Genetically engineered bacterium and method for producing omega-amino fatty acid by using genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium and a method for producing omega-amino fatty acid by using the genetically engineered bacterium. The genetically engineered bacterium has the effects of reducing the activity of fatty aldehyde dehydrogenase and increasing the activity of omega-transaminase. The genetically engineered bacterium provided by the invention has the capability of producing omega-amino fatty acid, and the yield is remarkably improved. The method for producing the omega-amino fatty acid through fermentation of the genetically engineered bacterium has the potential of industrial large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and relates to a genetically engineered bacterium and a method for producing ω-amino fatty acids using the same. Background Art

[0002] ω-amino fatty acids can be used to synthesize long-chain polyamides, which have good mechanical properties, high temperature resistance, wear resistance, chemical corrosion resistance, and low friction coefficient, etc., and can be widely applied in many fields such as 3D printing, automobile manufacturing, aerospace, electronic and electrical, medical devices, etc.

[0003] Taking polyamide 12 (also known as poly-dodecanolactam or poly-laurolactam) as an example, it is a long-chain polyamide. The average number of carbon atoms in its monomer unit is 12, and the molecular formula is [NH-(CH2) 11 -CO] n . Polyamide 12 is widely used in various fields such as 3D printing, automobile manufacturing, aerospace, electronic and electrical, medical devices, etc. due to its good mechanical properties, high temperature resistance, wear resistance, chemical corrosion resistance, and low friction coefficient. Polyamide 12 can be obtained by polymerizing two monomers, 12-aminolauric acid (or ω-aminolauric acid) or ω-dodecanolactam. The commonly used chemical synthesis methods include the oxidation-oximation method, the photo-nitrosation method, the Snia method, or the peroxyamine method. Taking the oxidation-oximation method as an example, it uses butadiene as a raw material to synthesize ω-dodecanolactam through multiple chemical reactions, and then obtains polyamide 12 through ring-opening polymerization; or uses cyclohexanone as a raw material to synthesize 12-aminolauric acid by the peroxyamine method, and then obtains polyamide 12 through polycondensation reaction. The development of these traditional chemical industry methods is limited due to the consumption of a large amount of non-renewable crude oil resources, the generation of environmental hazards, and process safety problems. The biosynthesis method using renewable resources is attracting more and more attention due to its green and sustainable nature and mild reaction conditions, and is gradually becoming a promising alternative to the chemical synthesis method.

[0004] Currently, the biosynthesis of long-chain amino acid-based polyamide monomers is mainly through the cleavage of natural oils and fats or the whole-cell catalytic synthesis using vegetable oil-derived compounds as raw materials. For example, the earliest bio-based industrialization of ω-undecyl amino acid was synthesized using castor oil as a raw material through a series of catalytic steps such as cleavage and alcoholysis. In 2011, the Schmid research group at the Technical University of Dortmund in Germany first reported the enzymatic synthesis of ω-dodecanolactam, using ω-dodecanolactam hydrolase to convert methyl ω-aminolaurate into ω-dodecanolactam. However, this reaction needs to be carried out under strong alkaline conditions, and there are serious side reactions, and the yield is only 13%, and the source problem of the substrate methyl ω-aminolaurate has not been fundamentally solved.

[0005] Therefore, in order to improve the biosynthesis efficiency of long-chain polyamide monomers and simplify the biological modification pathway, it is an urgent technical problem to provide a genetically engineered bacterium that can produce ω-amino fatty acids. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the resource and environmental problems generated in the chemical synthesis of long-chain polyamide monomers in the prior art, as well as the problems of complex gene modification and low production efficiency in the biological synthesis of long-chain polyamide monomers. Through metabolic engineering modification, a genetically engineered bacterium for producing ω-amino fatty acids and a method for producing ω-amino fatty acids using the same are provided. When using the genetically engineered bacterium of the present invention and the method of biological fermentation to produce ω-amino fatty acids, especially 12-aminolauric acid, the monomer yield is significantly increased, and it has strong industrial application prospects.

[0007] The term "ω-amino fatty acid" in the present invention may include all ω-amino fatty acids produced by microorganisms from many different types of carbon sources through metabolic processes. Specifically, the general molecular formula of the ω-amino fatty acid is CH2NH2(CH2) n COOH, where n is 7-16, for example, n = 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. More specifically, it includes ω-aminononanoic acid, ω-aminodecanoic acid, ω-aminoundecanoic acid, 12-aminolauric acid, ω-aminotridecanoic acid, ω-aminomyristic acid, ω-aminopentadecanoic acid or ω-aminopalmitic acid, but is not limited thereto.

[0008] The term "fatty aldehyde dehydrogenase" in the present invention may be abbreviated as FALDH; the term "fatty alcohol oxidase" may be abbreviated as FAO; the term "fatty alcohol dehydrogenase" may be abbreviated as ADH.

[0009] The first aspect of the present invention provides a genetically engineered bacterium for producing ω-amino fatty acids, the genetically engineered bacterium having reduced fatty aldehyde dehydrogenase activity and increased ω-transaminase activity, and the genetically engineered bacterium is selected from the genus Candida.

[0010] The "genetically engineered bacterium for producing ω-amino fatty acids" described in the present invention refers to a genetically engineered bacterium capable of producing ω-amino fatty acids, which can be a Candida strain with enhanced ω-amino fatty acid production ability by enhancing or inactivating the activity of genes involved in the ω-amino fatty acid production mechanism, or a Candida strain with enhanced ω-amino fatty acid production ability by introducing or enhancing the activity of foreign genes. The inventors found that by metabolically engineering Candida strains, activating the activity of ω-transaminase and simultaneously reducing the activity of fatty aldehyde dehydrogenase, the yield of ω-amino fatty acids produced by Candida strains was effectively increased. In the present invention, the genetically engineered bacterium maintains the β-oxidation metabolic pathway, that is, the β-oxidation metabolic pathway in the genetically engineered bacterium is not disrupted, and it does not lack the genes related to the β-oxidation metabolic pathway.

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

[0012] In some preferred embodiments, the genetically engineered bacterium is Candida viswanathii CATH2301, which has reduced fatty aldehyde dehydrogenase activity and increased ω-transaminase activity, and is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20232154.

[0013] In some embodiments, the amino acid sequence of the fatty aldehyde dehydrogenase is as shown in SEQ ID NO: 35, SEQ ID NO: 37 and / or SEQ ID NO: 39.

[0014] In some embodiments, the amino acid sequence of the fatty aldehyde dehydrogenase has at least 70% amino acid sequence identity or homology with any one of the sequences of SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher amino acid sequence identity or homology.

[0015] In some preferred embodiments, the nucleotide sequence of the encoding gene of the fatty aldehyde dehydrogenase is as shown in SEQ ID NO: 36, SEQ ID NO: 38, and / or SEQ ID NO: 40.

[0016] In some embodiments, the nucleotide sequence of the encoding gene of the fatty aldehyde dehydrogenase has at least 70% nucleotide sequence identity or homology with any one of the sequences of SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher nucleotide sequence identity or homology.

[0017] In some embodiments, the amino acid sequence of the ω-transaminase is as shown in SEQ ID NO: 43.

[0018] In some embodiments, the amino acid sequence of the ω-transaminase has at least 70% amino acid sequence identity or homology with SEQ ID NO: 43, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher amino acid sequence identity or homology.

[0019] In some preferred embodiments, the nucleotide sequence of the encoding gene of the ω-transaminase is as shown in SEQ ID NO: 13.

[0020] In some embodiments, the nucleotide sequence of the encoding gene of the ω-transaminase has at least 70% nucleotide sequence identity or homology with SEQ ID NO: 13, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher nucleotide sequence identity or homology.

[0021] In some preferred embodiments, the promoter sequence for expressing the ω-transaminase is as shown in SEQ ID NO: 18, SEQ ID NO: 41, or SEQ ID NO: 42.

[0022] In some preferred embodiments, the terminator sequence for expressing the ω-transaminase is as shown in SEQ ID NO: 21.

[0023] In some embodiments, in the genetically engineered bacterium, one or more fatty aldehyde dehydrogenase genes encoding endogenous fatty aldehyde dehydrogenase are inactivated, and a gene encoding exogenous ω-transaminase is introduced.

[0024] In some preferred embodiments, in the genetically engineered bacterium, the fatty aldehyde dehydrogenase with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37, and / or SEQ ID NO: 39 is inactivated, and the gene of the ω-transaminase with the amino acid sequence shown in SEQ ID NO: 43 is introduced.

[0025] In some embodiments, the reduced fatty aldehyde dehydrogenase activity or the inactivated fatty aldehyde dehydrogenase can be achieved by replacing the promoter of the fatty aldehyde dehydrogenase with a weak promoter, knocking out the promoter sequence of the fatty aldehyde dehydrogenase, knocking out part or all of the coding genes of the fatty aldehyde dehydrogenase, or inhibiting the expression of the fatty aldehyde dehydrogenase using the CRISPR-Cas9 tool or RNA interference technology. Specifically, for example, knocking out part or all of the coding genes in the coding genes of the fatty aldehyde dehydrogenase with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39.

[0026] In some embodiments, the ω-transaminase activity can be increased by overexpressing the ω-transaminase gene. Specifically, for example, introducing a plasmid carrying the gene encoding the ω-transaminase, and / or integrating the gene encoding the ω-transaminase into the genome of the genetically engineered bacterium.

[0027] As used in the present invention, "inactivated" means that the genetically engineered bacterium expresses less polypeptide than the corresponding strain without inactivating the enzyme, or the polypeptide of the enzyme is not expressed in the strain that usually expresses the enzyme; "overexpressed" means that the genetically engineered bacterium expresses more polypeptide than the corresponding strain without overexpressing the enzyme, or the polypeptide of the enzyme is expressed in the strain that usually does not express the enzyme, or overexpression is achieved by expressing a variant polypeptide with higher activity.

[0028] In some specific embodiments, in the genetically engineered bacterium, the genes of the fatty aldehyde dehydrogenase with the nucleotide sequences shown in SEQ ID NO: 36, SEQ ID NO: 38, and / or SEQ ID NO: 40 are inactivated, and the gene of the ω-transaminase with the nucleotide sequence shown in SEQ ID NO: 13 is introduced.

[0029] In some embodiments, in the genetically engineered bacterium, the gene encoding the ω-transaminase is inserted into the locus of any one or more genes encoding the fatty aldehyde dehydrogenase in the starting bacterium.

[0030] In some embodiments, in the genetically engineered bacterium, the gene encoding the ω-transaminase replaces any one gene encoding the fatty aldehyde dehydrogenase in the starting bacterium, and the remaining one or more genes encoding the fatty aldehyde dehydrogenase are knocked out.

[0031] In some preferred embodiments, the genetically engineered bacterium also has increased fatty alcohol oxidase activity and / or fatty alcohol dehydrogenase activity.

[0032] In some preferred embodiments, the genetically engineered bacterium also overexpresses one or two recombinant nucleotide sequences encoding the following polypeptides: a polypeptide having fatty alcohol oxidase activity, and a polypeptide having fatty alcohol dehydrogenase activity.

[0033] In some embodiments, the genetically engineered bacterium has reduced fatty aldehyde dehydrogenase activity, as well as increased ω-transaminase activity and fatty alcohol oxidase activity.

[0034] In some embodiments, the genetically engineered bacterium has reduced fatty aldehyde dehydrogenase activity, as well as increased ω-transaminase activity and fatty alcohol dehydrogenase activity.

[0035] In some embodiments, the genetically engineered bacterium has reduced fatty aldehyde dehydrogenase activity, as well as increased ω-transaminase activity, fatty alcohol oxidase activity and fatty alcohol dehydrogenase activity.

[0036] In some embodiments, the fatty alcohol oxidase activity or fatty alcohol dehydrogenase activity can be increased by overexpressing the fatty alcohol oxidase gene or fatty alcohol dehydrogenase gene respectively, for example, by introducing a plasmid carrying the gene encoding each enzyme, and / or integrating the gene encoding each enzyme into the genome of the engineered bacterium.

[0037] In some embodiments, the amino acid sequence of the fatty alcohol oxidase is as shown in SEQ ID NO:44.

[0038] In some embodiments, the amino acid sequence of the fatty alcohol oxidase has at least 70% amino acid sequence identity or homology with SEQ ID NO:44, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher amino acid sequence identity or homology.

[0039] In some preferred embodiments, in the genetically engineered bacterium, the fatty alcohol oxidase activity is increased by introducing the nucleotide sequence as shown in SEQ ID NO:34. Among them, the nucleotide sequence shown in SEQ ID NO:34 is used to express the fatty alcohol oxidase shown in SEQ ID NO:44.

[0040] In some embodiments, the amino acid sequence of the fatty alcohol dehydrogenase is as shown in SEQ ID NO: 45. In some embodiments, the amino acid sequence of the fatty alcohol dehydrogenase has at least 70% amino acid sequence identity or homology with SEQ ID NO: 45, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher amino acid sequence identity or homology.

[0041] In some preferred embodiments, in the genetically engineered bacterium, the activity of fatty alcohol dehydrogenase is increased by introducing the nucleotide sequence as shown in SEQ ID NO: 31. Among them, the nucleotide sequence shown in SEQ ID NO: 31 is used to express the fatty alcohol dehydrogenase shown in SEQ ID NO: 45.

[0042] In some embodiments, the starting bacterium of the genetically engineered bacterium is Candida viswanathii CAES2113, which is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M2020048.

[0043] In the present invention, using the genetically engineered bacterium described in the first aspect of the present invention to produce ω-amino fatty acid has an increased yield of ω-amino fatty acid.

[0044] The second aspect of the present invention provides a method for producing ω-amino fatty acid using the genetically engineered bacterium described in the first aspect of the present invention, which includes culturing the genetically engineered bacterium in a culture medium;

[0045] Optionally, separate, extract and / or purify ω-amino fatty acid from the genetically engineered bacterium or the culture medium to obtain a ω-amino fatty acid product.

[0046] Among them, the genetically engineered bacterium is selected from the genus Candida, preferably selected from one of Candida viswanathii, Candida tropicalis, Candida sake or Candida albicans. More preferably, the genetically engineered bacterium is selected from one of Candida viswanathii, Candida tropicalis or Candida sake.

[0047] 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 should meet the requirements of the specific strain in an appropriate manner.

[0048] In some embodiments, the temperature of the cultivation is 28 to 33 °C, such as 28 °C, 29 °C, 30 °C, 31 °C, 32 °C or 33 °C.

[0049] In some embodiments, the cultivation further includes adding an L-alanine solution during the fermentation process, for example, adding 100 - 300 μL of an L-alanine solution with a concentration of 100 - 200 g / L every 24 - 48 h during the fermentation process.

[0050] In some embodiments, the fermentation substrate for the cultivation is selected from at least one of fatty acids, fatty acid esters, and fatty acid salts.

[0051] In some 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.

[0052] In some embodiments, the fatty acid ester is selected from at least one of nonanoic acid ester, decanoic acid ester, undecanoic acid ester, lauric acid ester, tridecanoic acid ester, myristic acid ester, pentadecanoic acid ester, palmitic acid ester, heptadecanoic acid ester, and stearic acid ester.

[0053] In some embodiments, the fatty acid ester is selected from at least one of methyl fatty acid ester, ethyl fatty acid ester, propyl fatty acid ester, and butyl fatty acid ester. 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. Further preferably, the fatty acid ester is selected from at least one of methyl laurate, methyl myristate, and ethyl palmitate.

[0054] In some embodiments, the fatty acid salt is selected from at least one of the sodium salt, potassium salt, ammonium salt, and calcium salt of the fatty acid.

[0055] In some embodiments, when producing ω-aminodecanoic acid, the fermentation substrate for the cultivation is decanoic acid or a decanoic acid ester. The decanoic acid ester is selected from one or more of methyl decanoate, ethyl decanoate, propyl decanoate, and butyl decanoate, preferably methyl decanoate.

[0056] In some embodiments, when producing ω-aminoundecanoic acid, the fermentation substrate for the cultivation is undecanoic acid or an undecanoic acid ester. The undecanoic acid ester is selected from one or more of methyl undecanoate, ethyl undecanoate, propyl undecanoate, and butyl undecanoate, preferably methyl undecanoate.

[0057] In some embodiments, when producing 12 - aminolauric acid, the fermented substrate for cultivation is lauric acid or a laurate. The laurate is selected from one or more of methyl laurate, ethyl laurate, propyl laurate, and butyl laurate, preferably methyl laurate.

[0058] In some embodiments, when producing ω - aminotridecanoic acid, the fermented substrate for cultivation is tridecanoic acid or a tridecanoate. The tridecanoate is selected from one or more of methyl tridecanoate, ethyl tridecanoate, propyl tridecanoate, and butyl tridecanoate, preferably methyl tridecanoate.

[0059] In some embodiments, when producing ω - aminomyristic acid, the fermented substrate for cultivation is myristic acid or a myristate. The myristate is selected from one or more of methyl myristate, ethyl myristate, propyl myristate, and butyl myristate, preferably methyl myristate.

[0060] In some embodiments, when producing ω - aminopentadecanoic acid, the fermented substrate for cultivation is pentadecanoic acid or a pentadecanoate. The pentadecanoate is selected from one or more of methyl pentadecanoate, ethyl pentadecanoate, propyl pentadecanoate, and butyl pentadecanoate, preferably methyl pentadecanoate.

[0061] In some embodiments, when producing ω - aminopalmitic acid, the fermented substrate for cultivation is palmitic acid or a palmitate. The palmitate is selected from one or more of methyl palmitate, ethyl palmitate, propyl palmitate, and butyl palmitate, preferably methyl palmitate and butyl palmitate.

[0062] In some embodiments, the content of the cultivated substrate in the culture medium is 0.1 - 10% (volume percentage).

[0063] In some embodiments, when the fermented substrate is solid, it is first heated to dissolve it and then added to the fermentation broth; wherein, the temperature for heating the fermented substrate can be 40 - 100°C.

[0064] In some embodiments, the inoculation 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, such as 5:100, 10:100, 15:100, 20:100, 25:100, 30:100, 35:100, 40:100, 45:100, 50:100 or any value within this range.

[0065] In some embodiments, the culture medium contains a carbon source, a nitrogen source, and / or inorganic salts.

[0066] In some embodiments, the cultivation includes cultivating the genetically engineered bacterium successively in a seed culture medium and a fermentation culture medium, and the seed culture medium or the fermentation culture medium contains a carbon source, a nitrogen source, and / or inorganic salts.

[0067] In the present invention, the carbon source is selected from one or more of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, and preferably glucose, lactose, and sucrose.

[0068] In the present invention, the nitrogen source is selected from one or more of yeast extract, peptone, corn steep liquor, urea, ammonium salts, and nitrates.

[0069] In the present invention, the inorganic salts are selected from one or more of sulfates, hydrochlorides, nitrates, and phosphates; preferably, it includes one or more 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.

[0070] In some embodiments, the fermentation culture medium contains: 10 - 40 g / L of sucrose, 1 - 5 g / L of corn steep liquor, 1 - 12 g / L of yeast extract, 0 - 3 g / L of NaCl, 2 - 12 g / L of KNO3, 2 - 12 g / L of KH2PO4, and 0.5 - 5 g / L of urea.

[0071] The genetically engineered bacterium of the present invention can be used for fermentative production of ω-amino fatty acids and production of long carbon chain polyamides, such as polyamide 12.

[0072] On the basis of conforming to common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0073] The reagents and raw materials used in the present invention are all commercially available.

[0074] The positive and progressive effects of the present invention are as follows:

[0075] The genetically engineered bacterium of the Candida genus provided by the present invention, through metabolic engineering transformation, reduces the activity of fatty aldehyde dehydrogenase and increases the activity of ω-transaminase, enabling the strain to have the ability to produce ω-amino fatty acids, and the yield is significantly improved. Moreover, the yield is further increased by increasing the activity of fatty alcohol oxidase or fatty alcohol dehydrogenase. The genetically engineered bacterium provided by the present invention and the method for fermentatively producing ω-amino fatty acids using the genetically engineered bacterium have the potential for industrial scale-up production.

[0076] Strain preservation information:

[0077] The Candida viswanathii strain CATH2301 of the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2023. The deposit address is Wuhan University, Luojia Hill, Wuchang, Hubei Province, China, zip code: 430072, and the deposit number is CCTCC NO: M 20232154. The deposit date is November 8, 2023. The name of the culture is CATH2301, and the taxonomic name is Candida viswanathii. Detailed implementation manners

[0078] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples described herein. For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0079] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples described herein. For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0080] The strains and media used in the following examples are as follows:

[0081] I. Strains and vectors

[0082] Starting strain: Candida viswanathii CAES2113 (deposit number: CCTCC NO: M2020048, derived from patent CN 111748480A), and the starting strain maintains the β-oxidation metabolic pathway; plasmid vector pC31: prepared according to the Chinese invention patent application CN116218696A; TOP10 was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0083] II. Media for engineering bacteria

[0084] YPD medium (w / v): 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP0021). 1.5% agar powder needs to be added to the solid medium. 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. 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.

[0085] III. Product detection methods

[0086] The ω-amino 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.

[0087] Example 1 Construction of engineering bacterium CATH208 with reduced fatty aldehyde dehydrogenase activity

[0088] This was achieved by inactivating (knocking out) a partial FALDH-encoding gene of fatty aldehyde dehydrogenase.

[0089] 1. Construction of gene knockout plasmid

[0090] A mixed solution of oligonucleotide sequences SEQ ID NO:1 and SEQ ID NO:2 was prepared with a final concentration of 10 μM and annealed to 25 °C to form double-stranded DNA. The plasmid vector pC31 was digested with the restriction endonuclease PaqCI, and the vector backbone was recovered and purified. The annealed double-stranded DNA was diluted and ligated with the recovered and purified pC31 vector backbone using T4 DNA ligase (ThermFisher) at room temperature, and then transformed into TOP10 chemically competent cells. The next day, the transformants were picked and verified by colony PCR and sequencing. The constructed vector was pC31-partFALDH.

[0091] 2. Preparation of knockout template

[0092] Using oligonucleotides SEQ ID NO:3 and SEQ ID NO:4 as templates for each other, the template required for knocking out the partial FALDH was amplified, and the amplified PCR product was Donor1.

[0093] 3. Electroporation into yeast competent cells

[0094] (1) Preparation of yeast competent cells

[0095] A fresh single colony of Candida viswanathii CAES2113 was picked and inoculated into 2 ml of YPD medium, cultured at 30 °C and 200 rpm until the OD620 reached 1.3, and the cells were collected by centrifugation at 1500 g and 4 °C. The cells were washed twice with ice-cold sterile water, resuspended in 10 mL of 1 M sorbitol solution pre-cooled on ice, and after centrifugation at 4 °C and 1500 g to collect the cells, they were resuspended in 1 mL of the above sorbitol solution. 100 μL of the cell suspension was aliquoted for genetic transformation.

[0096] (2) Electroporation of yeast competent cells

[0097] 0.2 μg of pC31-partFALDH plasmid DNA and 1 μg of the recovered and purified Donor1 were added to the above-mentioned competent cells, gently mixed, placed on ice for 5 min, and then quickly transferred to a 0.2 cm electroporation cuvette for electroporation (BioRad, Micropulser TMAfter adding 1 mL of a mixture of YPD medium and 1 M sorbitol (1:1, v / v) after electroporation (1.5 - 2.4 kV, 4 - 5 ms), the bacterial solution was cultured at 30 °C with 200 rpm for 2 hours. After collecting the bacterial solution, it was spread on a YPD medium plate containing 100 mg / L hygromycin B and statically cultured at 30 °C until single colonies grew.

[0098] After the transformants were identified by colony PCR and verified by sequencing, they were inoculated into YPD liquid culture and cultured at 30 °C with 200 rpm for 48 h. One loop of bacteria was picked and streaked on a YPD solid plate and statically cultured at 30 °C for 3 days. Then, single colonies were picked and inoculated on YPD plates with and without antibiotics respectively. The clones that could not grow on the plate with antibiotics but could grow on the plate without antibiotics were the strains that had lost the plasmid, and this strain was named CATH208.

[0099] Example 2 Construction of engineering bacterium CATH320 with reduced fatty aldehyde dehydrogenase activity and increased ω - transaminase activity

[0100] It was achieved by inactivating (knocking out) a partial FALDH - encoding gene of fatty aldehyde dehydrogenase and overexpressing (introducing) the encoding gene of ω - transaminase.

[0101] 1. Construction of gene - editing plasmid

[0102] The construction method of the gene - editing plasmid for overexpressing ω - transaminase referred to Example 1, with the difference that the oligonucleotide sequences used were SEQ ID NO:5 and SEQ ID NO:6, and the constructed vector was pC31 - INTa.

[0103] 2. Preparation of homologous recombination template

[0104] Using primers SEQ ID NO:7 and SEQ ID NO:8, the genomic DNA of Candida viswanathii CAES2113 as the template to amplify the upstream homologous sequence SEQ ID NO:9 for integration. Using primers SEQ ID NO:10 and SEQ ID NO:11, the genomic DNA as the template to amplify the downstream homologous sequence SEQ ID NO:12 for integration.

[0105] The gene sequence encoding ω - transaminase is shown as SEQ ID NO:13, and the amino acid sequence is shown as SEQ ID NO:43. Using primers SEQ ID NO:14 and SEQ ID NO:15, the plasmid DNA carrying SEQ ID NO:13 as the template to amplify the nucleotide sequence encoding ω - transaminase, namely SEQ ID NO:13.

[0106] Using SEQ ID NO:16 and SEQ ID NO:17 as primers and genomic DNA as a template, the promoter sequence SEQ ID NO:18 for expressing ω-transaminase was amplified (other available promoters include SEQ ID NO:41, 42, etc.); using primers SEQ ID NO:19 and SEQ ID NO:20, the terminator sequence SEQ ID NO:21 for expressing ω-transaminase was amplified from genomic DNA.

[0107] The upstream and downstream homologous arms, promoter, terminator and the target gene were overlapped and extended by SOE-PCR as follows:

[0108] Using primers SEQ ID NO:5 and SEQ ID NO:11, and equimolar ratios of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:18 and SEQ ID NO:21 as templates, the template for homologous recombination was amplified. After being confirmed correct by sequencing, the sequence is as shown in SEQ ID NO:22.

[0109] 3. Construction of overexpression strain

[0110] The method for preparing electrocompetent yeast cells was the same as in Example 1, except that the starting strain used was CATH208, the plasmid was pC31-INTa, and the nucleotide sequence of the recombinant template was as shown in SEQ ID NO:22. After the transformants were identified by colony PCR and confirmed correct by sequencing, the plasmid was deleted, and the obtained genetically engineered strain was CATH320.

[0111] Example 3 Construction of engineered strain CATH2301 with reduced fatty aldehyde dehydrogenase activity and increased ω-transaminase activity

[0112] This was achieved by inactivating (knocking out) the FALDH-encoding gene of fatty aldehyde dehydrogenase and overexpressing (introducing) the encoding gene of ω-transaminase.

[0113] The process for constructing the genetically engineered strain referred to Example 1, with the differences being:

[0114] 1. The vector pC31-FALDH was constructed using the oligonucleotide sequences SEQ ID NO:23 and SEQ ID NO:24, as described in Example 2.

[0115] 2. Using oligonucleotides SEQ ID NO:25 and SEQ ID NO:26 as templates for each other, the template required for knocking out FALDH was amplified, and the amplified PCR product was Donor2.

[0116] 3. The competent cells used were CATH320 obtained in Example 2, the plasmid used was pC31-FALDH, and the knockout template DNA was Donor2. After the transformants were identified by colony PCR, sequencing confirmed that FALDH was knocked out and the plasmid was deleted. The constructed genetically engineered strain was CATH2301, which was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232154.

[0117] Example 4 Constructed engineered bacteria with increased alcohol dehydrogenase or alcohol oxidase activity based on CATH2301

[0118] This was achieved by overexpressing (introducing) the coding gene of alcohol dehydrogenase ADH or the gene of alcohol oxidase FAO.

[0119] 1. Construction of gene editing plasmids

[0120] The method for constructing the gene editing plasmids for overexpressing ADH and FAO was the same as that described in Example 1, except that the oligonucleotide sequences used were SEQ ID NO: 27 and SEQ ID NO: 28, and the constructed vector was pC31-INTz. The expression cassette sequence of the ADH gene was as shown in SEQ ID NO: 31, and the expression cassette sequence of FAO was as shown in SEQ ID NO: 34.

[0121] 2. Preparation of homologous recombination templates

[0122] Using primers SEQ ID NO: 29 and SEQ ID NO: 30, genomic DNA as the template to amplify the ADH expression cassette. After sequencing confirmation, the sequence was as shown in SEQ ID NO: 31.

[0123] Using primers SEQ ID NO: 32 and SEQ ID NO: 33, genomic DNA as the template to amplify the FAO expression cassette. After sequencing confirmation, the sequence was as shown in SEQ ID NO: 34.

[0124] 3. Construction of the strain overexpressing ADH

[0125] The method for preparing yeast electrotransformation competent cells was the same as that in Example 2, except that the starting strain used was CATH2301, the plasmid was pC31-INTz, and the recombinant template was SEQ ID NO: 31. After the transformants were identified by colony PCR, sequencing confirmed that they were correct and the plasmid was deleted. The obtained genetically engineered strain was CATH322.

[0126] 4. Construction of the strain overexpressing FAO

[0127] The method for preparing electrocompetent yeast cells was the same as in Example 2, except that the starting strain used was CATH2301, the plasmid was pC31-INTz, and the recombinant template was SEQ ID NO:34. After the transformants were identified by colony PCR, sequencing confirmed that they were correct and the plasmid was deleted, and the obtained genetically engineered strain was CATH323.

[0128] Example 5 Laboratory shake-flask fermentation for the production of ω-aminodecanoic acid

[0129] The strains Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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%, and the shaker speed was 250 rpm and the temperature was 30 °C. When the culture reached OD620 of 0.8 (after dilution 30 times) after 36 - 48 h. The seed liquid was inoculated into a shake flask containing 15 mL of fermentation medium, with an inoculation amount of 20%. The substrate in the fermentation medium was 0.5 mL of methyl decanoate. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, 150 μL of L-alanine solution with a concentration of 100 g / L was added every 24 h. After the fermentation was completed, the concentration of the target product ω-aminodecanoic acid was detected and calculated by high performance gas chromatography.

[0130] The genetic modifications in the above examples did not involve the modification of the β-oxidation metabolic pathway. Therefore, the obtained genetically engineered strains CATH208, CATH320, CATH2301, CATH322, and CATH323 all maintained the β-oxidation metabolic pathway. The results of their product concentrations are shown in Table 1. In the engineering strain of Example 2 in which part of the FALDH gene was knocked out and ω-transaminase was overexpressed, when methyl decanoate was used as the substrate, the accumulation of the target product ω-aminodecanoic acid was detected in the shake-flask fermentation, and the product concentration was 80 mg / L. On this basis, after further knocking out FALDH, as shown in Example 3, the concentration of ω-aminodecanoic acid increased significantly to 1.29 g / L, indicating that knocking out the FALDH gene is more helpful for improving the product yield than knocking out part of the FALDH gene. As shown in Example 4, for the engineering bacteria in which alcohol dehydrogenase ADH or alcohol oxidase FAO was further overexpressed, the concentrations of the target products all increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0131] Table 1 Genetically engineered strains constructed in the present invention and their yields of ω-aminodecanoic acid

[0132]

[0133] Example 6 Laboratory shake-flask fermentation for the production of ω-aminoundecanoic acid

[0134] The strains Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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 solution was inoculated into a 500 mL shake flask containing 30 mL of seed medium, with an inoculation amount of 3%, and the shaker speed was 250 rpm and the temperature was 30 °C. It was cultured for 36 - 48 h until the OD 620 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 substrate in the fermentation medium was 0.5 mL of methyl undecanoate. It was continuously cultured on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, 150 μL of L-alanine solution with a concentration of 100 g / L was added every 24 h. After the fermentation was completed, high performance gas chromatography was used to detect and calculate the concentration of the target product ω-aminoundecanoic acid.

[0135] The results of the product concentration are shown in Table 2. Using methyl undecanoate as the substrate, the accumulation of the target product ω-aminoundecanoic acid was detected in the shake flask fermentation, and the product concentration was 120 mg / L. On this basis, after further knocking out FALDH, the concentration of ω-aminoundecanoic acid increased significantly to 1.59 g / L. For the engineered bacteria with further overexpression of alcohol dehydrogenase ADH or alcohol oxidase FAO, the concentration of the target product all increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0136] Table 2 The genetically engineered strains constructed in the present invention and the yields of their ω-aminoundecanoic acid

[0137]

[0138]

[0139] Example 7 Laboratory shake flask fermentation for the production of 12-aminolauric acid

[0140] The strains Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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 solution was inoculated into a 500 mL shake flask containing 30 mL of seed medium, with an inoculation amount of 3%, and the shaker speed was 250 rpm and the temperature was 30 °C. It was cultured for 36 - 48 h until the OD 620When it reaches 0.8 (after dilution by 30 times), the seed liquid is inoculated into a shake flask containing 15 mL of fermentation medium, and the inoculation amount is 20%. The substrate in the fermentation medium is 0.5 mL of methyl laurate. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, 150 μL of L-alanine solution with a concentration of 100 g / L is supplemented every 24 h. After the fermentation is completed, high-performance gas chromatography is used to detect and calculate the concentration of the target product 12-aminolauric acid.

[0141] The results of the product concentration are shown in Table 3. Using methyl laurate as the substrate, the accumulation of the target product 12-aminolauric acid was detected in shake flask fermentation, and the product concentration was 280 mg / L. On this basis, after further knocking out FALDH, the concentration of 12-aminolauric acid increased significantly to 3.59 g / L, indicating that knocking out the FALDH gene is more helpful for improving the product yield than knocking out some FALDH genes. For the engineered bacteria with further overexpression of alcohol dehydrogenase ADH or alcohol oxidase FAO, the concentrations of the target products all increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0142] Table 3 Genetically engineered strains constructed in the present invention and their yields of 12-aminolauric acid

[0143]

[0144]

[0145] Example 8 Laboratory shake flask fermentation for the production of ω-aminotridecanoic acid

[0146] Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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. Take the above bacterial liquid and inoculate it into a 500 mL shake flask containing 30 mL of seed medium, with an inoculation amount of 3%, and the shaker speed is 250 rpm and 30 °C. Culture for 36 - 48 h until OD 620 When it reaches 0.8 (after dilution by 30 times), the seed liquid is inoculated into a shake flask containing 15 mL of fermentation medium, and the inoculation amount is 20%. The substrate in the fermentation medium is 0.5 ml of methyl tridecanoate. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, 150 μL of L-alanine solution with a concentration of 100 g / L is supplemented every 24 h. After the fermentation is completed, high-performance gas chromatography is used to detect and calculate the concentration of the target product ω-aminotridecanoic acid.

[0147] The results of the product concentration are shown in Table 4. Using methyl tridecanoate as the substrate, the accumulation of the target product ω-aminotridecanoic acid was detected in shake flask fermentation, and the product concentration was 550 mg / L. On this basis, after further knocking out FALDH, the concentration of ω-aminotridecanoic acid increased significantly to 4.68 g / L. For the engineered strains with further overexpression of alcohol dehydrogenase ADH or fatty alcohol oxidase FAO, the concentrations of the target products all increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0148] Table 4 Genetically engineered strains constructed in the present invention and their yields of ω-aminotridecanoic acid

[0149]

[0150]

[0151] Example 9 Laboratory shake flask fermentation for the production of ω-aminomyristic acid

[0152] Respectively inoculate the strains Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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 liquid and inoculate it into a 500 mL shake flask 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 OD 620 reaches 0.8 (after dilution 30 times). Inoculate the seed liquid into a shake flask containing 15 mL of fermentation medium, with an inoculation amount of 20%. The substrate in the fermentation medium is 0.5 mL of methyl myristate. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, add 150 μL of L-alanine solution with a concentration of 100 g / L every 24 h. After the fermentation is completed, use high performance gas chromatography to detect and calculate the concentration of the target product ω-aminomyristic acid.

[0153] The results of the product concentration are shown in Table 5. Using methyl myristate as the substrate, the accumulation of the target product ω-aminomyristic acid was detected in shake flask fermentation, and the product concentration was 451 mg / L. On this basis, after further knocking out FALDH, the concentration of ω-aminomyristic acid increased significantly to 4.32 g / L. For the engineered strains with further overexpression of alcohol dehydrogenase ADH or fatty alcohol oxidase FAO, the concentrations of the target products all increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0154] Table 5 Genetically engineered strains constructed in the present invention and their yields of ω-aminomyristic acid

[0155]

[0156] Example 10 Laboratory shake-flask fermentation for the production of ω-aminopentadecanoic acid

[0157] Respectively inoculate the strains Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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 liquid and inoculate it into a 500 mL shake flask containing 30 mL of seed medium, with an inoculation amount of 3%. The shaker speed is 250 rpm and the temperature is 30 °C. Culture for 36 - 48 h until the OD 620 reaches 0.8 (after dilution 30 times). Inoculate the seed liquid into a shake flask containing 15 mL of fermentation medium, with an inoculation amount of 20%. The substrate in the fermentation medium is 0.5 mL of methyl pentadecanoate. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, add 150 μL of L-alanine solution with a concentration of 100 g / L every 24 h. After the fermentation is completed, use high-performance gas chromatography to detect and calculate the concentration of the target product ω-aminopentadecanoic acid.

[0158] The results of its product concentration are shown in Table 6. Using methyl pentadecanoate as the substrate, the accumulation of the target product ω-aminopentadecanoic acid was detected in shake-flask fermentation, and the product concentration was 150 mg / L. On this basis, after further knocking out FALDH, the concentration of ω-aminopentadecanoic acid increased significantly to 1.32 g / L. For the engineered bacteria with further overexpression of alcohol dehydrogenase ADH or fatty alcohol oxidase FAO, the concentration of the target product increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0159] Table 6 Genetically engineered strains constructed in the present invention and their yields of ω-aminopentadecanoic acid

[0160]

[0161] Example 11 Laboratory shake-flask fermentation for the production of ω-aminopalmitic acid

[0162] Respectively inoculate the strains Candida viswanathii CAES2113, CATH208, CATH320, CATH2301, CATH322, and CATH323 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 liquid and inoculate it into a 500 mL shake flask containing 30 mL of seed medium, with an inoculation amount of 3%. The shaker speed is 250 rpm and the temperature is 30 °C. Culture for 36 - 48 h until the OD 620When it reaches 0.8 (after dilution 30 times), the seed liquid is inoculated into a shake flask containing 15 mL of fermentation medium, and the inoculation amount is 20%. The substrate in the fermentation medium is 0.5 mL of butyl palmitate. Continue to culture on a shaker at 250 rpm and 30 °C for 136 h, with the pH natural. During the fermentation process, 150 μL of L-alanine solution with a concentration of 100 g / L is supplemented every 24 h. After the fermentation is completed, high-performance gas chromatography is used to detect and calculate the concentration of the target product ω-aminopalmitic acid.

[0163] The results of its product concentration are shown in Table 7. Using butyl palmitate as the substrate, the accumulation of the target product ω-aminopalmitic acid was detected in shake flask fermentation, and the product concentration was 80 mg / L. On this basis, after further knocking out FALDH, the concentration of ω-aminopalmitic acid increased significantly to 0.99 g / L. For the engineered bacteria with further overexpression of alcohol dehydrogenase ADH or alcohol oxidase FAO, the concentration of the target product increased to varying degrees, indicating that further modification is beneficial to improving the product yield.

[0164] Table 7 Genetically engineered strains constructed in the present invention and their yields of ω-aminopalmitic acid

[0165]

[0166] SEQ ID NO:1

[0167] TACCaagatctcgcctatcgtaaa

[0168] SEQ ID NO:2

[0169] AAACtttacgataggcgagatctt

[0170] SEQ ID NO:3

[0171] atacaagttgccaaaatcatctaagatctcgcctatcgtTAGCTAACTGCAGCTAATCA

[0172] SEQ ID NO:4

[0173] tttgagctactttttgattttgccgaggtcttaccctttTGATTAGCTGCAGTTAGCTA

[0174] SEQ ID NO:5

[0175] TACCACCCAAAAGATAGTTACAAA

[0176] SEQ ID NO:6

[0177] AAACTTTGTAACTATCTTTTGGGT

[0178] SEQ ID NO:7

[0179] tacttggtacaaactggaaaagcatatacaaaatgaag

[0180] SEQ ID NO:8

[0181] tatcttttgggtttcgcccgc

[0182] SEQ ID NO:9

[0183] tacttggtacaaactggaaaagcatatacaaaatgaagaaaaagagaacaattgcacaaagcagaacaatcaacgactgctaccccttttataccgcgtttcctgtttactatcattttactcgcttaagttcgacgcaaatcagacgaaaatcccaacttcccccaactcccccttcaattcacgtcaaaccaccacaaacaggggaaccatctcctgatggcccatgtcggagaaaccaaaaaaaatcaatccgacagcggcaggattcgaacctgcgcgggcgaaacccaaaagata

[0184] SEQ ID NO:10

[0185] CTAATCTTTCTCTTTAACCACTCAGACACG

[0186] SEQ ID NO:11

[0187] gttgccttctgtgcatgtttagag

[0188] SEQ ID NO:12

[0189] ctaatctttctctttaaccactcagacacgctgcctcattcgtggaaatttatccttactgaagtgaccaccgaagtctggtagaatctaccaaccagcatcacacgcgtcaacatcacgtcagtaacaactcacatttcctctctgtgtttatattatcttattgcttactttctagcgtgtgttgttaaggttttcggtagtatacaggggtcttctacattcagtttgtcacgtgatcagcctctcgaaatcctcgtaatcaacctgtaaatcctctaaacatgcacagaaggcaac

[0190] SEQ ID NO:13

[0191]

[0192] SEQ ID NO:14

[0193] ATGCAAAAACAAAGAACCACGTCC

[0194] SEQ ID NO:15

[0195] ctaagccaacccacgagc

[0196] SEQ ID NO:16

[0197] ctgcgcgggcgaaacccaaaagataCTCGCAACACAAGGCTAACG

[0198] SEQ ID NO:17

[0199] gggacgtggttctttgtttttgcatggtcgtgaggtgtgtg

[0200] SEQ ID NO:18

[0201]

[0202] SEQ ID NO:19

[0203] cctcaaggctcgtgggttggcttaGAGGATCATGTGTTATTTTTGATTGGTTTAGTC

[0204] SEQ ID NO:20

[0205] ctgagtggttaaagagaaagattagcaggttgcaaaaaccacaatcac

[0206] SEQ ID NO:21

[0207] cctcaaggctcgtgggttggcttaGaggatcatgtgttatttttgattggtttagtctgtttgtagctattgattaggttaattcacggattgttatttattgatagggggtgcgtgtgtgtgtgtgtgttgcattcacatgggatcgttccaggttgttgtttccttccatcctgttgagtcaaaaggagttttgttttgtaactccggacgatgtcttagatagaaggtcgatctccatgtgattgtttgactgctactctgattatgtaatctgtaaagcctagacgttatgcaagcatgtgattgtggtttttgcaacctgctaatctttctctttaaccactcag

[0208] SEQ ID NO:22

[0209]

[0210] SEQ ID NO:23

[0211] taccTAACCTTTACTTTGCGGTCC

[0212] SEQ ID NO:24

[0213] AAACggaccgcaaagtaaaggtta

[0214] SEQ ID NO:25

[0215] agttccgcttgaaccaattgcgtaacctttactttgcggTAGCTAACTGCAGCTAATCA

[0216] SEQ ID NO:26

[0217] tgtccaaggcagcacagagcgcgtcggcattgtcctggaTGATTAGCTGCAGTTAGCTA

[0218] SEQ ID NO:27

[0219] TACCgaattggaaagtgctagatg

[0220] SEQ ID NO:28

[0221] AAACcatctagcactttccaattc

[0222] SEQ ID NO:29

[0223] gtacattaattgaaacaagtggtcatcaattcaatgatccCCATTGGCTACTCGGTCG

[0224] SEQ ID NO:30

[0225] ttcctatttggagtagaccttgaaccatttactcactatgGGcggaatcaagtggctttac

[0226] SEQ ID NO:31

[0227]

[0228] SEQ ID NO:32

[0229] gtacattaattgaaacaagtggtcatcaattcaatgatccGGCTATCATCAAGGCTACCG

[0230] SEQ ID NO:33

[0231] ttcctatttggagtagaccttgaaccatttactcactatgGGCAAACTAGCCAAGGATGT

[0232] SEQ ID NO:34

[0233]

[0234] SEQ ID NO:35

[0235] >FALDH1_prot Protein sequence 1

[0236] MPKGVVNIVPGFGATAGSAIASHPRIDKVAFTGSTATGKIIMKLAAESNLKKVTLELGGKSPNIVFNDADLDKTIGNLVVSIFYNSGEVCCAGSRLLVQSEIYDDVVARFKKAAETIKVGDPFNEETFMGAQANENQLSKILKYIEQGKEQGATVVTGGARANDKGYFIKPTIFADVNRDMSIVKEEIFGPVVTLIKFDTVEEAIALANDSDYGLAAGIHSTNVNKCIDVANRLKAGTVWINTYNDFHPMVPFGGFSASGIGREMGEEVLKEYTQVRAVRMKLNPLP

[0237] SEQ ID NO:36

[0238] >FALDH1_nucl Nucleotide sequence 1

[0239] ATGCCAAAGGGTGTCGTCAACATTGTCCCAGGTTTTGGTGCTACCGCTGGTTCTGCCATTGCTAGTCACCCAAGGATCGACAAGGTTGCATTCACTGGATCTACTGCTACTGGTAAAATCATCATGAAATTGGCTGCTGAatcgaacttgaagaaggtcaCCTTGGAATTGGGAGGTAAGTCTCCAAACATTGTTTTCAACGACgctgacttggacaagactATTGGCAACTTGGTTGTTTCGATCTTCTACAACTCTGGTGAAGTCTGCTGTGCTGGTTCTCGTCTCTTGGTTCAATCTGAGATCTACGACGATGTTGTTGCTAGATTCAAGAAGGCCGCTGAAACTATCAAGGTCGGTGATCCATTTAACGAAGAAACTTTCATGGGTGCTCAAGCTAACGAAAAccaattgtccaagattttGAAGTACATTGAACAAGGTAAGGAACAAGGTGCTACTGTTGTTACTGGTGGTGCTAGGGCCAACGACAAGGGATACTTCATCAAGCCAACTATTTTTGCTGACGTCAACAGAGACATGAGCAttgtcaaggaagaaatTTTCGGCCCTGTTGttactttgatcaagttcgaCACCGTTGAAGAAGCTATCGCTTTGGCCAACGACTCCGACTATGGTTTGGCTGCTGGTATCcactccaccaacgtcaacaagtGTATTGATGTTGCCAACAGACTCAAGGCTGGTACTGTCTGGATCAACACTTACAATGACTTCCACCCAATGGTTCCATTTGGTGGTTTCAGTGCCTCTGGTATCGGTAGAGAAATGGGTGAAGAAGTCTTGAAGGAATACACTCAAGTCAGAGCTGTCAGAATGAAGCTCAACCCTCTCCCATAA

[0240] SEQ ID NO:37

[0241] >FALDH2_prot Protein sequence 2

[0242] MSPPSKLEDSSSATTAADTLGDSWYTKVSDIAPGVQRLTESFHRDQKTHDIQFRLNQLRNLYFAVQDNADALCAALDKDFYRPPSETKNLELVGGLNELVHTISSLHEWMKPEKVTDLPLTLRSNPIYIERIPLGVVLIISPFNYPFFLSFSAVVGAIAGGNAVVLKGSELTPNFSSLFSKILTKALDPDIFFAVDGAIPETTELLEQKFDKIMYTGNNTVGKIIAKKAAETLTPVILELGGKSPAFILDDVKDKNLEVIARRIAWGRFTNAGQTCVAVDYVLVPTKLHKKFIAALTKVLSQEFYPNLTKDTKGYTHVIHDRAFNNLSKIISTTKGDIVFGGDTDAATRFIAPTVIDNATWEDSSMKGEIFGPILPVLTYDKLTTAIRQVVSTHDTPLAQYIFTSGSTSRKYNRQLDQILTGVRSGGVIVNDVLMHVALINAPFGGVGDSGYGSYHGKFSFRSFTHERTTMEQKLWNDGMVKVRYPPYNSNKDKLIQVSQQNYNGKVWFDRNGDVPVNGPGALFSAWTTFTGVFHLLGEFITNKQ

[0243] SEQ ID NO:38

[0244] >FALDH_2_nucl Nucleotide sequence 2

[0245]

[0246] SEQ ID NO:39

[0247] >FALDH3_prot Protein sequence 3

[0248] MSPPSKLEDSSSSNTAADTLGDSWYTKVSDIAPGVQRLTESFHRDQKTHDIQFRLNQLRNLYFAVQDNADALCAALDKDFYRPPSETKNLELVGGLIELVHTMSSLHEWMKPEKVTDLPLTLKSNPIYIERIPLGVVLIISPFNYPFFLSFSAVVGAIAGGNAVVLKGSELTPNFSSLFTKILTKALDPDIFFAVDGGIPETTELLEQKFDKIMYTGNNTVGKIVAKKAAETLTPVILELGGKSPAFILDDVKDNDLEVIARRIAWGRFTNAGQTCVAVDYVLVPSKLHKKFIDALTKVLSQEFYPNLTKDTKGYTHVIHDRAFNNLSKIISTTKGDIVFGGETDAATRFIAPTVIDNATWEDSSMKGEIFGPILPVLTYDKLTTAIRQVVSTHDTPLAQYIFTSGSTSRKYNRQLDQILTGVRSGGVIVNDVLMHVALINAPFGGVGDSGYGSYHGKFSFRSFTHERTTMEQKLWNDGLVKVRYPPYNSNKDKLIQVSQQNYNGKVWFDRKGDVPVRGPGALFSAWTTFTGVFHLLGEFITNKQ

[0249] SEQ ID NO:40

[0250] >FALDH3_nucl Nucleotide sequence 3

[0251]

[0252] SEQ ID NO:41

[0253] >promoter 2 of transaminase 2 nucleotide sequence

[0254]

[0255] SEQ ID NO:42

[0256] >promoter 3 nucleotide sequence of prom3 aminotransferase

[0257] Tgacaagatctcgcaacacaaggctaacgcctggttgttgaacaccggttgggttggttcttctgctgctagaggtggtaagagatgctcattgaagtacaccagagccattttggacgctatccactctggtgaattgtccaaggttgaatacgaaactttcccagtcttcaacttgaatgtcccaacctcctgtccaggtgtcccaagtgaaatcttgaacccaaccaaggcctggaccggaaggtgttgactccttcaacaaggaaatcaagtctttggctggtaagtttgctgaaaacttcaagacctatgctgaccaagctaccgctgaagtgagagctgcaggtccagaagcttaaagatatttattcattatttagtttgcctatttatttctcattacccatcatcattcaacactatatataaagttacttcggatatcattgtaatcgtgcgtgtcgcaattggatgatttggaactgcgcttgaaacggattcatgcacgaagcggagataaaagattacgtaatttatctcctgagacaattttagccgtgttcacacgcccttctttgttctgagcgaaggataaataattagacttccacagctcattctaatttccgtcacgcgaatattgaaggggggtacatgtggccgctgaatgtgggggcagtaaacgcagtctctcctctcccaggaatagtgcaacggaggaaggataacggatagaaagcggaatgcgaggaaaattttgaacgcgcaagaaaagcaatatccgggctaccaggttttgagccagggaacacactcctatttctgctcaatgactgaacatagaaaaaacaccaagacgcaatgaaacgcacatggacatttagacctccccacatgtgatagtttgtcttaacagaaaagtataataagaacccatgccgtcccttttctttcgccgcttcaacttttttttttttatcttacacacatcacgacc。

[0258] SEQ ID NO:43

[0259] MQKQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFFKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMVQAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRACGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA

[0260] SEQ ID NO:44

[0261] MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLGKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLTNDGFKSLVLEKGRYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL

[0262] SEQ ID NO:45

[0263] MSVPTTQKAVIFETNGGKLEYKDVPVPVPKPNELLVNVKYSGVCHSDLHVWKGDWPIPAKLPLVGGHEGAGVVVGMGDNVKGWKVGDLAGIKWLNGSCMNCEFCQQGAEPNCSRADMSGYTHDGTFQQYATADAVQAAKIPEGADMASIAPILCAGVTVYKALKNADLLAGQWVAISGAGGGLGSLGVQYAKAMGYRVLAIDGGDERGEFVKSLGAEVYIDFLKEQDIVSAIRKATGGGPHGVINVSVSEKAINQSVEYVRTLGKVVLVSLPAGGKLTAPLFESVARSIQIRTTCVGNRKDTTEAIDFFVRGLIDCPIKVAGLSEVPEIFDLMEQGKILGRYVVDTSK

[0264] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples. Without departing from the principle and essence of the present invention, various changes or modifications can be made to these embodiments. Therefore, the protection scope of the present invention is defined by the appended claims.

Claims

1. A genetically engineered bacterium for producing ω-amino fatty acids, characterized in that: The genetically engineered bacteria has reduced fatty aldehyde dehydrogenase activity and increased ω-aminotransferase activity, and the genetically engineered bacteria is selected from the genus Candida.

2. The genetically engineered bacterium according to claim 1, characterized in that The genetically engineered bacteria is selected from one of Candida viswanathii, Candida tropicalis, Candida sake or Candida albicans; the genetically engineered bacteria is preferably Candida viswanathii CATH2301, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20232154; and / or The molecular formula of the ω-amino fatty acid is CH2NH2(CH2) n COOH, wherein n is 7 to 16, and the ω-amino fatty acid preferably includes ω-aminononanoic acid, ω-aminodecanoic acid, ω-aminoundecanoic acid, 12-aminolauric acid, ω-aminotridecanoic acid, ω-aminomyristic acid, ω-aminopentadecanoic acid or ω-aminopalmitic acid.

3. The genetically engineered bacterium according to claim 1, characterized in that The amino acid sequence of the fatty aldehyde dehydrogenase is shown in SEQ ID NO:35, SEQ ID NO:37 and / or SEQ ID NO:39; Preferably, the nucleotide sequence of the gene encoding the fatty aldehyde dehydrogenase is shown in SEQ ID NO:36, SEQ ID NO:38 and / or SEQ ID NO:

40.

4. The genetically engineered bacterium according to claim 1, characterized in that The amino acid sequence of the ω-aminotransferase is shown in SEQ ID NO:43; Preferably, the nucleotide sequence of the gene encoding the ω-transaminase is shown as SEQ ID NO: 13; and / or, the promoter sequence used to express the ω-transaminase is shown as SEQ ID NO: 18, SEQ ID NO: 41 or SEQ ID NO: 42; and / or, the terminator sequence used to express the ω-transaminase is shown as SEQ ID NO:

21.

5. The genetically engineered bacterium according to claim 1, characterized in that One or more fatty aldehyde dehydrogenase genes encoding endogenous fatty aldehyde dehydrogenase are inactivated, and a gene encoding an exogenous ω-aminotransferase is introduced; Preferably, in the genetically engineered bacteria, the fatty aldehyde dehydrogenase having an amino acid sequence as shown in SEQ ID NO:35, SEQ ID NO:37 and / or SEQ ID NO:39 is inactivated, and a gene encoding an ω-aminotransferase having an amino acid sequence as shown in SEQ ID NO:43 is introduced.

6. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The genetically engineered bacteria also have increased fatty alcohol oxidase activity and / or fatty alcohol dehydrogenase activity; Preferably, the genetically engineered bacteria also overexpress one or two recombinant nucleotide sequences encoding the following polypeptides: a polypeptide having fatty alcohol oxidase activity, and a polypeptide having fatty alcohol dehydrogenase activity.

7. The genetically engineered bacterium according to claim 6, characterized in that The amino acid sequence of the fatty alcohol oxidase is shown in SEQ ID NO:

44.

8. The genetically engineered bacterium according to claim 6, characterized in that The amino acid sequence of the fatty alcohol dehydrogenase is shown in SEQ ID NO:

45.

9. A method for producing ω-amino fatty acids, characterized in that Cultivating the genetically engineered bacteria according to any one of claims 1 to 8 in a culture medium; Optionally, ω-amino fatty acids are separated, extracted and / or purified from the genetically engineered bacteria or culture medium to obtain ω-amino fatty acid products.

10. The method according to claim 9, characterized in that The culture satisfies one or both of the following conditions: (1) The culture temperature is 28 to 33° C.; (2) the fermentation substrate of the culture is selected from at least one of fatty acids, fatty acid esters, and fatty acid salts; Preferably, the fatty acid is selected from at least one of nonadecanoic acid, deadecanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid and octadecanoic acid; The fatty acid ester is selected from at least one of nonadecanoate, decadecanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, heptadecanoate and octadecanoate; for example, 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; or The fatty acid salt is selected from at least one of sodium salt, potassium salt, ammonium salt and calcium salt of fatty acid.

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