Genetically engineered bacterium and production method of omega-hydroxy fatty acid

Abstract

The invention provides a genetically engineered bacterium and a production method of omega-hydroxy fatty acid. The genetically engineered bacterium provided by the invention has reduced fatty alcohol oxidase activity, reduced fatty alcohol dehydrogenase activity and reduced monoacylglyceride enzyme activity, can efficiently utilize fatty acid or derivatives thereof, and can significantly improve the yield of the product when producing omega-hydroxy fatty acid.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a genetically engineered bacterium for producing ω-hydroxy fatty acids and a method for producing ω-hydroxy fatty acids. Background Art

[0002] ω-hydroxy fatty acids (ω-HFAs) are fatty acids with a hydroxyl group (-OH) at the terminal carbon position. Compared with non-polar fatty acids, they have the characteristics of high melting point, high boiling point, and high viscosity, and are more likely to form dimers and are insoluble in water. The polymers synthesized from ω-hydroxy fatty acids not only have higher tolerance to high temperature, chemical reagents, and impact, but also are green and safe, have good biocompatibility, and can be widely used in chemical industry, food, pharmacy, etc.

[0003] The traditional chemical synthesis method of ω-hydroxy fatty acids has a complex route and difficult separation, while the microbial fermentation method is green and environmentally friendly, but there is no industrialization report yet, and related technologies need to be further studied and developed. Summary of the Invention

[0004] The purpose of the present invention is to provide a genetically engineered bacterium through metabolic engineering transformation, which can not only use alkanes as substrates, but also efficiently utilize fatty acids or their derivatives as substrates for producing ω-hydroxy fatty acids. Especially when using fatty acids or their derivatives as fermentation substrates, ω-hydroxy fatty acids with significantly increased yields are obtained. The genetically engineered bacterium provided by the present invention and the method for fermenting and producing ω-hydroxy fatty acids using the genetically engineered bacterium have the potential for industrial scale-up production.

[0005] The term "ω-hydroxy fatty acids" in the present invention may include all ω-hydroxy fatty acids produced by microorganisms through metabolic processes from many different types of carbon sources. Specifically, the general formula of the ω-hydroxy fatty acids is CH2OH(CH2) n COOH, where n can be 7-16, for example, n = 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. More specifically, it includes ω-hydroxynonanoic acid, ω-hydroxydecanoic acid, ω-hydroxoundecanoic acid, ω-hydroxydodecanoic acid, ω-hydroxytridecanoic acid, ω-hydroxytetradecanoic acid, ω-hydroxypentadecanoic acid, and ω-hydroxypalmitic acid, but is not limited thereto.

[0006] The term "fatty alcohol oxidase" in the present invention may be abbreviated as FAO, and the term "fatty alcohol dehydrogenase" may be abbreviated as ADH.

[0007] In the terms of the present invention, "monoacylglycerol lipase" can be abbreviated as MGL, which is a serine hydrolase that mainly catalyzes the hydrolysis of monoacylglycerol, decomposing it into glycerol and free fatty acids. The structure of MGL includes an N-terminal signal peptide, a catalytic region, a subcellular organelle localization region, and a C-terminal region. Among them, the catalytic region includes a catalytic triad and a subunit binding region, which jointly participate in the catalytic reaction.

[0008] The inventors of the present invention found that reducing the enzyme activities of FAO, ADH, and MGL can improve the utilization rate of fatty acid or its derivatives as fermentation substrates during the fermentation of Candida strains to produce ω-hydroxy fatty acids.

[0009] Specifically, on the one hand, the present invention provides a genetically engineered bacterium for producing ω-hydroxy fatty acids, which has reduced fatty alcohol oxidase activity, reduced fatty alcohol dehydrogenase activity, and reduced monoacylglycerol lipase activity, and the genetically engineered bacterium is selected from the genus Candida.

[0010] The "genetically engineered bacterium for producing ω-hydroxy fatty acids" in the present invention means that the genetically engineered bacterium has the ability to produce ω-hydroxy fatty acids. It can be either the natural wild-type Candida strain itself, or a Candida strain with enhanced ω-hydroxy fatty acid production ability by enhancing or inactivating the activity of genes involved in the ω-hydroxy fatty acid production mechanism, or a Candida strain with enhanced ω-hydroxy fatty acid production ability by introducing or enhancing the activity of foreign genes.

[0011] In some preferred embodiments, the genetically engineered bacterium is selected from Candidaviswanathii, Candida tropicalis, Candida sake, or Candida albicans. More preferably, the genetically engineered bacterium is selected from Candidaviswanathii or Candida tropicalis.

[0012] In some embodiments, the EC number of the fatty alcohol oxidase is EC 1.1.3.20.

[0013] In some embodiments, the amino acid sequence of the fatty alcohol oxidase is as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0014] In some embodiments, the coding nucleotide sequence of the fatty alcohol oxidase is as shown in SEQ ID NO:3 or SEQ ID NO:4.

[0015] In some embodiments, the EC number of the fatty alcohol dehydrogenase is EC 1.1.1.1.

[0016] In some embodiments, the amino acid sequence of the fatty alcohol dehydrogenase is as shown in SEQ ID NO: 5.

[0017] In some embodiments, the coding nucleotide sequence of the fatty alcohol dehydrogenase is as shown in SEQ ID NO: 6.

[0018] In some embodiments, the EC number of the monoglyceride lipase is EC 3.1.1.23.

[0019] In some embodiments, the amino acid sequence of the monoglyceride lipase is as shown in SEQ ID NO: 7 or SEQ ID NO: 8.

[0020] In some embodiments, the coding nucleotide sequence of the monoglyceride lipase is as shown in SEQ ID NO: 9 or SEQ ID NO: 10.

[0021] In some embodiments, the genetically engineered bacterium can be obtained by inactivating the coding genes of endogenous fatty alcohol oxidase, fatty alcohol dehydrogenase, and monoglyceride lipase. Specifically, inactivation can be achieved by knockout, replacement, or insertion of the coding genes.

[0022] In some preferred embodiments, in the genetically engineered bacterium, the fatty alcohol oxidase with the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2 is inactivated, the fatty alcohol dehydrogenase with the amino acid sequence shown in SEQ ID NO: 5 is inactivated, and the monoglyceride lipase with the amino acid sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 8 is inactivated.

[0023] In some embodiments, the reduced fatty alcohol oxidase activity, reduced fatty alcohol dehydrogenase activity, and reduced monoglyceride lipase activity are achieved by any one or more of the following methods:

[0024] (i) Knocking out one or more genes of the enzyme;

[0025] (ii) Reducing the activity of the corresponding enzyme by mutating the coding region of the enzyme gene;

[0026] (iii) Reducing the expression of the corresponding enzyme by replacing the promoter of the enzyme gene;

[0027] (iv) Reducing the expression level of the enzyme gene by RNA interference technology; and / or,

[0028] (v) Reducing the expression level of the enzyme by CRISPRi technology.

[0029] In some specific embodiments, the reduced fatty alcohol oxidase activity can be achieved by any one or more of the following methods:

[0030] (i) Knocking out one or more genes of fatty alcohol oxidase;

[0031] (ii) Reducing the activity of fatty alcohol oxidase by mutating the coding region of the fatty alcohol oxidase gene;

[0032] (iii) Reducing the expression of fatty alcohol oxidase by replacing the promoter of the fatty alcohol oxidase gene;

[0033] (iv) Reducing the expression level of the fatty alcohol oxidase gene by RNA interference technology; and / or

[0034] (v) Reducing the expression level of fatty alcohol oxidase by CRISPRi technology.

[0035] In some preferred embodiments, the coding gene of fatty alcohol oxidase with the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2 is knocked out; preferably, the gene with the coding nucleotide sequence shown in SEQ ID NO:3 and / or SEQ ID NO:4 is knocked out; more preferably, the genes with the coding nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 are knocked out.

[0036] In some specific embodiments, the reduced fatty alcohol dehydrogenase activity can be achieved by any one or more of the following methods:

[0037] (i) Knocking out one or more genes of fatty alcohol dehydrogenase;

[0038] (ii) Reducing the activity of fatty alcohol dehydrogenase by mutating the coding region of the fatty alcohol dehydrogenase gene;

[0039] (iii) Reducing the expression of fatty alcohol dehydrogenase by replacing the promoter of the fatty alcohol dehydrogenase gene;

[0040] (iv) Reducing the expression level of the fatty alcohol dehydrogenase gene by RNA interference technology; and / or

[0041] (v) Reducing the expression level of fatty alcohol dehydrogenase by CRISPRi technology.

[0042] In some preferred embodiments, the coding gene of fatty alcohol dehydrogenase with the amino acid sequence shown in SEQ ID NO:5 is knocked out; preferably, the gene with the coding nucleotide sequence shown in SEQ ID NO:6 is knocked out.

[0043] In some specific embodiments, the reduced monoacylglycerol lipase activity can be achieved by any one or more of the following methods:

[0044] (i) Knocking out one or more genes of monoacylglycerol lipase;

[0045] (ii) Reducing the activity of monoacylglycerol lipase by mutating the coding region of the monoacylglycerol lipase gene;

[0046] (iii) Reducing the expression of monoacylglycerol lipase by replacing the promoter of the monoacylglycerol lipase gene;

[0047] (iv) Reducing the expression level of the monoacylglycerol lipase gene by RNA interference technology; and / or

[0048] (v) Reducing the expression level of the monoacylglycerol lipase by CRISPRi technology.

[0049] In some preferred embodiments, the method for reducing monoacylglycerol lipase activity is to reduce the activity of monoacylglycerol lipase by mutating the coding region of the monoacylglycerol lipase gene, reduce the expression of monoacylglycerol lipase by replacing the promoter of the monoacylglycerol lipase gene, and / or reduce the expression level of the monoacylglycerol lipase gene by RNA interference technology.

[0050] In some preferred embodiments, the coding gene of monoacylglycerol lipase with the amino acid sequence shown in SEQ ID NO:7 and / or SEQ ID NO:8 is knocked out; preferably, the gene with the coding nucleotide sequence shown in SEQ ID NO:9 and / or SEQ ID NO:10 is knocked out.

[0051] In some preferred embodiments, the method for reducing monoacylglycerol lipase activity is to reduce the expression of monoacylglycerol lipase by replacing the promoter of the monoacylglycerol lipase gene to reduce the monoacylglycerol lipase activity. Among them, the promoter of the monoacylglycerol lipase is replaced with a weak promoter; preferably, the promoter of the monoacylglycerol lipase is replaced with a first promoter or a second promoter, the sequence of the first promoter is shown in SEQ ID NO:11, and the sequence of the second promoter is shown in SEQ ID NO:12.

[0052] In another aspect, the present invention provides a method for producing ω-hydroxy fatty acid, which includes: culturing the genetically engineered bacterium of the present invention in a culture medium, and separating and extracting ω-hydroxy fatty acid from the genetically engineered bacterium or the culture medium to obtain an ω-hydroxy fatty acid product.

[0053] As used in the present invention, the term "culturing" means allowing the strain to grow under appropriate and artificially controlled environmental conditions. The culture medium used for culturing should meet the requirements of a specific strain in an appropriate manner.

[0054] In some embodiments, the culture medium contains a carbon source, a nitrogen source, and / or inorganic salts.

[0055] In some preferred embodiments, the carbon source is selected from at least one of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose.

[0056] In some preferred embodiments, the nitrogen source is selected from at least one of yeast extract, peptone, corn steep liquor, urea, ammonium salts, and nitrates.

[0057] In some preferred embodiments, the inorganic salts are selected from at least one of sulfates, hydrochlorides, nitrates, and phosphates, and more preferably from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride, and potassium nitrate.

[0058] In some preferred embodiments, the culture medium includes a seed culture medium and a fermentation culture medium, both of which contain the carbon source, the nitrogen source, and the inorganic salts. Exemplarily, the seed culture medium contains: sucrose 10 - 40 g / L, yeast extract 1 - 12 g / L, corn steep liquor 1 - 5 g / L, KH2PO4 2 - 12 g / L, urea 0.5 - 5 g / L; the fermentation culture medium contains: sucrose 10 - 40 g / L, corn steep liquor 1 - 5 g / L, yeast extract 1 - 12 g / L, NaCl 0 - 3 g / L, KNO3 2 - 12 g / L, KH2PO4 2 - 12 g / L, urea 0.5 - 5 g / L.

[0059] In some embodiments, the culture medium contains a fermentation substrate, and the fermentation substrate is selected from at least one of fatty acids, fatty acid esters, fatty acid salts, and alkanes.

[0060] In some embodiments, the content of the fermentation substrate in the culture medium is 50 - 300 g / L, such as 50 g / L, 80 g / L, 100 g / L, 120 g / L, 150 g / L, 180 g / L, 200 g / L, 220 g / L, 250 g / L, 280 g / L, 300 g / L, or any value within the range.

[0061] In some preferred embodiments, the fatty acid is selected from at least one of nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, and stearic acid.

[0062] In some preferred embodiments, the fatty acid ester is selected from at least one of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, and fatty acid butyl ester.

[0063] In some preferred embodiments, the fatty acid ester is selected from at least one of nonanoate, decanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, heptadecanoate, and octadecanoate. More preferably, the fatty acid ester is selected from at least one of methyl laurate, ethyl laurate, butyl laurate, methyl myristate, ethyl myristate, butyl myristate, methyl palmitate, ethyl palmitate, and butyl palmitate.

[0064] In some preferred embodiments, the fatty acid salt is selected from at least one of the sodium salt, potassium salt, ammonium salt, and calcium salt of a fatty acid.

[0065] In some preferred embodiments, the alkane is selected from at least one of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, and octadecane, and more preferably a normal alkane.

[0066] In some embodiments, when the fermentation substrate is solid, it is first heated to dissolve it and then added to the fermentation broth; wherein, the temperature for heating the fermentation substrate can be 40 - 100 °C.

[0067] In some embodiments, the culture temperature is 28 °C - 33 °C, such as 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, or 33 °C.

[0068] In some embodiments, the inoculum amount of the genetically engineered bacterium is 5 - 50% by volume of the culture medium, that is, the volume ratio between the seed liquid and the culture medium is (5 - 50):100.

[0069] In another aspect, the present invention provides the use of the genetically engineered bacterium in the production of ω-hydroxy fatty acids.

[0070] The genetically engineered bacterium provided by the present invention, by reducing the activities of fatty alcohol oxidase, fatty alcohol dehydrogenase, and monoacylglycerol lipase, can not only use alkanes as substrates, but also efficiently utilize fatty acids or their derivatives. While expanding the range of selectable substrates, it also significantly increases the yield of ω-hydroxy fatty acids. The genetically engineered bacterium provided by the present invention and the method for fermenting and producing ω-hydroxy fatty acids using the genetically engineered bacterium have the potential for industrial scale-up production. Detailed implementation manners

[0071] The present invention will be further described below through specific examples. The examples described in the present invention are only for the purpose of illustrating the present invention and do not limit the scope of the present invention. Unless otherwise specified, the raw materials and materials used in the following examples are commercially available products, and the methods and conditions used are known methods and conventional conditions in the art.

[0072] Materials and methods:

[0073] I. Strains and Vectors

[0074] Starting strain: Strain 1, Candida viswanathii CAES2113 (Deposit number: CCTCC NO: M2020048, disclosed in patent application CN111748480A).

[0075] The plasmid vector pC31 was constructed according to the method described in patent application CN116218696A, and TOP10 was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0076] II. Medium for Engineered Bacteria

[0077] YPD medium (w / v): 2% peptone, 2% glucose and 1% yeast extract (OXOID, number LP0021), and 1.5% agar powder is also added to the solid medium.

[0078] Seed medium: 15 g / L sucrose, 5 g / L yeast extract, 3 g / L corn steep liquor, 10 g / L KH2PO4, 1.5 g / L urea.

[0079] Fermentation medium: 35 g / L sucrose, 3 g / L corn steep liquor, 8 g / L yeast extract, 1.5 g / L NaCl, 6 g / L KNO3, 6 g / L KH2PO4, 2 g / L urea.

[0080] III. Product Detection Method: ω-Hydroxy Fatty Acids in the Fermentation Broth were Detected by High-Performance Gas Chromatography, and the Product Concentration was Calculated According to the Ratio of the Peak Area of the Product to the Peak Area of the Internal Standard with a Known Concentration.

[0081] Example 1: Construction of a Genetically Engineered Bacterium with Reduced Fatty Alcohol Oxidase and Fatty Alcohol Dehydrogenase Activities

[0082] The coding genes of fatty alcohol oxidase and fatty alcohol dehydrogenase in Strain 1 were knocked out. The amino acid sequence of fatty alcohol oxidase FAO1 is SEQ ID NO:1, and the coding nucleotide sequence is SEQ ID NO:3; the amino acid sequence of fatty alcohol oxidase FAO2 is SEQ ID NO:2, and the coding nucleotide sequence is SEQ ID NO:4; the amino acid sequence of fatty alcohol dehydrogenase ADH is SEQ ID NO:5, and the coding nucleotide sequence is SEQ ID NO:6.

[0083] 1. Construction of Gene Knockout Plasmid

[0084] Prepare a mixed solution of upstream and downstream primers for oligonucleotide sequences. The primers for knocking out FAO1 are N20_FAO1-F (SEQ ID NO: 13) and N20_FAO1-R (SEQ ID NO: 14), the primers for knocking out FAO2 are N20_FAO2-F (SEQ ID NO: 15) and N20_FAO2-R (SEQ ID NO: 16), and the primers for knocking out ADH are N20_ADH-F (SEQ ID NO: 17) and N20_ADH-R (SEQ ID NO: 18). The final concentration is 10 μM, and anneal to 25 °C. Digest the vector pC31 (the sequence refers to CN116218696A) with the restriction endonuclease PaqCI, and recover and purify the vector backbone. Dilute the annealed double-stranded DNA and ligate it with the recovered and purified pC31 vector backbone at room temperature using T4 DNA ligase (ThermoFisher), and transform it into TOP10 chemically competent cells. The next day, pick the transformants and verify them by colony PCR and sequencing. The constructed vectors are pC31-FAO1, pC31-FAO2, and pC31-ADH respectively.

[0085] 2. Preparation of knockout templates

[0086] Amplify the templates required for knocking out each gene. For FAO1, use oligonucleotides donor_FAO1-F (SEQ ID NO: 19) and donor_FAO1-R (SEQ ID NO: 20) as templates for each other, and the amplified PCR product is Donor_FAO1; for FAO2, use oligonucleotides donor_FAO2-F (SEQ ID NO: 21) and donor_FAO2-R (SEQ ID NO: 22) as templates for each other, and the amplified PCR product is Donor_FAO2; for ADH, use oligonucleotides donor_ADH-F (SEQ ID NO: 23) and donor_ADH-R (SEQ ID NO: 24) as templates for each other, and the amplified PCR product is Donor_ADH.

[0087] 3. Construction of strains by yeast electroporation

[0088] The method is as follows:

[0089] Step 1: Pick a single colony of Candida viswanathii 1 (starting strain: Candida viswanathii CAES2113) freshly streaked and inoculate it into 2 ml of YPD medium. Culture at 30 °C and 200 rpm until the OD620 reaches 1.3, then centrifuge at 1500 g and 4 °C to collect the cells. Wash the cells twice with ice-cold sterile water, resuspend them in 10 mL of ice-precooled 1 M sorbitol solution, centrifuge at 4 °C and 1500 g to collect the cells, and then resuspend them in 1 mL of the above sorbitol solution. Aliquot 100 μL of the competent cell suspension for electroporation.

[0090] Step 2: Electrotransformation of yeast competent cells

[0091] Add 0.2 μg of pC31-FAO1 plasmid DNA and 1 μg of the purified Donor_FAO1 to the competent cells obtained in Step 1, gently mix, place on ice for 5 min, then quickly transfer to a 0.2 cm electroporation cuvette. After electrotransformation (BioRad, MicropulserTM Electroporator, 1.5 - 2.4 kv, 4 - 5 ms), quickly add 1 mL of a mixture of YPD medium and 1 M sorbitol (1:1, v / v). Incubate at 30 °C and 200 rpm for 2 hours, then collect the bacterial solution and spread it on a YPD medium plate containing 100 mg / L hygromycin B. Incubate statically at 30 °C until single colonies grow.

[0092] After the transformants are identified by colony PCR and verified by sequencing, inoculate them into YPD liquid culture, incubate at 30 °C and 200 rpm for 48 h. Pick one loop of bacteria and streak it on a YPD solid medium plate. After incubating statically at 30 °C for 3 days, pick monoclonal colonies and inoculate them on YPD plates with and without antibiotics respectively. The clones that cannot grow on the plate containing antibiotics but can grow on the plate without antibiotics are the strains that have lost the plasmid and can be used for the next yeast electrotransformation.

[0093] Step 3: Knock out FAO2 and ADH genes by yeast electrotransformation

[0094] Successively knock out the remaining genes, and use the transformants obtained from the previous successful knockout as the chassis bacteria each time. The methods for preparing competent cells and electrotransformation are the same as in Steps 1 and 2, except that the plasmids and recombinant templates introduced are different. For knocking out FAO2, use pC31-FAO2 plasmid and Donor_FAO2; for knocking out ADH, use pC31-ADH plasmid and Donor_ADH. The verification of transformants is the same as in Step 2, and the finally obtained strain is named strain 1-1 (FAO1, FAO2, and ADH are knocked out).

[0095] Example 2: Construction of an engineered bacterium with reduced monoacylglycerol lipase activity by knockout

[0096] Knock out the coding genes of monoacylglycerol lipase in strain 1-1. The amino acid sequence of monoacylglycerol lipase MGLa is shown in SEQ ID NO:7, and the coding nucleotide sequence is shown in SEQ ID NO:9; the amino acid sequence of monoacylglycerol lipase MGLb is shown in SEQ ID NO:8, and the coding nucleotide sequence is shown in SEQ ID NO:10.

[0097] 1. Construction of gene editing plasmid

[0098] Prepare a mixed solution of upstream and downstream primers of the oligonucleotide sequence. The primers for knocking out MGLa are N20_MGLa-F (SEQ ID NO: 25) and N20_MGLa-R (SEQ ID NO: 26), and the primers for knocking out MGLb are N20_MGLb-F (SEQ ID NO: 27) and N20_MGLb-R (SEQ ID NO: 28). The final concentration is 10 μM, and anneal to 25 °C. Digest the vector pC31 (the sequence refers to CN116218696A) with the restriction endonuclease PaqCI, and recover and purify the vector backbone. The annealed double-stranded DNA after dilution is ligated with the recovered and purified pC31 vector backbone using T4 DNA ligase (ThermoFisher) at room temperature, and then transformed into TOP10 chemically competent cells. The next day, pick the transformants and verify them by colony PCR and sequencing. The constructed vectors are pC31-MGLa and pC31-MGLb.

[0099] 2. Preparation of homologous recombination template

[0100] Amplify the templates required for knocking out MGLa and MGLb. For MGLa, the oligonucleotides donor_MGLa-F (SEQ ID NO: 29) and donor_MGLa-R (SEQ ID NO: 30) are used as templates for each other, and the amplified PCR product is Donor_MGLa; for MGLb, the oligonucleotides donor_MGLb-F (SEQ ID NO: 31) and donor_MGLb-R (SEQ ID NO: 32) are used as templates for each other, and the amplified PCR product is Donor_MGLb.

[0101] 3. Construction of strains

[0102] The method is as follows:

[0103] Step 1: Pick a single colony of freshly streaked bacteria 1-1 and inoculate it into 2 ml of YPD medium. Culture at 30 °C and 200 rpm until the OD620 reaches 1.3, then centrifuge at 1500 g and 4 °C to collect the cells. Wash the cells twice with ice-cold sterile water, resuspend them in 10 mL of ice-precooled 1 M sorbitol solution, centrifuge at 4 °C and 1500 g to collect the cells, and then resuspend them in 1 mL of the above sorbitol solution. Aliquot 100 μL of the competent cell suspension for electroporation.

[0104] Step 2: Electroporation of yeast competent cells

[0105] Add 0.2 μg of pC31-MGLa plasmid DNA and 1 μg of purified Donor_MGLa obtained in Step 1 to the competent cells obtained in Step 1, gently mix, place on ice for 5 min, then quickly transfer to a 0.2 cm electroporation cuvette. After electroporation (BioRad, MicropulserTM Electroporator, 1.5 - 2.4 kv, 4 - 5 ms), quickly add 1 mL of a mixture of YPD medium and 1 M sorbitol (1:1, v / v). Incubate at 30 °C and 200 rpm for 2 hours, then collect the bacterial solution and spread it on a YPD medium plate containing 100 mg / L hygromycin B. Incubate statically at 30 °C until single colonies grow.

[0106] After the transformants were identified by colony PCR and verified by sequencing, and the sequencing was confirmed to be correct and the plasmid was deleted, the obtained genetically engineered strain was strain 1-1-1 (MGLa knockout in strain 1-1).

[0107] Similarly, replace the pC31-MGLa plasmid in Step 2 with the pC31-MGLb plasmid and Donor_MGLa with Donor_MGLb, and perform the same operations as in Step 2 above. The obtained genetically engineered strain is strain 1-1-2 (MGLb knockout in strain 1-1).

[0108] Example 3: Construction of an engineered bacterium with reduced monoglyceride lipase activity by replacing weak promoters (QDZ1 and QDZ2)

[0109] The first promoter is denoted as QDZ1, and its sequence is as shown in SEQ ID NO:11; the second promoter is denoted as QDZ2, and its sequence is as shown in SEQ ID NO:12.

[0110] 1. Construction of gene editing plasmid

[0111] The construction method of the gene editing plasmid for replacing the promoter is the same as that described in Example 2, except that the oligonucleotide sequences used are N20_MGLp-F (SEQ ID NO:33) and N20_MGLp-R (SEQ ID NO:34), and the constructed vector is pC31-MGLp. The sgRNA sequence of this vector can target the original promoter regions of both MGLa and MGLb simultaneously, replacing both promoters. The original promoter region of MGLa is MGLap (SEQ ID NO:35), and the original promoter region of MGLb is MGLbp (SEQ ID NO:36).

[0112] 2. Preparation of homologous recombination template

[0113] The first promoter QDZ1 (SEQ ID NO: 11) and the second promoter QDZ2 (SEQ ID NO: 12) were selected and amplified using the genomic DNA of strain 1-1 as a template. The primers were donor_QDZ1-F (SEQ ID NO: 37) and donor_QDZ1-R (SEQ ID NO: 38), donor_QDZ2-F (SEQ ID NO: 39) and donor_QDZ2-R (SEQ ID NO: 40). The amplified templates were Donor_QDZ1 and Donor_QDZ2 respectively.

[0114] 3. Construction of strains

[0115] The method for preparing electrocompetent yeast cells was the same as in Example 2, except that the plasmids and recombinant templates introduced were pC31-MGLp and Donor_QDZ1, pC31-MGLp and Donor_QDZ2 respectively. After the transformants were identified by colony PCR and confirmed by sequencing, the plasmids were deleted. The obtained genetically engineered strains were strain 1-1-3 and strain 1-1-4.

[0116] Example 4: Shake flask fermentation for the production of ω-hydroxydecanoic acid

[0117] Strains 1, 1-1, 1-1-1, 1-1-2, 1-1-3 and 1-1-4 were respectively inoculated into 2 ml centrifuge tubes containing 1 ml of YPD medium and cultured on a shaker at 30 °C and 250 rpm for 1 day. The above-mentioned bacterial solutions were taken and inoculated into 500 mL shake flasks containing 30 ml of seed medium, with an inoculation amount of 3%. The shaker speed was 250 rpm and the temperature was 30 °C, and the culture was continued for 36 - 48 h until the OD620 reached 0.8 (after dilution 30 times). The seed liquid was inoculated into a shake flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. The culture was continued on a shaker at 250 rpm and 30 °C for 136 h, and the pH was natural. The fermentation medium contained 3 g of the substrate methyl decanoate for the production of ω-hydroxydecanoic acid. After the fermentation was completed, high performance gas chromatography was used to detect and calculate the concentration of ω-hydroxydecanoic acid in the medium.

[0118] As shown in Table 1, after knocking out FAO1, FAO2 and ADH in strain 1, the yield of ω-hydroxydecanoic acid increased by 7 times. On the basis of strain 1-1, engineering strains with reduced monoglyceride lipase (MGL) activity were further obtained by gene knockout or promoter replacement. Using methyl decanoate as the substrate, the yield of ω-hydroxydecanoic acid in shake flask fermentation also increased, indicating that reducing MGL activity significantly improved the substrate utilization rate. Among them, the yield of strain 1-1-4 was the highest, and the increase in the concentration of the target product ω-hydroxydecanoic acid was 44.71%.

[0119] Table 1

[0120] Strain Name Strain Information and Modification Method ω-Hydroxydecanoic Acid (g / L) Candida viswanathii CAES2113 Strain 1 (Starting Strain) 1.11 Strain 1-1 Strain 1, with FAO1, FAO2, and ADH knocked out 7.65 Strain 1-1-1 Strain 1-1, with MGLa knocked out 8.45 Strain 1-1-2 Strain 1-1, with MGLb knocked out 8.83 Strain 1-1-3 Strain 1-1, with the MGL promoter replaced by QDZ1 10.39 Strain 1-1-4 Strain 1-1, with the MGL promoter replaced by QDZ2 11.07

[0121] Example 5: Shake-flask fermentation for the production of ω-hydroxyundecanoic acid

[0122] Inoculate strains 1, 1-1, 1-1-1, 1-1-2, 1-1-3, and 1-1-4 into 2-ml centrifuge tubes containing 1 ml of YPD medium, and culture them on a shaker at 30 °C and 250 rpm for 1 day. Take the above bacterial solutions and inoculate them into 500-mL shake flasks containing 30 ml of seed medium, with an inoculation amount of 3%, a shaker speed of 250 rpm, and a temperature of 30 °C. Culture for 36 - 48 h until the OD620 reaches 0.8 (after dilution 30-fold). Inoculate the seed liquid into a shake flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. The fermentation medium contains 3 g of the substrate methyl undecanoate for the production of ω-hydroxyundecanoic acid. After fermentation, use high-performance gas chromatography to detect and calculate the concentration of ω-hydroxyundecanoic acid in the medium.

[0123] As shown in Table 2, after knocking out FAO1, FAO2, and ADH in strain 1, the yield of ω-hydroxyundecanoic acid increased by 6-fold. Based on strain 1-1, engineering strains with reduced monoglyceride lipase (MGL) activity were further obtained by gene knockout or promoter replacement. Using methyl undecanoate as the substrate, the yield of ω-hydroxyundecanoic acid also increased in shake-flask fermentation, indicating that reducing MGL activity significantly improved the substrate utilization rate. Among them, the yield of strain 1-1-4 was the highest, and the increase in the concentration of the target product ω-hydroxyundecanoic acid was 20.63%.

[0124] Table 2

[0125]

[0126] Example 6: Shake-flask fermentation for the production of ω-hydroxylauric acid

[0127] Inoculate strains 1, 1-1, 1-1-1, 1-1-2, 1-1-3, and 1-1-4 into 2-ml centrifuge tubes containing 1 ml of YPD medium, and culture them on a shaker at 30 °C and 250 rpm for 1 day. Take the above bacterial solutions and inoculate them into 500-mL shake flasks containing 30 ml of seed medium, with an inoculation amount of 3%, a shaker speed of 250 rpm, and a temperature of 30 °C. Culture for 36 - 48 h until the OD620 reaches 0.8 (after dilution 30-fold). Inoculate the seed liquid into a shake flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. The fermentation medium contains 3 g of the substrate methyl laurate for the production of ω-hydroxylauric acid. After fermentation, use high-performance gas chromatography to detect and calculate the concentration of ω-hydroxylauric acid in the medium.

[0128] As shown in Table 3, after knocking out FAO1, FAO2, and ADH of bacterium 1, the yield of ω-hydroxy lauric acid increased by 6 times. On the basis of bacterium 1-1, an engineered strain with reduced monoglyceride lipase (MGL) activity by continued gene knockout or promoter replacement was used. With methyl laurate as the substrate, the yield of ω-hydroxy lauric acid also increased in shake flask fermentation, indicating that reducing MGL activity significantly improved the substrate utilization rate. Among them, the yield of bacterium 1-1-4 was the highest, and the increase in the concentration of the target product ω-hydroxy lauric acid was 36.91%.

[0129] Table 3

[0130]

[0131]

[0132] Example 7: Shake flask fermentation for the production of ω-hydroxytetradecanoic acid

[0133] Bacterium 1, bacterium 1-1, bacterium 1-1-1, bacterium 1-1-2, bacterium 1-1-3, and bacterium 1-1-4 were respectively inoculated into 2 ml centrifuge tubes containing 1 ml of YPD medium and cultured on a shaker at 30 °C and 250 rpm for 1 day. The above bacterial liquid was taken and inoculated into a 500 mL shake flask containing 30 ml of seed medium, with an inoculation amount of 3%. The shaker speed was 250 rpm and the temperature was 30 °C, and the culture was continued for 36 - 48 h until OD620 reached 0.8 (after dilution 30 times). The seed liquid was inoculated into a shake flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. The culture was continued on a shaker at 250 rpm and 30 °C for 136 h, and the pH was natural. The fermentation medium contained 3 g of the substrate methyl tridecanoate for the production of ω-hydroxytetradecanoic acid. After fermentation, high performance gas chromatography was used to detect and calculate the concentration of ω-hydroxytetradecanoic acid in the medium.

[0134] As shown in Table 4, after knocking out FAO1, FAO2, and ADH of bacterium 1, the yield of ω-hydroxytetradecanoic acid increased by 5 times. On the basis of bacterium 1-1, an engineered strain with reduced monoglyceride lipase (MGL) activity by continued gene knockout or promoter replacement was used. With methyl tridecanoate as the substrate, the yield of ω-hydroxytetradecanoic acid also increased in shake flask fermentation, indicating that reducing MGL activity significantly improved the substrate utilization rate. Among them, the yield of bacterium 1-1-4 was the highest, and the increase in the concentration of the target product ω-hydroxytetradecanoic acid was 23.32%.

[0135] Table 4

[0136]

[0137] Example 8: Shake flask fermentation for the production of ω-hydroxypentadecanoic acid

[0138] Inoculate strain 1, strain 1-1, strain 1-1-1, strain 1-1-2, strain 1-1-3, and strain 1-1-4 respectively into 2-ml centrifuge tubes containing 1 ml of YPD medium, and culture them on a shaker at 30 °C and 250 rpm for 1 day. Take the above bacterial solutions and inoculate them into 500-mL flasks containing 30 ml of seed medium, with an inoculation amount of 3%, a shaker speed of 250 rpm, and a temperature of 30 °C, and culture for 36 - 48 h until the OD620 reaches 0.8 (after dilution 30 times). Inoculate the seed solution into a flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. The fermentation medium contains 3 g of the substrate methyl myristate for the production of ω-hydroxy myristic acid. After fermentation, use high-performance gas chromatography to detect and calculate the concentration of ω-hydroxy myristic acid in the medium.

[0139] As shown in Table 5, after knocking out FAO1, FAO2, and ADH in strain 1, the yield of ω-hydroxy myristic acid increased by 5 times. Based on strain 1-1, engineering strains with reduced monoglyceride lipase (MGL) activity were further obtained by gene knockout or promoter replacement. Using methyl myristate as the substrate, the yield of ω-hydroxy myristic acid also increased in flask fermentation, indicating that reducing MGL activity significantly improved the substrate utilization rate. Among them, strain 1-1-4 had the highest yield, and the increase in the concentration of the target product ω-hydroxy myristic acid was 31.43%.

[0140] Table 5

[0141]

[0142] Example 9: Flask fermentation for the production of ω-hydroxypentadecanoic acid

[0143] Inoculate strain 1, strain 1-1, strain 1-1-1, strain 1-1-2, strain 1-1-3, and strain 1-1-4 respectively into 2-ml centrifuge tubes containing 1 ml of YPD medium, and culture them on a shaker at 30 °C and 250 rpm for 1 day. Take the above bacterial solutions and inoculate them into 500-mL flasks containing 30 ml of seed medium, with an inoculation amount of 3%, a shaker speed of 250 rpm, and a temperature of 30 °C, and culture for 36 - 48 h until the OD620 reaches 0.8 (after dilution 30 times). Inoculate the seed solution into a flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. The fermentation medium contains 3 g of the substrate methyl pentadecanoate for the production of ω-hydroxypentadecanoic acid. After fermentation, use high-performance gas chromatography to detect and calculate the concentration of ω-hydroxypentadecanoic acid in the medium.

[0144] As shown in Table 5, after knocking out FAO1, FAO2, and ADH in Bacterium 1, the yield of ω-hydroxypentadecanoic acid increased by 4-fold. Based on Bacterium 1-1, an engineered strain with reduced monoglyceride lipase (MGL) activity by further gene knockout or promoter replacement was used. With methyl pentadecanoate as the substrate, the yield of ω-hydroxypentadecanoic acid also increased in shake flask fermentation, indicating that reducing MGL activity significantly improved substrate utilization. Among them, Bacterium 1-1-4 had the highest yield, and the increase in the concentration of the target product ω-hydroxypentadecanoic acid was 56.63%.

[0145] Table 5

[0146]

[0147]

[0148] Example 10: Shake Flask Fermentation for Production of ω-Hydroxypalmitic Acid

[0149] Bacterium 1, Bacterium 1-1, Bacterium 1-1-1, Bacterium 1-1-2, Bacterium 1-1-3, and Bacterium 1-1-4 were respectively inoculated into 2-ml centrifuge tubes containing 1 ml of YPD medium and cultured on a shaker at 30 °C and 250 rpm for 1 day. The above bacterial liquid was taken and inoculated into a 500-mL shake flask containing 30 ml of seed medium, with an inoculation amount of 3%. The shaker speed was 250 rpm and the temperature was 30 °C. After culturing for 36 - 48 h, the OD620 reached 0.8 (after dilution 30 times). The seed liquid was inoculated into a shake flask containing 15 ml of fermentation medium, with an inoculation amount of 20%. Culturing was continued on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. The fermentation medium contained 3 g of the substrate butyl palmitate for the production of ω-hydroxypalmitic acid. After fermentation, high-performance gas chromatography was used to detect and calculate the concentration of ω-hydroxypalmitic acid in the medium.

[0150] As shown in Table 7, after knocking out FAO1, FAO2, and ADH in Bacterium 1, the yield of ω-hydroxypalmitic acid increased by 5-fold. Based on Bacterium 1-1, an engineered strain with reduced monoglyceride lipase (MGL) activity by further gene knockout or promoter replacement was used. With butyl palmitate as the substrate, the yield of ω-hydroxypalmitic acid also increased in shake flask fermentation, indicating that reducing MGL activity significantly improved substrate utilization. Among them, Bacterium 1-1-4 had the highest yield, and the increase in the concentration of the target product ω-hydroxypalmitic acid was 44.72%.

[0151] Table 7

[0152]

[0153] Sequence Information:

[0154] Amino Acid Sequence of Fatty Alcohol Oxidase FAO1 (SEQ ID NO: 1)

[0155] MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLGKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLTNDGFKSLVLEKGRYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL

[0156] Amino acid sequence of fatty alcohol oxidase FAO2 (SEQ ID NO:2)

[0157] MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIKDAIAPDFPEDQYEEYLKTFTKPSETPGFREAVYDTINATPTDAVHMCIVLTTALDSRILAPTLTNSLTPIKDMTLKEREQLLASWRDSPIAAKRRLFRLISSLTLTTFTRLASELHLKAIHYPGRDLREKAYETQVVDPFRYLFMEKPKFDGAELYLPDIDVIIIGSGAGAGVMAHTLANDGFKTLVLEKGKYFSNSELNFNDADGVKELYQGKGALATTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTDGITHSLANEVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILNKNGVAYGILCEDVETGVRFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYEINKFDKNAILQAFLITSDMLYIEGAKRILSPQPWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMSTCRMSGKGPKYGAVDTDGRLFECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL

[0158] Coding nucleotide sequence of fatty alcohol oxidase FAO1 (SEQ ID NO: 3)

[0159]

[0160] Coding nucleotide sequence of fatty alcohol oxidase FAO2 (SEQ ID NO: 4)

[0161]

[0162] Amino acid sequence of fatty acid desaturase (SEQ ID NO: 5)

[0163] MSVPTTQKAVIFETNGGKLEYKDVPVPVPKPNELLVNVKYSGVCHSDLHVWKGDWPIPAKLPLVGGHEGAGVVVGMGDNVKGWKVGDLAGIKWLNGSCMNCEFCQQGAEPNCSRADMSGYTHDGTFQQYATADAVQAAKIPEGADMASIAPILCAGVTVYKALKNADLLAGQWVAISGAGGGLGSLGVQYAKAMGYRVLAIDGGDERGEFVKSLGAEVYIDFLKEQDIVSAIRKATGGGPHGVINVSVSEKAINQSVEYVRTLGKVVLVSLPAGGKLTAPLFESVARSIQIRTTCVGNRKDTTEAIDFFVRGLIDCPIKVAGLSEVPEIFDLMEQGKILGRYVVDTSK

[0164] Coding nucleotide sequence of fatty acid desaturase (SEQ ID NO: 6)

[0165]

[0166] Amino acid sequence of monoacylglycerol lipase MGLa (SEQ ID NO:7)

[0167] MTGKVEIPYTSKGEPTVEFIDHNNAKFKTVTWKVPKDIPYKGKIIYVHGFAEQSSVYTEFFDNLSQNGYEVFFFDQRGAGETSPGSLVGQTNEFHVFDDLEFFIKRLLDLRVNKDEKFYLMGHSMGGGIILNYGIRGKYVENIRGIIACGPLIQLHPDTQPNFILRAAMPYANKIVPGFKIDSKLNYDYITSNERWKDYIKRSDKLIGSIRQFHDMFARGEELLKPEYVAKFDKNVPLLIVHGDDDHINDVRASRKFIALLKDQENKKLVEIKEGRHSLFIENDELFKDIFKIVLEFLDTN

[0168] Amino acid sequence of monoacylglycerol lipase MGLb (SEQ ID NO:8)

[0169] MTGKVEIPYTPKGEPIVEFIDHNNAKFKTVTWKVPKDIPYKGKIIYVHGFAEQSSVYTEFFDNLSQNGYEVFFFDQRGAGETSPGSLVGQTDEFHVFDDLEFFIKRLLDLRVNKDEKFYLMGHSMGGGIILNYGIRGKYVEDIRGIIACGPLIQLHPDTQPNFIMRAAMPYANKIVPGFKIDSKLNYDYITSNERWKDYIKSSDRLIGSIRQFHDMFARGEELLKPEYAAKFDKNVPLLVVHGDDDHINDVRASRKFIALLKDQENKKLVEIKEGRHSLFIENDELFKDIFKIVLEFLDAN

[0170] Coding nucleotide sequence of monoacylglycerol lipase MGLa (SEQ ID NO:9)

[0171] atgactggtaaggttgagatcccatacacctccaagggcgaaccaactgttgaattcatcgaccacaacaacgccaagttcaaaacggtcacttggaaagtccccaaagacattccttacaagggcaaaatcatttacgtccacggttttgctgaacagtcgaacgtctacaccgagttctttgacaacttgtcacaaaacggatacgaagtgttctttttcgaccaaagaggagctggtgagacctctcctggaagcttggtgggacaaactaatgagttccatgtgtttgacgatttggagttcttcatcaagagattattggacttgagagtcaacaaagacgagaagttctacttgatgggtcactccatgggtggtggtatcatcttgaactacggtattagaggtaaatatgtcgagaatatcagaggtattattgcctgtggcccattgatccaattgcacccagacactcagccaaacttcatcttgcgtgccgctatgccgtatgccaacaagattgttcctggtttcaagatcgacagtaagttgaactacgactacatcacctccaacgaaagatggaaggactacatcaagagaagtgacaaattgattggttctattcgtcaattccacgacatgtttgccagaggcgaagagttgttgaagcctgaatacgtagctaagttcgacaaaaatgtgccattgttgattgttcacggtgacgatgaccacatcaatgacgttagagcaagtagaaagttcattgctttgctcaaagatcaagagaacaagaaattggttgagattaaagagggtcgccactctttgtttattgagaacgacgagttgtttaaagatatctttaagattgtcttagagttcttagataccaactag

[0172] Coding nucleotide sequence of monoglyceride lipase MGLb (SEQ ID NO:10)

[0173] atgactggtaaggttgagatcccatacaccccaaagggtgagccaattgttgaattcatcgaccacaacaacgccaagttcaaaaccgtcacttggaaagtccccaaagacattccttacaagggcaaaatcatttacgtccatggtttcgctgagcagtcgagcgtctacacagagttctttgacaacttgtcacaaaacggatacgaagtgttctttttcgaccaaagaggagctggtgagacgtctccgggaagcttggtgggacaaacggacgagtttcatgtgtttgacgatttggagttcttcatcaagagattattggacttgagagtcaacaaagacgagaagttctacttgatgggtcactccatgggtggtggtatcatcttgaactacggtattagaggtaaatacgtcgaggatatcagaggcattatcgcctgtggcccattgatccaattgcacccagacactcagccaaacttcatcatgcgcgccgctatgccgtacgctaacaagatcgttcctggtttcaagatcgacagtaagttgaactacgattacatcacctccaacgaaagatggaaggactacatcaagagcagtgacagattgattggttctattcgtcaattccacgacatgtttgccagaggtgaagagttgttgaagcctgaatacgcagctaagttcgacaagaatgtgccattgttggttgttcacggtgacgacgaccacatcaatgacgttagagcaagtagaaagttcattgctttgctcaaggatcaagagaacaagaaattggttgagatcaaagaaggtcgccactctttgtttattgagaacgacgagttgtttaaagatatctttaagattgtcttagagttcttagatgccaactag

[0174] Sequence of the first promoter QDZ1 (SEQ ID NO: 11)

[0175] tgttgttgtgtaatgtaaattatttgataaggaaaagaaaaggagtaaattgaaaaaaaaatttcagaaagggaagatttggctgagtttaaatatgggaattcttgtttgttggtgagtggtttctttgtgttttgagtttggggtttgtttgttgttttttgcagtcttgtgttgtcgtgacgcacaagagagaagagagagaagagaagagaagagaagagagagggagagggaagtgcgagtcttttgtgcgcaacaaaagaactttggcgcgggtgttgatattcttcggtgggcaacaaaagaacactggtgttcctctcttgtattgagaaaaaccctgtgtgccttctctctacccaacaagaacacccacaagcgagggtaaagcagttggtagaagtgatcctagtaccactatacagccctcaccacctcatatatctactcaaaccacaacatatccctcaccacaccgtgtttctcctccaagaacgccacaaaccgttgattttgcagtcttttgttgcttggaaaagatgcgtttttaactatgttcgtgctcatatttacgcacacgcaccatacaagccctcgctctcgcgttctcagttttgctcctctctctcttctgtcctccccccaccaagtttcgcggcacgcgactgcccaccgcgcaagaaacaaaaccaaaacaatcccacaaagatcacgtttcgcttcagctatccctcgcccccatcgtttcaaatcattataaatatctccaggactccctccttagcaaatttttcaaaccttcctttctttctttttctcctttatcattacaacaaattcaaattataatcatatacaaca

[0176] Sequence of the second promoter QDZ2 (SEQ ID NO: 12)

[0177]

[0178] Sequence of N20_FAO1-F (SEQ ID NO:13)

[0179] tacctgaaaaggcttatgaaaccc

[0180] Sequence of N20_FAO1-R (SEQ ID NO:14)

[0181] aaacgggtttcataagccttttca

[0182] Sequence of N20_FAO2-F (SEQ ID NO:15)

[0183] tacccaactcgttgacgcctatca

[0184] Sequence of N20_FAO2-R (SEQ ID NO:16)

[0185] aaactgataggcgtcaacgagttg

[0186] Sequence of N20_ADH-F (SEQ ID NO:17)

[0187] taccaactgtgagttttgccaaca

[0188] Sequence of N20_ADH-R (SEQ ID NO:18)

[0189] aaactgttggcaaaactcacagtt

[0190] Sequence of donor_FAO1-F (SEQ ID NO:19)

[0191] tatccaggaagagaagaccgtgaaaaggcttatgaaatagctaactgcagctaatca

[0192] Sequence of donor_FAO1-R (SEQ ID NO:20)

[0193] ttcccaaaaactggtacttaaaagggtcaatctcctgggtgattagctgcagttagcta

[0194] Sequence of donor_FAO2-F (SEQ ID NO:21)

[0195] gaatcttggcccccacgttgaccaactcgttgacgcctatagctaactgcagctaatca

[0196] Sequence of donor_FAO2-R (SEQ ID NO:22)

[0197] ccaacaattgttcacgctccttcaaggtcatatccttgatgattagctgcagttagcta

[0198] Sequence of donor_ADH-F (SEQ ID NO:23)

[0199] gtggttgaatggttcgtgtatgaactgtgagttttgccatagctaactgcagctaatca

[0200] Sequence of donor_ADH-R (SEQ ID NO:24)

[0201] gacatgtcggctcttgaacagttaggttctgcgccctgttgattagctgcagttagcta

[0202] Sequence of N20_MGLa-F (SEQ ID NO:25)

[0203] tacccaacaacgccaagttcaaaa

[0204] Sequence of N20_MGLa-R (SEQ ID NO:26)

[0205] aaacttttgaacttggcgttgttg

[0206] Sequence of N20_MGLb-F (SEQ ID NO:27)

[0207] tacctattagaggtaaatacgtcg

[0208] Sequence of N20_MGLb-R (SEQ ID NO:28)

[0209] aaaccgacgtatttacctctaata

[0210] Sequence of donor_MGLa-F (SEQ ID NO:29)

[0211] caactgttgaattcatcgaccacaacaacgccaagttcatagctaactgcagctaatca

[0212] Sequence of donor_MGLa-R (SEQ ID NO:30)

[0213] tgtaaggaatgtctttggggactttccaagtgaccgttttgattagctgcagttagcta

[0214] Sequence of donor_MGLb-F (SEQ ID NO:31)

[0215] gtggtatcatcttgaactacggtattagaggtaaatacgtagctaactgcagctaatca

[0216] Sequence of donor_MGLb-R (SEQ ID NO:32)

[0217] tcaatgggccacaggcgataatgcctctgatatcctcgatgattagctgcagttagcta

[0218] Sequence of N20_MGLp-F (SEQ ID NO:33)

[0219] taccgatgaggtatctctgggatc

[0220] N20_MGLp-R (SEQ ID NO:34)

[0221] aaacgatcccagagatacctcatc

[0222] MGLap (SEQ ID NO:35)

[0223]

[0224] MGLbp (SEQ ID NO:36)

[0225]

[0226] donor_QDZ1-F (SEQ ID NO:37)

[0227] tctgtaacgagtttgtcaagtcgtacttgttgtcaatgttgttgtgtaatgtaaattat

[0228] donor_QDZ1-R (SEQ ID NO:38)

[0229] ggtgtatgggatctcaaccttaccagtcattgttgtatatgattataatttgaatttg

[0230] donor_QDZ2-F (SEQ ID NO:39)

[0231] tctgtaacgagtttgtcaagtcgtacttgttgtcaaccgttcgtcttgtctgaaaatggtc

[0232] donor_QDZ2-R (SEQ ID NO:40)

[0233] ggtgtatgggatctcaaccttaccagtcatttcggggtctatggaatattcca

[0234] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for producing ω-hydroxy fatty acids, characterized in that: The genetically engineered bacteria have reduced fatty alcohol oxidase activity, reduced fatty alcohol dehydrogenase activity and reduced monoacylglycerol esterase activity, and the genetically engineered bacteria are selected from the genus Candida; Preferably, the genetically engineered bacteria is selected from Candida visweiss, Candida tropicalis, Candida sakei or Candida albicans; more preferably, the genetically engineered bacteria is selected from Candida visweiss or Candida tropicalis.

2. The genetically engineered bacterium according to claim 1, characterized in that: The amino acid sequence of the fatty alcohol oxidase is shown in SEQ ID NO: 1 or SEQ ID NO: 2; preferably, the encoding nucleotide sequence of the fatty alcohol oxidase is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

3. The genetically engineered bacterium according to claim 1, characterized in that The amino acid sequence of the fatty alcohol dehydrogenase is shown in SEQ ID NO:5; preferably, the encoding nucleotide sequence of the fatty alcohol dehydrogenase is shown in SEQ ID NO:

6.

4. The genetically engineered bacterium according to claim 1, characterized in that: The amino acid sequence of the monoacylglycerol esterase is shown in SEQ ID NO:7 or SEQ ID NO:8; preferably, the encoding nucleotide sequence of the monoacylglycerol esterase is shown in SEQ ID NO:9 or SEQ ID NO:

10.

5. The genetically engineered bacterium according to claim 1, characterized in that: In the genetically engineered bacteria, the fatty alcohol oxidase with the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2 is inactivated, the fatty alcohol dehydrogenase with the amino acid sequence shown in SEQ ID NO:5 is inactivated, and the monoacylglycerol esterase with the amino acid sequence shown in SEQ ID NO:7 and / or SEQ ID NO:8 is inactivated.

6. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The reduced fatty alcohol oxidase activity, reduced fatty alcohol dehydrogenase activity and reduced monoacylglycerol esterase activity are achieved by any one or more of the following methods: (i) knocking out one or more genes of an enzyme; (ii) reducing the activity of the corresponding enzyme by mutating the coding region of the enzyme gene; (iii) reducing the expression of the corresponding enzyme by replacing the promoter of the enzyme gene; (iv) reducing the expression level of the gene of the enzyme by RNA interference technology; and / or, (v) Reducing the expression level of the enzyme by CRISPRi technology.

7. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The gene encoding the fatty alcohol oxidase whose amino acid sequence is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2 is knocked out; preferably, the gene encoding the nucleotide sequence is shown in SEQ ID NO: 3 and / or SEQ ID NO: 4 is knocked out; The gene encoding fatty alcohol dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 5 is knocked out; preferably, the gene encoding nucleotide sequence as shown in SEQ ID NO: 6 is knocked out.

8. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The gene encoding the monoacylglycerol esterase whose amino acid sequence is shown in SEQ ID NO: 7 and / or SEQ ID NO: 8 is knocked out; preferably, the gene encoding the nucleotide sequence is shown in SEQ ID NO: 9 and / or SEQ ID NO: 10 is knocked out; or, The promoter of monoacylglycerol esterase is replaced with a promoter with a sequence as shown in SEQ ID NO:11 or SEQ ID NO:

12.

9. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The general formula of the ω-hydroxy fatty acid is CH2OH(CH2) n COOH, wherein n is 7 to 16; Preferably, the ω-hydroxy fatty acid includes ω-hydroxynonanoic acid, ω-hydroxydecanoic acid, ω-hydroxyundecanoic acid, ω-hydroxylauric acid, ω-hydroxytridecanoic acid, ω-hydroxymyristic acid, ω-hydroxypentadecanoic acid and ω-hydroxypalmitic acid.

10. A method for producing ω-hydroxy fatty acid, characterized in that: include: Cultivating the genetically engineered bacteria according to any one of claims 1 to 9 in a culture medium, and separating and extracting ω-hydroxy fatty acids from the genetically engineered bacteria or the culture medium to obtain an ω-hydroxy fatty acid product; The culture medium contains a fermentation substrate, and the fermentation substrate is selected from at least one of fatty acids, fatty acid esters, fatty acid salts, and alkanes.

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