41) as primers; HOA-PGK1P-F (SEQ ID NO.44), CYP52M1-PGK1P-R (SEQ ID NO.45), CYP52M1-ADH1t-F (SEQ ID NO.46) and TDH3P-ADH1t-R (SEQ ID NO.47) are respectively used as primers to amplify the promoter P_PGK1(SEQ ID NO.7) and a terminator T_ADH1t(SEQ ID NO. 8); amplifying cytochrome P450 oxidase gene CYP52M1(SEQ ID No.4) by taking PGK1P-CYP52M1-F (SEQ ID No.48) and ADH1t-CYP52M1-R (SEQ ID No.49) as primers; ADH1t-TDH3P-F (SEQ ID NO.50), SbCPR-TDH3P-R (SEQ ID NO.51), SbCPR-TDH2t-F (SEQ ID NO.52) and HIS3-TDH2t-R (SEQ ID NO.53) are respectively used as primers to amplify promoter P_TDH3(SEQ ID NO.9) and a terminator T_TDH2t(SEQ ID NO. 10); the cytochrome P450 reductase gene SbCPR (SEQ ID NO.5) is amplified by using TDH3P-SbCPR-F (SEQ ID NO.54) and TDH2t-SbCPR-R (SEQ ID NO.55) as primers. The marker gene HIS3(SEQ ID NO.31) was amplified using plasmid PRS403 (ATCC, USA) as a template, TDH2t-HIS3-F (SEQ ID NO.56), HOB-HIS3-R (SEQ ID NO.57) as primers. The PCR method was performed as in example 1 to obtain the DNA fragments for transforming Saccharomyces cerevisiae, which are the upstream homology arm HOAup and the gene CYP52M1 expression cassette P_PGK1-CYP52M1-T_ADH1Gene SbCPR expression cassette P_TDH3-SbCPR-T_TDH2And a downstream homology arm his3-HOBdown containing a selectable marker gene his 3. The sequence is shown in a sequence table.

The transformation method is the same as example 1, and the DNA fragment is introduced into saccharomyces cerevisiae recombinant bacteria 1 to obtain recombinant saccharomyces cerevisiae (II) for producing the hydroxy fatty acid.

Example 3 construction method of recombinant Saccharomyces cerevisiae (III) for producing Hydroxyfatty acids

Respectively amplifying homologous arms HOup (SEQ ID NO.42) and HOdown (SEQ ID NO.43) BY taking a Saccharomyces cerevisiae BY4741 genome as a template and HOA-F (SEQ ID NO.38), PGK1p-HOA-R (SEQ ID NO.39), HIS3-HOB-F (SEQ ID NO.40) and HOB-R (SEQ ID NO.41) as primers; HOA-PGK1P-F (SEQ ID NO.44), CYP52M1-PGK1P-R (SEQ ID NO.45), CYP52M1-ADH1t-F (SEQ ID NO.46) and TDH3P-ADH1t-R (SEQ ID NO.47) are respectively used as primers to amplify the promoter P_PGK1(SEQ ID NO.7) and a terminator T_ADH1t(SEQ ID NO. 8); amplifying cytochrome P450 oxidase gene CYP52M1(SEQ ID No.4) by taking PGK1P-CYP52M1-F (SEQ ID No.48) and ADH1t-CYP52M1-R (SEQ ID No.49) as primers; ADH1t-TDH3P-F (SEQ ID NO.50), AtCPR1-TDH3P-R (SEQ ID NO.58), AtCPR1-TDH2t-F (SEQ ID NO.59) and HIS3-TDH2t-R (SEQ ID NO.53) are respectively used as primers to amplify the promoter P_TDH3(SEQ ID NO.9) and a terminator T_TDH2t(SEQ ID NO. 10); TDH3P-AtCPR1-F (SEQ ID NO.60) and TDH2t-AtCPR1-R (SEQ ID NO.61) are used as primers to amplify cytochrome P450 reductase gene AtCPR1(SEQ ID NO. 6). The marker gene HIS3(SEQ ID NO.31) was amplified using plasmid PRS403 (ATCC, USA) as a template, TDH2t-HIS3-F (SEQ ID NO.56), HOB-HIS3-R (SEQ ID NO.57) as primers. The PCR method was performed as in example 1 to obtain the DNA fragments for transforming Saccharomyces cerevisiae, which are the upstream homology arm HOAup and the gene CYP52M1 expression cassette P_PGK1-CYP52M1-T_ADH1Gene AtCPR1 expression cassette P_TDH3-AtCPR1-T_TDH2And a downstream homology arm his3-HOBdown containing a selectable marker gene his 3. The sequence is shown in a sequence table.

The transformation method is the same as example 1, and the DNA fragment is introduced into saccharomyces cerevisiae recombinant bacteria 1 to obtain recombinant saccharomyces cerevisiae (III) for producing the hydroxy fatty acid.

Example 4 construction method of recombinant Saccharomyces cerevisiae (IV) for producing hydroxy fatty acids

Respectively amplifying homologous arms HOup (SEQ ID NO.42) and HOdown (SEQ ID NO.43) BY taking a Saccharomyces cerevisiae BY4741 genome as a template and HOA-F (SEQ ID NO.38), PGK1p-HOA-R (SEQ ID NO.39), HIS3-HOB-F (SEQ ID NO.40) and HOB-R (SEQ ID NO.41) as primers; HOA-PGK1p-F (SEQ ID NO.44) and CYP52M1-PGK1p-R (SEQ ID NO.44) respectivelyNO.45), AtCPR1-TDH2t-F (SEQ ID NO.59), HIS3-TDH2t-R (SEQ ID NO.53) as primers, and an amplification promoter P_PGK1(SEQ ID NO.7) and a terminator T_TDH2t(SEQ ID NO. 8); PGK1P-CYP52M1-F (SEQ ID NO.48) and TDH2t-AtCPR1-R (SEQ ID NO.61) are used as primers to amplify cytochrome P450 oxidase gene and cytochrome P450 reductase gene fusion protein gene CYP52M1-AtCPR1(SEQ ID NO. 7); the marker gene HIS3(SEQ ID NO.31) was amplified using plasmid PRS403 (ATCC, USA) as a template, TDH2t-HIS3-F (SEQ ID NO.56), HOB-HIS3-R (SEQ ID NO.57) as primers. The PCR method was performed as in example 1 to obtain the DNA fragments for the transformed Saccharomyces cerevisiae, which were the upstream homology arm HOAup and the gene CYP52M1-AtCPR1 expression cassette P_PGK1-CYP52M1-AtCPR1-T_TDH2The downstream homology arm his3-HOBdown containing the selectable marker gene his 3. The sequence is shown in a sequence table.

The transformation method is the same as example 1, and the DNA fragment is introduced into saccharomyces cerevisiae recombinant bacteria 1 to obtain recombinant saccharomyces cerevisiae (IV) for producing the hydroxy fatty acid.

Example 5 fermentation of recombinant Saccharomyces cerevisiae and GC-MS detection of the fermentation product

The recombinant Saccharomyces cerevisiae strains (I), 2, 3 and 4 obtained in examples 1 to 4 were cultured in YPD medium (30 ℃ C., 220rmp), respectively. After fermentation for 4 days, taking the fermentation liquor, centrifuging for 10min at the normal temperature of 12000rpm, adding glass beads and ethyl acetate into the lower-layer thallus, oscillating for 30min, centrifuging for 10min at the normal temperature of 12000rpm, taking an organic phase for derivatization, selecting N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) as a derivatization reagent, and then carrying out GC-MS (gas chromatography-mass spectrometry) qualitative and quantitative detection after passing through a 0.22-micrometer organic film.

And (3) detecting conditions of hydroxy fatty acid GC-MS: the GC-TQ 8030 was produced by Shimadzu corporation, Japan. The gas chromatography column was an HP-5MS (30 m. times.0.250 mm. times.0.25 μm) type column from Agilent, USA, with helium as the carrier gas.

The sample introduction conditions and the detection conditions are as follows:

sample introduction amount: 1 mul; the split ratio is as follows: 10; sample inlet temperature: 300 ℃; column temperature: keeping at 100 deg.C for 2min, increasing to 200 deg.C at 40 deg.C/min for 1min, increasing to 245 deg.C/min for 1min at 4 deg.C/min, and increasing to 290 deg.C at 30 deg.C/min for 10 min. Ion source temperature: at 310 ℃; solvent retention time: and 5 min.

The GC-MS detection results are shown in FIG. 2. Comparing the four hydroxy fatty acid mass spectrograms with the corresponding mass spectrograms in the Wiley mass spectrograms database, the four peaks can be determined to be omega-1-hydroxy-hexadecanoic acid, omega-1-hydroxy-oleic acid and omega-hydroxy-oleic acid respectively.

The yield results of each recombinant strain are shown in FIG. 3. The recombinant bacterium 2 generates 37.8mg/L hydroxy fatty acid; the recombinant bacterium 3 generates 83.2mg/L hydroxy fatty acid; the recombinant bacterium 4 produces 148.7mg/L of hydroxy fatty acid.

Example 6 feeding fermentation of recombinant bacterium 4 in 5L fermenter

And (3) performing a 5L fermentation tank feeding fermentation experiment on the recombinant bacterium 4 with the highest yield of the hydroxy fatty acid. The feed solution comprises 500g/L glucose, 300g/L sodium glutamate and inorganic salt solution (0.9g KH)₂PO4，0.512g MgSO₄·7H₂O，0.35gK₂SO₄，0.028g Na₂SO₄And adding 10mL/L of trace element solution and 12mL/L of vitamin solution after distilled water is added to 50mL and sterilized by moist heat at 115 ℃ for 20 min). Wherein the vitamin solution is 0.005g of biotin, 0.1g of calcium pantothenate, 2.5g of inositol, 0.1g of nicotinic acid, 0.02g of p-aminobenzoic acid, 0.1g of pyridoxal hydrochloride and 0.1g of thiamine hydrochloride, and distilled water is added to the solution to reach a constant volume of 100 mL; the microelement solution is ZnSO₄·7H₂O 0.575g，MnCl₂·4H₂O0.032 g, anhydrous CuSO₄0.032g，CoCl₂·6H₂O0.047g，Na₂MoO₄·2H₂O 0.048g，FeSO₄·7H₂O 0.28g，CaCl₂·2H₂O0.29 g, 0.5M EDTA solution 8mL, distilled water to 100 mL.

The concrete operation of feeding is as follows: the recombinant Saccharomyces cerevisiae strain 4 was inoculated into YPD liquid medium, cultured at 30 ℃ and 220rmp for 20 hours, and then inoculated at 10% inoculum size into a 5L fermentor containing 2L of YPD liquid medium. Feeding materials after fermenting for 24h, feeding 80mL of glucose, 50mL of sodium glutamate and 50mL of inorganic salt solution every 10h, feeding materials after 120h, and continuing fermenting for 144h to finish fermentation.

As shown in FIG. 4, 347mg/L of hydroxy fatty acid was produced by the recombinant strain 4 after 144 hours.

Sequence listing

<110> Tianjin university

<120> recombinant saccharomyces cerevisiae, construction method thereof and application thereof in production of hydroxy fatty acid

<160>61

<170>SIPOSequenceListing 1.0

<210>62

<211>2247

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>62

atgacgagac gtactactat taatcccgat tcggtggttc tgaatcctca aaaatttatc 60

cagaaagaaa gggcggattc gaaaatcaaa gttgaccaag ttaacacatt tttagagtca 120

tccccggaga ggagaactct gacgcacgcc ttaatagacc aaatagtgaa tgatcctata 180

ttgaaaactg atacggacta ttacgatgct aaaaaaatgc aagagagaga aattactgcc 240

aaaaaaatag ctaggcttgc tagttatatg gagcacgata tcaaaacagt gcgcaaacac 300

tttcgcgaca ctgacctgat gaaagagttg caagcaaatg atccagacaa agcttcgcct 360

ttaacaaaca aagacctttt tatattcgat aagagattgt cacttgtagc aaatattgat 420

cctcaattgg gtacgcgcgt gggtgtacac ttggggctat ttggtaattg tatcaagggc 480

aatggtactg atgagcaaat ccggtattgg ttgcaggaga gaggtgccac tttgatgaaa 540

ggtatatatg gctgttttgc aatgactgag ttaggacatg gttccaatgt tgcccagctg 600

cagactaggg ctgtgtacga taagcaaaat gatacttttg taattgatac acctgatcta 660

actgccacca aatggtggat tggtggggct gcccattctg ccacgcacgc tgccgtgtac 720

gccagattga tcgttgaagg taaagactac ggtgtaaaaa cattcgttgt tcctctgaga 780

gacccttcga ctttccaact gttagctggt gtttccatag gggatattgg agcgaagatg 840

ggtcgtgacg gtattgataa tggctggatc cagttcagaa acgtagttat ccctagagaa 900

tttatgctaa gtagatttac caaagttgtc cgttctccag atggttcagt caccgtcaaa 960

actgagccac aattggatca aatttctggt tatagtgcat tgttaagtgg tagagttaac 1020

atggtcatgg attcatttag gtttggctcc aaatttgcta ctattgctgt acgttacgcg 1080

gttggtcgtc agcaattcgc acctagaaag ggattgtctg aaacacaatt aatcgactat 1140

ccccttcacc aatatcgtgt tttaccacaa ttgtgtgttc catatttggt gtcacctgta 1200

gcttttaagt taatggacaa ctattattcc actttggacg agttatacaa cgcttcctca 1260

tctgcataca aagctgctct ggttaccgtg agtaaaaagt tgaagaattt atttattgat 1320

agcgccagct tgaaagccac caatacttgg ttaattgcta cactgattga tgagttgaga 1380

cagacttgcg gaggacatgg gtattcacag tataacggat ttggtaaagg ctatgacgac 1440

tgggtggttc agtgcacatg ggagggtgat aataatgttt tatctttaac ttcagcaaaa 1500

tcaatattga aaaaatttat cgattcagcc acaaagggta gatttgacaa cacactggat 1560

gtggactcat tctcttactt aaaacctcag tacataggat ctgtggtttc tggagaaata 1620

aagagtggtt taaaggagtt gggtgattat actgaaattt ggtctatcac cttaatcaaa 1680

ttactggcac atattggtac tttagttgaa aaatcaagaa gtattgatag cgtttctaag 1740

cttttagtct tagtatccaa atttcatgcc ttgcgctgca tgttgaaaac ctattacgac 1800

aagttaaact ctcgtgattc acatatttcc gatgaaatta caaaggaatc tatgtggaat 1860

gtttataagt tattttcctt gtattttatt gacaagcatt ccggagaatt ccaacaattc 1920

aagatcttca ctcctgatca gatctctaaa gttgtgcagc cacaactatt ggctcttttg 1980

ccaattgtga ggaaagactg tataggtctg acagactcct ttgaattacc tgacgcgatg 2040

ttaaattctc ctataggtta ctttgatggc gatatctatc acaattactt caatgaagtt 2100

tgccgcaata atccagtgga ggcagatggg gcagggaagc cttcttatca tgcgctgttg 2160

agcagcatgc tcggtagagg tttcgaattt gaccaaaagt taggtggtgc agctaatgcg 2220

gaaattttat cgaaaataaa caagtga 2247

<210>2

<211>2103

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>2

atggttgctc aatataccgt tccagttggg aaagccgcca atgagcatga aactgctcca 60

agaagaaatt atcaatgccg cgagaagccg ctcgtcagac cgcctaacac aaagtgttcc 120

actgtttatg agtttgttct agagtgcttt cagaagaaca aaaattcaaa tgctatgggt 180

tggagggatg ttaaggaaat tcatgaagaa tccaaatcgg ttatgaaaaa agttgatggc240

aaggagactt cagtggaaaa gaaatggatg tattatgaac tatcgcatta tcattataat 300

tcatttgacc aattgaccga tatcatgcat gaaattggtc gtgggttggt gaaaatagga 360

ttaaagccta atgatgatga caaattacat ctttacgcag ccacttctca caagtggatg 420

aagatgttct taggagcgca gtctcaaggt attcctgtcg tcactgccta cgatactttg 480

ggagagaaag ggctaattca ttctttggtg caaacggggt ctaaggccat ttttaccgat 540

aactctttat taccatcctt gatcaaacca gtgcaagccg ctcaagacgt aaaatacata 600

attcatttcg attccatcag ttctgaggac aggaggcaaa gtggtaagat ctatcaatct 660

gctcatgatg ccatcaacag aattaaagaa gttagacctg atatcaagac ctttagcttt 720

gacgacatct tgaagctagg taaagaatcc tgtaacgaaa tcgatgttca tccacctggc 780

aaggatgatc tttgttgcat catgtatacg tctggttcta caggtgagcc aaagggtgtt 840

gtcttgaaac attcaaatgt tgtcgcaggt gttggtggtg caagtttgaa tgttttgaag 900

tttgtgggca ataccgaccg tgttatctgt tttttgccac tagctcatat ttttgaattg 960

gttttcgaac tattgtcctt ttattggggg gcctgcattg gttatgccac cgtaaaaact 1020

ttaactagca gctctgtgag aaattgtcaa ggtgatttgc aagaattcaa gcccacaatc 1080

atggttggtg tcgccgctgt ttgggaaaca gtgagaaaag ggatcttaaa ccaaattgat 1140

aatttgccct tcctcaccaa gaaaatcttc tggaccgcgt ataataccaa gttgaacatg 1200

caacgtctcc acatccctgg tggcggcgcc ttaggaaact tggttttcaa aaaaatcaga 1260

actgccacag gtggccaatt aagatatttg ttaaacggtg gttctccaat cagtcgggat 1320

gctcaggaat tcatcacaaa tttaatctgc cctatgctta ttggttacgg tttaaccgag 1380

acatgcgcta gtaccaccat cttggatcct gctaattttg aactcggcgt cgctggtgac 1440

ctaacaggtt gtgttaccgt caaactagtt gatgttgaag aattaggtta ttttgctaaa 1500

aacaaccaag gtgaagtttg gatcacaggt gccaatgtca cgcctgaata ttataagaat 1560

gaggaagaaa cttctcaagc tttaacaagc gatggttggt tcaagaccgg tgacatcggt 1620

gaatgggaag caaatggcca tttgaaaata attgacagga agaaaaactt ggtcaaaaca 1680

atgaacggtg aatatatcgc actcgagaaa ttagagtccg tttacagatc taacgaatat 1740

gttgctaaca tttgtgttta tgccgaccaa tctaagacta agccagttgg tattattgta 1800

ccaaatcatg ctccattaac gaagcttgct aaaaagttgg gaattatgga acaaaaagac 1860

agttcaatta atatcgaaaa ttatttggag gatgcaaaat tgattaaagc tgtttattct 1920

gatcttttga agacaggtaa agaccaaggt ttggttggca ttgaattact agcaggcata 1980

gtgttctttg acggcgaatg gactccacaa aacggttttg ttacgtccgc tcagaaattg 2040

aaaagaaaag acattttgaa tgctgtcaaa gataaagttg acgccgttta tagttcgtct 2100

taa 2103

<210>3

<211>2085

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>3

atgaccgaac aatattccgt tgcagttggc gaagccgcca atgagcatga aaccgctcca 60

agaagaaata tcagggttaa agaccagcct ttgattagac ccataaactc ctcagcatct 120

acactgtacg aattcgccct ggaatgtttt accaagggtg gtaagagaga cggtatggca 180

tggagagata ttatagatat acatgagacg aaaaaaacca tagtcaagag ggtggatggt 240

aaggataagc ccatcgaaaa aacatggttg tactacgaac tgactcccta cataaccatg 300

acatacgagg agatgatctg cgtaatgcac gacattggac gtgggctgat aaagattggt 360

gttaaaccta acggtgagaa caagttccac atctttgcct ctacttctca caagtggatg 420

aaaacttttc ttggttgcat gtcacaaggt attcctgtgg tcaccgcgta cgacactttg 480

ggtgagagcg gtttgattca ctccatggtg gaaacagatt ccgtcgccat tttcacggac 540

aaccagctgt tgtccaaatt agcagttcct ttgaaaaccg ccaagaacgt aaaattcgtc 600

attcacaacg aacccatcga tccaagtgac aaaagacaaa atggtaagct ttacaaggct 660

gccaaggatg ctgttgacaa aatcaaggaa gttagaccgg acataaaaat ctacagtttc 720

gatgaaatta ttgagatagg taaaaaggcc aaggacgagg ttgaattgca tttccccaag 780

cctgaagatc cagcttgtat catgtacact tctggttcca ctggtacacc aaagggtgtg 840

gtattgacac attacaacat tgtagctggt attggtggtg tgggccataa cgttatcgga 900

tggattggcc caacagaccg tattatcgca ttcttgccat tggctcatat ttttgaatta 960

acctttgaat tcgaagcgtt ctactggaat ggtatcctag ggtacgccaa tgtcaagact 1020

ttaaccccaa cttctacacg taattgccaa ggtgacctga tggagtttaa acctaccgta 1080

atggtgggtg tcgccgcagt ttgggaaaca gtgagaaaag gtatcctggc caagatcaac 1140

gaattgcccggttggtctca aacgcttttc tggactgtct atgctttgaa agagagaaat 1200

ataccatgta gcggcttgct gagtgggttg atcttcaaga gaatcagaga agcaaccggt 1260

ggaaacttaa ggtttattct gaacggtggg tctgcaatca gcatagacgc ccaaaaattc 1320

ctctccaacc ttctatgtcc tatgctcatt ggatatgggc taactgaggg tgtggctaat 1380

gcctgtgtcc tggagcctga acattttgat tacggtattg ctggtgacct tgtcggaact 1440

attacagcta aattggtgga tgtcgaagat ttgggctatt ttgccaagaa taatcaaggt 1500

gaattgctgt ttaagggtgc acccatctgt tctgaatact ataagaatcc tgaagaaact 1560

gctgcggcct ttaccgatga tggctggttc cgtaccggtg atatcgctga atggaccccc 1620

aagggacaag ttaagatcat tgatagaaag aaaaatttgg ttaagacctt aaatggtgag 1680

tacattgcat tggaaaaatt agaatccatt tacagatcaa atccttacgt ccaaaacatc 1740

tgtgtctacg ctgatgaaaa caaagttaag cctgtcggta ttgtggtccc taacttagga 1800

cacttgtcta agctggctat cgaattaggt ataatggtac caggtgaaga tgtcgaaagc 1860

tatatccatg aaaagaagct acaggatgcc gtttgcaaag atatgctgtc aactgccaaa 1920

tctcaaggct tgaatggtat tgaattatta tgtggcattg ttttctttga agaagaatgg 1980

actccagaaa atggccttgt tacatccgcc caaaaattaa agagaagaga tattctagcg 2040

gctgtcaagc cagatgtgga aagagtttat aaagaaaaca cttaa 2085

<210>4

<211>1617

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>4

atgctgatca aggatattat cttgacgccc atgtctttgt ctgctgttgc tggtttgttg 60

cctttgttgt ttgttgcttt tttggtcttg catgagccaa tttggttgtt gtggtataga 120

tatgctgcta gaaggcataa gtgttccatg ccaagattca tcgaaaagtc tttcccattg 180

ggtatccaaa gaaccatgga tatgattaag accgctaagt cttacacctt gttggaagtt 240

caatacgaca gggtgtttaa caagttcaag gctagaactt acttgagaca agctccattg 300

caataccaaa tcttcactat cgaaccagag aacatcaaga ctattttggc taccaagttc 360

aacgattttg gtttgggtgc tagattccat actgttggta aagttttcgg tcaaggtatc 420

ttcactttgt ctggtaatgg ttggaagcag tccagatcta tgttaagacc acaattcact 480

aaggaccaag tctgcagaat tgaccaaatt tcttctcatg ctgccgaact gatcaaagaa 540

atgaatagag ctatgaaggt cgaccaattc atcgatgttc aacattactt ccacaagttg 600

accttggata ctgctactga atttttgttc ggtgagtctt gcgaatcttt gaatccagaa 660

aatcaatcct gcatcgttgc tagagatggt tctgaaatta ctgccgaaca attcgtcgaa 720

tcctacaact ttttgttgaa ctacgctttc aagaggacct tgtcctctaa agtttactgg 780

ctgtttaact ccaaagaatt cagggatcat aagaagagag cccaatccta cattgattac 840

tatgttgata aggccttgta cgctacttct tttgctgctg aaaattccat tgctgaaaaa 900

gatgctgctg cagaatcttc tggtatctac gttttttctt tggaaatggc caaggttacc 960

agagatccag ttactatcag agatcagatt ttcaacattt tgatcgccgg tagagataca 1020

actgctgcta ctttgtcttt tgccattcat tttttggcta gaaaccccga cgttttcaac 1080

aagttgagag aagaagtttt ggaccatttc ggcaccaaag aagaacaaag accattgtcc 1140

ttcgagttgt tgaaacaagc accatacttg aagcaagtca tcaacgaagt tttgagattg 1200

gctccagttt tgcctttgaa ctttagaact gctgttagag ataccacctt gccaattggt 1260

ggtggtccag aacaaaaaga tccaattttc gttccaaaag gtactgccgt ttactactct 1320

atctacatgg ttcacagaga tatcaaatac tggggtccag atgctcatga attcaatcca 1380

aatagatggg agaacctgaa gttggataat gtttgggctt tcttgccttt taatggtggc 1440

ccaagaattt gtttgggtca acaatttgct ttgaccgagt tgtctttgac tttggttaga 1500

ctgttgcaag agtactccaa gattgaaatg ggtcctgatt ttccagaatc tcctagattc 1560

tctactactt tgactgctca acatgctcca ccaggtgttg ttgttagatt ttcttga 1617

<210>5

<211>2064

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>5

atggccgata ttaacttcat tgcctctgtt gttgttgctt tggctgttgt ttttgttgcc 60

tacaagtact ttaatggtgg tccagatgtt caatcttcta aggctggtaa ttctactcca 120

ttcggtaact ctaaagctga tgaagatggt gattccagag atttcgttgc tttgatggaa 180

aagaacaaca agaacgtcat cgtgttctac ggttctcaaa ctggtactgc tgaagatttg 240

gcttctaagt tggctaaaga actgtcctct aaatacggtt tgaggactat gactgctgat 300

ccagaaaatt tcgacttcga aaagttggac actttcccag aatctcattt ggccgttttt 360

ttgatggctt cttatggtga tggtgaacct actgataatg ctcaagactt gtactctttc 420

ttgggtaact ctccatcttt ctcacaagac ggtgaaactt tggaaaactt gaactttgct 480

gttttcggtt tgggtaacgt cttgtacgaa ttttacaaca aagccggtaa ggacatgcat 540

aagtacttga ctgacttagg tggtcattct attggtccat acggtgaagg tgatgattct 600

aaaggtatgt tggaagagga ttacatggct tggaaggatg aatttttggc tgctttggtt 660

gctaaatggg gtttgactga aagagaagct gtttacgaac catccatctc cgtcaaagaa 720

attgaagagg atgctcattc ccacgatgtt tatttgggtg agccaaattt gaaacacttg 780

caagcttcta aagcccaaga aattccaaag ggtccataca atgcttctaa tccaatgttg 840

gctaagatta ctgctgctag agaattattc accaacaccg atagacattg catccacatg 900

gaatttgata caactggtgc tagatacact accggtgatc atttggcttt ttggttccaa 960

aacaacgaag aagaagtcca aagattcgtt aaggctttgg gtattgctaa tccacaacaa 1020

cctattgcca tttccgtttt ggataagact tccaccgtta gaattccatc tccaactact 1080

tacgaaacca tcatcagaca tttcttggaa atcaacggtc cagtttccag acaagttttg 1140

tcatctattg caccatttgc tccatccgaa gaagttaaga aagctactca acaactgggc 1200

tctaacaaag aattatttgc ttctcatgtt gccgccaaga agtttaacat tgctagattg 1260

ctattgcact tgtcaggtgg tcaaccatgg aaaaatgttc cattctcctt cgtcattgaa 1320

acgattccac acttgcagcc aagatattac tctatctctt cctcttctgt gcaatctcca 1380

aacactgttt ctattaccgc tgttgttgaa agacaaactt tgaccggtgt tgatcatgaa 1440

ttgagaggtg ttgctaccaa tcaaattttg gctttgtctg aagccttggt tggtcatcca 1500

tctatgactt atagattgca acaaccacac gacttcacca actcattatc ctctcaagat 1560

atcagagttc cagttcacat taggcacagc ttgtttaaat tgccaggtaa acctaccgtt 1620

ccaatcatta tggttggtcc aggtactggt gttgctcctt ttagaggttt tgttcacgaa 1680

agagcttctc aaaaagctgc cggtaaagaa gttggtaagg ctatgttgtt taccggttct 1740

agacatgcta acgaggattt cttgtatagg gatgaatgga agcaattctc cgacttcttg 1800

gatttggaaa ctgccttttc tagagactcc tctaagaagg tttacgtcca acacaaattg 1860

aaagaaagag ccaaggacgt tttcgccttg ttgaatgaag gtgctgtttt ttacgtttgt 1920

ggtgatgctg gtggtatgtc tcatgatgtt cattctgctt tattggaaat cgttgcccaa 1980

gaaggtaact tgtcatctga agatgctgat aagttcgtca gaaagatgag atctaggaac 2040

aagtaccaag aggatgtttg gtga 2064

<210>6

<211>2079

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>6

atgacttctg cattatacgc atcagactta tttaagcagt tgaaatctat aatgggaaca 60

gactcattgt cagacgacgt cgttttagtt attgctacta cttcattggc tttggttgct 120

ggatttgttg ttttattgtg gaaaaagaca acagctgata ggtctggtga attaaagcca180

ttaatgatac ctaaatcttt aatggctaag gacgaggacg acgacttgga tttaggatca 240

ggaaagacta gagtctctat atttttcgga actcagacag gaacagctga gggattcgca 300

aaggctttat cagaagagat taaagcaagg tacgagaagg ctgctgtcaa agttatagat 360

ttggatgact acgcagctga tgacgaccag tacgaggaaa agttgaaaaa ggaaactttg 420

gcatttttct gtgttgcaac atacggtgac ggtgagccaa ctgacaacgc tgctaggttc 480

tacaaatggt tcacagagga aaatgagaga gacattaaat tgcagcagtt ggcttacggt 540

gtcttcgcat tgggaaacag gcaatatgaa catttcaata agattggaat tgtcttggac 600

gaagaattat gcaaaaaagg agctaagagg ttgatagagg tcggtttggg tgacgatgac 660

cagtcaatag aggacgactt caatgcatgg aaagagtcat tgtggtcaga gttagataag 720

ttattaaaag acgaagacga caagtcagtc gcaacacctt acacagcagt catacctgag 780

tatagggtcg tcactcacga cccaagattc actactcaaa agtcaatgga gtcaaatgtc 840

gcaaacggaa atactactat tgacattcat cacccatgca gggttgacgt cgctgtccag 900

aaagagttac acactcacga gtctgacagg tcatgcattc acttggagtt cgatatttca 960

agaactggta ttacttacga aacaggtgac cacgttggtg tctacgctga gaaccacgtc 1020

gagattgtcg aggaagctgg aaagttgttg ggacattctt tagatttggt cttctcaatt 1080

catgctgaca aagaggacgg ttcaccattg gagtctgctg ttccaccacc attccctgga 1140

ccatgcactt taggtactgg tttggcaagg tacgcagact tattgaaccc acctaggaag 1200

tcagctttag ttgcattggc tgcatatgca acagaaccat ctgaggcaga gaaattaaag 1260

cacttgactt ctcctgacgg taaggacgag tactcacagt ggatagtcgc atctcagagg 1320

tcattgttgg aggtcatggc agcatttcca tcagcaaagc cacctttagg tgttttcttc 1380

gcagctatag cacctagatt gcagcctagg tattattcaa tatcttcttc acctaggttg 1440

gctccatcta gggtccacgt cacatcagct ttggtttacg gacctactcc tacaggaagg 1500

atacataaag gagtctgctc tacttggatg aagaacgctg tcccagcaga gaagtctcat 1560

gagtgctcag gagctcctat ttttattagg gcatcaaatt tcaaattgcc ttcaaaccca 1620

tctactccaa tagtcatggt cggaccagga acaggtttgg ctcctttcag gggatttttg 1680

caggagagga tggctttgaa ggaggatggt gaggaattgg gatcatcttt gttgttcttt 1740

ggttgtagga ataggcaaat ggacttcatt tatgaggacg aattgaacaa ctttgttgat 1800

caaggagtca tatcagagtt aattatggct ttctcaaggg agggtgcaca aaaggaatac 1860

gtccaacaca agatgatgga aaaggctgca caggtctggg acttgattaa ggaggaggga 1920

tacttatatg tctgcggtga cgcaaagggt atggcaagag acgtccacag gactttgcac 1980

acaattgtcc aggaacagga gggtgtttct tcatctgaag cagaggctat tgttaaaaag 2040

ttgcaaactg aaggtaggta cttgagggac gtctggtaa 2079

<210>7

<211>3582

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>7

atgctgatca aggatattat cttgacgccc atgtctttgt ctgctgttgc tggtttgttg 60

cctttgttgt ttgttgcttt tttggtcttg catgagccaa tttggttgtt gtggtataga 120

tatgctgcta gaaggcataa gtgttccatg ccaagattca tcgaaaagtc tttcccattg 180

ggtatccaaa gaaccatgga tatgattaag accgctaagt cttacacctt gttggaagtt 240

caatacgaca gggtgtttaa caagttcaag gctagaactt acttgagaca agctccattg 300

caataccaaa tcttcactat cgaaccagag aacatcaaga ctattttggc taccaagttc 360

aacgattttg gtttgggtgc tagattccat actgttggta aagttttcgg tcaaggtatc 420

ttcactttgt ctggtaatgg ttggaagcag tccagatcta tgttaagacc acaattcact 480

aaggaccaag tctgcagaat tgaccaaatt tcttctcatg ctgccgaact gatcaaagaa 540

atgaatagag ctatgaaggt cgaccaattc atcgatgttc aacattactt ccacaagttg 600

accttggata ctgctactga atttttgttc ggtgagtctt gcgaatcttt gaatccagaa 660

aatcaatcct gcatcgttgc tagagatggt tctgaaatta ctgccgaaca attcgtcgaa 720

tcctacaact ttttgttgaa ctacgctttc aagaggacct tgtcctctaa agtttactgg 780

ctgtttaact ccaaagaatt cagggatcat aagaagagag cccaatccta cattgattac 840

tatgttgata aggccttgta cgctacttct tttgctgctg aaaattccat tgctgaaaaa 900

gatgctgctg cagaatcttc tggtatctac gttttttctt tggaaatggc caaggttacc 960

agagatccag ttactatcag agatcagatt ttcaacattt tgatcgccgg tagagataca 1020

actgctgcta ctttgtcttt tgccattcat tttttggcta gaaaccccga cgttttcaac 1080

aagttgagag aagaagtttt ggaccatttc ggcaccaaag aagaacaaag accattgtcc 1140

ttcgagttgt tgaaacaagc accatacttg aagcaagtca tcaacgaagt tttgagattg 1200

gctccagttt tgcctttgaa ctttagaact gctgttagag ataccacctt gccaattggt 1260

ggtggtccag aacaaaaaga tccaattttc gttccaaaag gtactgccgt ttactactct 1320

atctacatgg ttcacagaga tatcaaatac tggggtccag atgctcatga attcaatcca 1380

aatagatggg agaacctgaa gttggataat gtttgggctt tcttgccttt taatggtggc 1440

ccaagaattt gtttgggtca acaatttgct ttgaccgagt tgtctttgac tttggttaga 1500

ctgttgcaag agtactccaa gattgaaatg ggtcctgatt ttccagaatc tcctagattc 1560

tctactactt tgactgctca acatgctcca ccaggtgttg ttgttagatt ttcttgaggt 1620

tctacttctt caggttcagg ttggaaaaag acaacagctg ataggtctgg tgaattaaag 1680

ccattaatga tacctaaatc tttaatggct aaggacgagg acgacgactt ggatttagga 1740

tcaggaaaga ctagagtctc tatatttttc ggaactcaga caggaacagc tgagggattc 1800

gcaaaggctt tatcagaaga gattaaagca aggtacgaga aggctgctgt caaagttata 1860

gatttggatg actacgcagc tgatgacgac cagtacgagg aaaagttgaa aaaggaaact 1920

ttggcatttt tctgtgttgc aacatacggt gacggtgagc caactgacaa cgctgctagg 1980

ttctacaaat ggttcacaga ggaaaatgag agagacatta aattgcagca gttggcttac 2040

ggtgtcttcg cattgggaaa caggcaatat gaacatttca ataagattgg aattgtcttg 2100

gacgaagaat tatgcaaaaa aggagctaag aggttgatag aggtcggttt gggtgacgat 2160

gaccagtcaa tagaggacga cttcaatgca tggaaagagt cattgtggtc agagttagat 2220

aagttattaa aagacgaaga cgacaagtca gtcgcaacac cttacacagc agtcatacct 2280

gagtataggg tcgtcactca cgacccaaga ttcactactc aaaagtcaat ggagtcaaat 2340

gtcgcaaacg gaaatactac tattgacatt catcacccat gcagggttga cgtcgctgtc 2400

cagaaagagt tacacactca cgagtctgac aggtcatgca ttcacttgga gttcgatatt 2460

tcaagaactg gtattactta cgaaacaggt gaccacgttg gtgtctacgc tgagaaccac 2520

gtcgagattg tcgaggaagc tggaaagttg ttgggacatt ctttagattt ggtcttctca 2580

attcatgctg acaaagagga cggttcacca ttggagtctg ctgttccacc accattccct 2640

ggaccatgca ctttaggtac tggtttggca aggtacgcag acttattgaa cccacctagg 2700

aagtcagctt tagttgcatt ggctgcatat gcaacagaac catctgaggc agagaaatta 2760

aagcacttga cttctcctga cggtaaggac gagtactcac agtggatagt cgcatctcag 2820

aggtcattgt tggaggtcat ggcagcattt ccatcagcaa agccaccttt aggtgttttc 2880

ttcgcagcta tagcacctag attgcagcct aggtattatt caatatcttc ttcacctagg 2940

ttggctccat ctagggtcca cgtcacatca gctttggttt acggacctac tcctacagga 3000

aggatacata aaggagtctg ctctacttgg atgaagaacg ctgtcccagc agagaagtct 3060

catgagtgct caggagctcc tatttttatt agggcatcaa atttcaaatt gccttcaaac 3120

ccatctactc caatagtcat ggtcggacca ggaacaggtt tggctccttt caggggattt 3180

ttgcaggaga ggatggcttt gaaggaggat ggtgaggaat tgggatcatc tttgttgttc 3240

tttggttgta ggaataggca aatggacttc atttatgagg acgaattgaa caactttgtt 3300

gatcaaggag tcatatcaga gttaattatg gctttctcaa gggagggtgc acaaaaggaa 3360

tacgtccaac acaagatgat ggaaaaggct gcacaggtct gggacttgat taaggaggag 3420

ggatacttat atgtctgcgg tgacgcaaag ggtatggcaa gagacgtcca caggactttg 3480

cacacaattg tccaggaaca ggagggtgtt tcttcatctg aagcagaggc tattgttaaa 3540

aagttgcaaa ctgaaggtag gtacttgagg gacgtctggt aa 3582

<210>8

<211>750

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>8

acgcacagat attataacat ctgcataata ggcatttgca agaattactc gtgagtaagg 60

aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120

tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180

cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240

gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300

gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360

caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420

gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480

tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540

tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600

atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660

agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720

ttatctactt tttacaacaa atataaaaca 750

<210>9

<211>437

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>9

gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60

tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120

ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180

tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata 240

tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttatgtcct cagaggacaa 300

cacctgttgt aatcgttctt ccacacggat ccacagccta gccttcagtt gggctctatc 360

ttcatcgtca ttcattgcat ctactagccc cttacctgag cttcaagacg ttatatcgct 420

tttatgtatc atgatct 437

<210>10

<211>800

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>10

atactagcgt tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca 60

aaaagattcc ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt 120

cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 180

agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 240

acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 300

acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 360

aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 420

ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 480

ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 540

atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 600

agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 660

atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 720

tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 780

aacacacata aacaaacaaa 800

<210>11

<211>400

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>11

atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60

acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120

gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180

aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240

cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300

gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360

ttcgataaag cacttagtat cacactaatt ggcttttcgc 400

<210>12

<211>24

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>12

tcctagcgac gaaaaatgcg agat 24

<210>13

<211>31

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>13

gatcctctag agtcgacgca tcgtaatagt c 31

<210>14

<211>683

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>14

tcctagcgac gaaaaatgcg agatctcgac caaaaaaagg gggtagggta ataaaattaa 60

ccctattatt ttttaacttt aaaacctata atgtgctaat attttattat aaacctcctt 120

tttttgcgtt caaaccctga cacattttaa gccctatatt tacggtatta gttgattaaa 180

ctccgaagcg aaaggaattc ggtcattagc ggctaatagc cgttggggta aatcacctac 240

aagcaagtac acaagagaac gttggcgttg ttaagtcaaa gcactaatac attggggctt 300

taagagtgtt tataaaggtc taacctgtaa aaattattta aacaacttga acaggcctta 360

aagttttcct cattccgctc atcatcacta atattgctct ccgtttttga atacacactt 420

gacactaata agtatcacag aaaaaaagaa aatataataa attagtattg cgatatgacg 480

agacgtacta ctattaatcc cgattcggtg gttctgaatc ctcaaaaatt tatccagaaa 540

gaaagggcgg attcgaaaat caaagttgac caagttaaca catttttaga gtcatccccg 600

gagaggagaa ctctgacgca cgccttaata gaccaaatag tgaatgatcc tatattgaaa 660

actgatacgg actattacga tgc 683

<210>15

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>15

gaattcatca cgtgcctcta aagttgtgca gc 32

<210>16

<211>28

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>16

agtaaacact caataattac ctgccatc 28

<210>17

<211>618

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>17

ctctaaagtt gtgcagccac aactattggc tcttttgcca attgtgagga aagactgtat 60

aggtctgaca gactcctttg aattacctga cgcgatgtta aattctccta taggttactt 120

tgatggcgat atctatcaca attacttcaa tgaagtttgc cgcaataatc cagtggaggc 180

agatggggca gggaagcctt cttatcatgc gctgttgagc agcatgctcg gtagaggttt 240

cgaatttgac caaaagttag gtggtgcagc taatgcggaa attttatcga aaataaacaa 300

gtgagtagag gtttcctgtt ttccttcgaa ccctctgttt tgcgactttt gtttcaattc 360

aactagtgtc gccaagtttt aacaaaaagt tacaaaatcc tagtgagagg ccatcttatg 420

tgcataacgg tactctctat ctatttacat atctaatact attcacataa ctatgacgaa 480

tcaatgacat gactacattt accaatgtat agtagtaaaa aaattgccat ctcctagaat 540

cgaaccaggg tttcatcggc cacaacgatg tgtactaacc actatactaa gatggcaggt 600

aattattgag tgtttact 618

<210>18

<211>21

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>18

atacgaatga cacaggggca c 21

<210>19

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>19

gatcctctag agtcgacatc cctccaaccc at 32

<210>20

<211>544

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>20

atacgaatga cacaggggca cccacccatc gcatatcagg agaacttccc tgtgcatacg 60

ggacagcaga gataggctgt tctcgcgcgt tttttttttt tttttgcttt ttttggttcc 120

tttcgcggat atcgtattat cgatattaca acaaaaatgt ttggagatct ccttcgctgt 180

ctttaagtaa aaggctttaa ccgcttattt ttctctcttt ttttcctttt acccctcact 240

tgtcgtgaga caattgtagt atatttagta agtatagaaa gttcttgttg ttaaaaaact 300

cgttaggata caataaaaac tagaacaaac acaaaagaca aaaaaagaca acaatatggt 360

tgctcaatat accgttccag ttgggaaagc cgccaatgag catgaaactg ctccaagaag 420

aaattatcaa tgccgcgaga agccgctcgt cagaccgcct aacacaaagt gttccactgt 480

ttatgagttt gttctagagt gctttcagaa gaacaaaaat tcaaatgcta tgggttggag 540

ggat 544

<210>21

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>21

catcacgtgc tagccgacca atctaagact aa 32

<210>22

<211>23

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>22

gctcctaatt agactgccat gcg 23

<210>23

<211>635

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>23

gccgaccaat ctaagactaa gccagttggt attattgtac caaatcatgc tccattaacg 60

aagcttgcta aaaagttggg aattatggaa caaaaagaca gttcaattaa tatcgaaaat 120

tatttggagg atgcaaaatt gattaaagct gtttattctg atcttttgaa gacaggtaaa 180

gaccaaggtt tggttggcat tgaattacta gcaggcatag tgttctttga cggcgaatgg 240

actccacaaa acggttttgt tacgtccgct cagaaattga aaagaaaaga cattttgaat 300

gctgtcaaag ataaagttga cgccgtttat agttcgtctt aatggatcaa catttccatg 360

ataggaaagc ctcatcatac taaagcactt tttcagtttt ttgctttaga actgctacca 420

attataaaat agaatgcaca ggttatcttg tttggtttgt tattatcctt ttttcttctt 480

taccagtcat atcacctcat tcacgtatgt tcgtaattta taatgttaaa aatggcatat 540

atagatatat ttatgaacat cctaaaaaat aactaaattt ttctacattt caaaaaaggt 600

aacgttgact gccgcatggc agtctaatta ggagc 635

<210>24

<211>22

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>24

tattctgggt gtttctcggc ct 22

<210>25

<211>29

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>25

ctctagagtc gactatgtag ggagtcagt 29

<210>26

<211>615

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>26

tattctgggt gtttctcggc cttcgttcat ctcgttcttt ctactcttag ccctcatctt 60

tctcttctgc attcttttca cctcattaag aacattcgcg gcgccgcttt accccaacaa 120

aaacagccca tcttttgttc tacctctttc agataaggct caacagatag acaataactg 180

atcattcttt cttttgtttg caacccctct gagttgacat aaaagcgaga atacaaaaga 240

accagcatta gtaacacatc atttttttct ctgttcttca ctatttcttg aaaaactaag 300

aagtacgcat caaaatgacc gaacaatatt ccgttgcagt tggcgaagcc gccaatgagc 360

atgaaaccgc tccaagaaga aatatcaggg ttaaagacca gcctttgatt agacccataa 420

actcctcagc atctacactg tacgaattcg ccctggaatg ttttaccaag ggtggtaaga 480

gagacggtat ggcatggaga gatattatag atatacatga gacgaaaaaa accatagtca 540

agagggtgga tggtaaggat aagcccatcg aaaaaacatg gttgtactac gaactgactc 600

cctacataac catga 615

<210>27

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>27

tcatcacgtg ctaccaaaac atctgtgtct ac 32

<210>28

<211>28

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>28

gtttgattac taaaagttgg ggcattcg 28

<210>29

<211>688

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>29

ccaaaacatc tgtgtctacg ctgatgaaaa caaagttaag cctgtcggta ttgtggtccc 60

taacttagga cacttgtcta agctggctat cgaattaggt ataatggtac caggtgaaga 120

tgtcgaaagc tatatccatg aaaagaagct acaggatgcc gtttgcaaag atatgctgtc 180

aactgccaaa tctcaaggct tgaatggtat tgaattatta tgtggcattg ttttctttga 240

agaagaatgg actccagaaa atggccttgt tacatccgcc caaaaattaa agagaagaga 300

tattctagcg gctgtcaagc cagatgtgga aagagtttat aaagaaaaca cttaaaggaa 360

gacatagttt tttactttcc cccctgccct tcataaacac tacgtttcat tttctaagag 420

catcaatttg caaacacctg aactacttgc acaatacata gtttcttctc ctacagatac 480

agttaataat taataaataa cattacacat aaacatgttt aagtttcatt tcagcaagac 540

aagattgaca ttattaagcg tgatctaaac tatgaatttc tcccgggtat gaaactacat 600

tctactattg attttaatag atgagcgatt aaacgggata tttgaattta tgacttctgt 660

aaaccatcga atgccccaac ttttagta 688

<210>30

<211>31

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>30

gactattacg atgcgtcgac tctagaggat c 31

<210>31

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>31

gctgcacaac tttagaggca cgtgatgaat tc 32

<210>32

<211>1299

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>32

gtcgactcta gaggatcccc gggataactt cgtatagcat acattatacg aagttatcgt 60

tttaagagct tggtgagcgc taggagtcac tgccaggtat cgtttgaaca cggcattagt 120

cagggaagtc ataacacagt cctttcccgc aattttcttt ttctattact cttggcctcc 180

tctgtacact ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct 240

agcggatgac tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta 300

tataaagtaa tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga 360

aggcaaagat gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc 420

agattgcgat ctctttaaag ggtggtcccc tagcgatagagcactcgatc ttcccagaaa 480

aagaggcaga agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag 540

gtatagggtt tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa 600

tcgttgagtg cattggtgac ttacacatag acgaccatca caccactgaa gactgcggga 660

ttgctctcgg tcaagctttt aaagaggccc taggggccgt gcgtggagta aaaaggtttg 720

gatcaggatt tgcgcctttg gatgaggcac tttccagagc ggtggtagat ctttcgaaca 780

ggccgtacgc agttgtcgaa cttggtttgc aaagggagaa agtaggagat ctctcttgcg 840

agatgatccc gcattttctt gaaagctttg cagaggctag cagaattacc ctccacgttg 900

attgtctgcg aggcaagaat gatcatcacc gtagtgagag tgcgttcaag gctcttgcgg 960

ttgccataag agaagccacc tcgcccaatg gtaccaacga tgttccctcc accaaaggtg 1020

ttcttatgta gtgacaccga ttatttaaag ctgcagcata cgatatatat acatgtgtat 1080

atatgtatac ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac 1140

aaggtaatgc atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt 1200

ttgctttttc tttttttttc tcttgaactc gaataacttc gtatagcata cattatacga 1260

agttatcccg ggtaccgagc tcgaattcat cacgtgcta 1299

<210>33

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>33

atgggttgga gggatgtcga ctctagagga tc 32

<210>34

<211>33

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>34

cttagtctta gattggtcgg ctagcacgtg atg 33

<210>35

<211>2285

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>35

gtcgactcta gaggatcccc gggataactt cgtatagcat acattatacg aagttatcgt 60

tttaagagct tggtgagcgc taggaaccta atattattgc cttattaaaa atggaatccc 120

aacaattaca tcaaaatcca cattctcttc aaaatcaatt gtcctgtact tccttgttca 180

tgtgtgttca aaaacgttat atttatagga taattatact ctatttctca acaagtaatt 240

ggttgtttgg ccgagcggtc taaggcgcct gattcaagaa atatcttgac cgcagttaac 300

tgtgggaata ctcaggtatc gtaagatgca agagttcgaa tctcttagca accattattt 360

ttttcctcaa cataacgaga acacacaggg gcgctatcgc acagaatcaa attcgatgac 420

tggaaatttt ttgttaattt cagaggtcgc ctgacgcata tacctttttc aactgaaaaa 480

ttgggagaaa aaggaaaggt gagagcgccg gaaccggctt ttcatataga atagagaagc 540

gttcatgact aaatgcttgc atcacaatac ttgaagttga caatattatt taaggaccta600

ttgttttttc caataggtgg ttagcaatcg tcttactttc taacttttct taccttttac 660

atttcagcaa tatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctaag 720

aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa tcacagccga agccattaag 780

gttcttaaag ctatttctga tgttcgttcc aatgtcaagt tcgatttcga aaatcattta 840

attggtggtg ctgctatcga tgctacaggt gttccacttc cagatgaggc gctggaagcc 900

tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg gtcctaaatg gggtaccggt 960

agtgttagac ctgaacaagg tttactaaaa atccgtaaag aacttcaatt gtacgccaac 1020

ttaagaccat gtaactttgc atccgactct cttttagact tatctccaat caagccacaa 1080

tttgctaaag gtactgactt cgttgttgtc agagaattag tgggaggtat ttactttggt 1140

aagagaaagg aagacgatgg tgatggtgtc gcttgggata gtgaacaata caccgttcca 1200

gaagtgcaaa gaatcacaag aatggccgct ttcatggccc tacaacatga gccaccattg 1260

cctatttggt ccttggataa agctaatgtt ttggcctctt caagattatg gagaaaaact 1320

gtggaggaaa ccatcaagaa cgaattccct acattgaagg ttcaacatca attgattgat 1380

tctgccgcca tgatcctagt taagaaccca acccacctaa atggtattat aatcaccagc 1440

aacatgtttg gtgatatcat ctccgatgaa gcctccgtta tcccaggttc cttgggtttg 1500

ttgccatctg cgtccttggc ctctttgcca gacaagaaca ccgcatttgg tttgtacgaa 1560

ccatgccacg gttctgctcc agatttgcca aagaataagg tcaaccctat cgccactatc 1620

ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc ctgaagaagg taaggccatt 1680

gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa ctggtgattt aggtggttcc 1740

aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag ttaagaaaat ccttgcttaa 1800

aaagattctc tttttttatg atatttgtac ataaacttta taaatgaaat tcataataga 1860

aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta tatacgcaat 1920

ctacatacat ttatcaagaa ggagaaaaag gaggatgtaa aggaatacag gtaagcaaat 1980

tgatactaat ggctcaacgt gataaggaaa aagaattgca ctttaacatt aatattgaca 2040

aggaggaggg caccacacaa aaagttaggt gtaacagaaa atcatgaaac tatgattcct 2100

aatttatata ttggaggatt ttctctaaaa aaaaaaaaat acaacaaata aaaaacactc 2160

aatgacctga ccatttgatg gagtttaagt caataccttc ttgaactctt gaactcgaat 2220

aacttcgtat agcatacatt atacgaagtt atcccgggta ccgagctcga attcatcacg 2280

tgcta 2285

<210>36

<211>31

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>36

tgactcccta catagtcgac tctagaggat c 31

<210>37

<211>33

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>37

acagatgttt tggtagcacg tgatgaattc gag 33

<210>38

<211>24

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>38

agtcgcggaa aaaagtaaac agct 24

<210>39

<211>34

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>39

taatatctgt gcgtactgac aatttatgac ctgc 34

<210>40

<211>38

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>40

attcatcacg tgctagaaag ggaatcaagt atttttat 38

<210>41

<211>34

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>41

gcttgataac tgttactgat atgtctgagg aaag 34

<210>42

<211>486

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>42

agtcgcggaa aaaagtaaac agctattgct actcaaatga ggtttgcaga agcttgttga 60

agcatgatga agcgttctaa acgcactatt catcattaaa tatttaaagc tcataaaatt 120

gtattcaatt cctattctaa atggctttta tttctattac aactattagc tctaaatcca 180

tatcctcata agcagcaatc aattctatct atactttaaa atgctttctg aaaacacgac 240

tattctgatg gctaacggtg aaattaaaga catcgcaaac gtcacggcta actcttacgt 300

tatgtgcgca gatggctccg ctgcccgcgt cataaatgtc acacagggct atcagaaaat 360

ctataatata cagcaaaaaa ccaaacacag agcttttgaa ggtgaacctg gtaggttaga 420

tcccaggcgt agaacagttt atcagcgtct tgcattacaa tgtactgcag gtcataaatt 480

gtcagt 486

<210>43

<211>516

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>43

agaaagggaa tcaagtattt ttataaagat tggagtggta aaaatcgagt atgtgctaga 60

tgctatggaa gatacaaatt cagcggtcat cactgtataa attgcaagta tgtaccagaa 120

gcacgtgaag tgaaaaaggc aaaagacaaa ggcgaaaaat tgggcattac gcccgaaggt 180

ttgccagtta aaggaccaga gtgtataaaa tgtggcggaa tcttacagtt tgatgctgtc 240

cgcgggcctc ataagagttg tggtaacaac gcaggtgcgc gcatctgcta aaatgtgtat 300

attagtttaa aaagttgtat gtaataaaag taaaatttaa tattttggat gaaaaaaacc 360

atttttagac tttttcttaa ctagaatgct ggagtagaaa tacgccatct caagatacaa 420

aaagcgttac cggcactgat ttgtttcaac cagtatatag attattattg ggtcttgatc 480

aactttcctc agacatatca gtaacagtta tcaagc 516

<210>44

<211>37

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>44

gtcataaatt gtcagtacgc acagatatta taacatc 37

<210>45

<211>41

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>45

atatccttga tcagcattgt tttatatttg ttgtaaaaag t 41

<210>46

<211>37

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>46

tgttagattt tcttgagcga atttcttatg atttatg 37

<210>47

<211>40

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>47

ctaacattca acgctagtat agatcatgat acataaaagc 40

<210>48

<211>41

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>48

caaatataaa acaatgctga tcaaggatat tatcttgacg c 41

<210>49

<211>35

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>49

cataagaaat tcgctcaaga aaatctaaca acaac 35

<210>50

<211>40

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>50

gcttttatgt atcatgatct atactagcgt tgaatgttag 40

<210>51

<211>38

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>51

taatatcggc cattttgttt gtttatgtgt gtttattc 38

<210>52

<211>40

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>52

aggatgtttg gtgaatttaa ctccttaagt tactttaatg 40

<210>53

<211>31

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>53

caggcatgca agcgcgaaaa gccaattagt g 31

<210>54

<211>33

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>54

acataaacaa acaaaatggc cgatattaac ttc 33

<210>55

<211>33

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>55

cttaaggagt taaattcacc aaacatcctc ttg 33

<210>56

<211>31

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>56

cactaattgg cttttcgcgc ttgcatgcct g 31

<210>57

<211>32

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>57

tgattccctt tctagcacgt gatgaattcg ag 32

<210>58

<211>38

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>58

atgcagaagtcattttgttt gtttatgtgt gtttattc 38

<210>59

<211>39

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>59

ggacgtctgg taaatttaac tccttaagtt actttaatg 39

<210>60

<211>34

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>60

acataaacaa acaaaatgac ttctgcatta tacg 34

<210>61

<211>33

<212>DNA

<213> a recombinant Saccharomyces cerevisiae producing hydroxy fatty acids

<400>61

ttaaggagtt aaatttacca gacgtccctc aag 33

Claims

1. A construction method of recombinant saccharomyces cerevisiae (I) for producing hydroxy fatty acid is characterized by comprising the following steps of knocking out acyl-CoA oxidase POX1 which is a key gene in β oxidation pathway related to fatty acid catabolism in saccharomyces cerevisiae by a homologous recombination method, and knocking out acyl-CoA activating enzymes FAA1 and FAA4 to obtain the recombinant saccharomyces cerevisiae (I);

the nucleotide sequence of the acyl-CoA oxidase gene POX1 is shown in SEQ ID NO. 1;

the nucleotide sequence of the acyl-CoA synthetase gene FAA1 is shown in SEQ ID NO. 2;

the nucleotide sequence of the acyl-CoA synthetase gene FAA4 is shown in SEQ ID NO. 3.

2. The recombinant Saccharomyces cerevisiae (I) for producing hydroxy fatty acid constructed by the method of claim 1.

3. A construction method of recombinant saccharomyces cerevisiae (II) for producing hydroxy fatty acid is characterized by comprising the following steps: introducing a cytochrome P450 oxidase gene CYP52M1 expression cassette and a cytochrome P450 reductase gene SbCPR expression cassette into an HO site of the recombinant saccharomyces cerevisiae (I) by a homologous recombination method to obtain a recombinant saccharomyces cerevisiae (II) for producing hydroxy fatty acid, wherein the obtained hydroxy fatty acid comprises omega-1-hydroxy-hexadecanoic acid and omega-1-hydroxy-oleic acid;

the nucleotide sequence of cytochrome P450 oxidase gene CYP52M1 is shown in SEQ ID NO. 4;

the nucleotide sequence of cytochrome P450 reductase gene SbCPR is shown in SEQ ID NO. 5.

4. The recombinant Saccharomyces cerevisiae producing hydroxy fatty acids constructed by the method of claim 3 (II).

5. A construction method of recombinant saccharomyces cerevisiae (III) for producing hydroxy fatty acid is characterized by comprising the following steps: introducing a cytochrome P450 oxidase gene CYP52M1 expression cassette and a cytochrome P450 reductase gene AtCPR1 expression cassette into an HO site of the recombinant saccharomyces cerevisiae (I) by a homologous recombination method to obtain the recombinant saccharomyces cerevisiae (III) for producing hydroxy fatty acid, wherein the obtained hydroxy fatty acid types comprise omega-hydroxy-hexadecanoic acid, omega-1-hydroxy-hexadecanoic acid, omega-hydroxy-oleic acid and omega-1-hydroxy-oleic acid;

the nucleotide sequence of cytochrome P450 reductase gene AtCPR1 is shown in SEQ ID NO. 6.

6. The recombinant Saccharomyces cerevisiae producing hydroxy fatty acids constructed by the method of claim 5 (III).

7. A construction method of recombinant saccharomyces cerevisiae (IV) for producing hydroxy fatty acid is characterized by comprising the following steps: introducing a cytochrome P450 oxidase gene expression cassette and a cytochrome P450 reductase gene fusion protein gene CYP52M1-AtCPR1 expression cassette into a recombinant saccharomyces cerevisiae strain (I) by a homologous recombination method to obtain recombinant saccharomyces cerevisiae (IV) for producing hydroxy fatty acid, wherein the obtained hydroxy fatty acid types comprise omega-hydroxy-hexadecanoic acid, omega-1-hydroxy-hexadecanoic acid, omega-hydroxy-oleic acid and omega-1-hydroxy-oleic acid;

the nucleotide sequence of the cytochrome P450 oxidase and cytochrome P450 reductase fusion protein gene CYP52M1-AtCPR1 is shown in SEQ ID NO. 7.

8. The recombinant Saccharomyces cerevisiae producing hydroxy fatty acids (IV) constructed by the method of claim 7.

9. Use of the recombinant saccharomyces cerevisiae yeast of any of claims 2, 4, 6 and 8 for the fermentative production of hydroxy fatty acids.