Search

CN-122012636-A - Method for biosynthesis of omega-hydroxy fatty acid and genetically engineered bacterium

CN122012636ACN 122012636 ACN122012636 ACN 122012636ACN-122012636-A

Abstract

The invention provides a method for biosynthesis of omega-hydroxy fatty acid and genetically engineered bacteria. The method comprises the step of carrying out catalytic reaction on fatty acid by using P450 hydroxylase to obtain omega-hydroxy fatty acid, wherein the P450 hydroxylase has any one of the following amino acid sequences of SEQ ID NO. 6, SEQ ID NOs 12-14, SEQ ID NO. 22, SEQ ID NOs 26-27, SEQ ID NOs 30-32 or SEQ ID NO. 37. The method has the advantages of high catalytic efficiency and high regioselectivity, has the characteristics of green and environment-friendly performance, meets the requirement of sustainable development, has wide application prospect and has higher economic value.

Inventors

  • ZHANG NA
  • LI RUI
  • LI MINGJI
  • ZHAO YING
  • CUI YI
  • YIN SHILE
  • ZHAO WANYING
  • LIU SIMIN
  • Hao Ruxin
  • YANG WENYI

Assignees

  • 天津凯莱英生物科技有限公司
  • 凯莱英生命科学技术(天津)有限公司
  • 辽宁凯莱英医药化学有限公司

Dates

Publication Date
20260512
Application Date
20260414

Claims (20)

  1. 1. A method of biosynthesizing omega-hydroxy fatty acids, the method comprising: catalyzing the fatty acid by using P450 hydroxylase to obtain omega-hydroxy fatty acid; The P450 hydroxylase has any one of the following amino acid sequences of SEQ ID NO 6, SEQ ID NOs 12-14, SEQ ID NO 22, SEQ ID NOs 26-27, SEQ ID NOs 30-32 or SEQ ID NO 37.
  2. 2. The method of claim 1, wherein the catalytic reaction further comprises a redox partner.
  3. 3. The method of claim 2, wherein the fatty acid is selected from any of a C12 saturated fatty acid, a C14 saturated fatty acid, a C16 monounsaturated fatty acid, and a C18 monounsaturated fatty acid.
  4. 4. A method according to claim 3, wherein the redox partner is selected from any one of: A reductase domain BMR derived from bacillus megatherium P450 enzyme CYP102 A1; a reductase domain RhFRed derived from rhodococcus P450 enzyme CYP116B 2; Redox partners CamA and CamB derived from pseudomonas putida; redox partners PetH and PetF derived from synechocystis; Or reductase AtATR2 derived from type II P450 of Arabidopsis thaliana.
  5. 5. The method of claim 4, wherein the amino acid sequence of BMR is shown as SEQ ID NO. 44, the amino acid sequence of RhFRed is shown as SEQ ID NO. 45, the amino acid sequence of CamA is shown as SEQ ID NO. 46, the amino acid sequence of CamB is shown as SEQ ID NO. 47, the amino acid sequence of PetH is shown as SEQ ID NO. 48, the amino acid sequence of PetF is shown as SEQ ID NO. 49, and the amino acid sequence of AtATR2 is shown as SEQ ID NO. 50.
  6. 6. The method according to claim 5, characterized in that the method comprises: Mixing thalli, the fatty acid and a carbon source and carrying out hydroxylation reaction to obtain omega-hydroxy fatty acid; wherein the bacterial body is capable of expressing the P450 hydroxylase and the redox partner.
  7. 7. The method according to claim 6, wherein the method for producing the bacterial cells comprises: Inoculating the first recombinant cells cultured overnight into a first culture medium for first expansion culture until the OD600 is 0.6-0.8, adding an inducer and a cofactor, inducing expression for 24-48 h, centrifuging and collecting precipitate to obtain the thalli; wherein the first recombinant cell is capable of expressing the P450 hydroxylase and the redox partner.
  8. 8. The method of claim 7, wherein the carbon source comprises glucose and glycerol, wherein the final concentration of glucose is 0.2g/L to 0.5g/L, and wherein the final concentration of glycerol is 5g/L to 10g/L.
  9. 9. The method of claim 8, wherein the final concentration of fatty acid is 0.5mM to 1mM.
  10. 10. The method of claim 8, wherein the first medium is LB medium.
  11. 11. The method of claim 10, wherein the conditions of the first expansion culture are 30 ℃ to 37 ℃ and 200rpm to 220rpm.
  12. 12. The method of claim 11, wherein the conditions for inducing expression are 28 ℃ to 30 ℃ and 200rpm to 220rpm.
  13. 13. The method according to claim 5, characterized in that the method comprises: Inoculating the second recombinant cells cultured overnight into a second culture medium for second expansion culture until the OD600 is 0.6-0.8, adding an inducer and a cofactor, and performing induced fermentation for 48-72 h to obtain omega-hydroxy fatty acid; wherein the second recombinant cell is capable of expressing the P450 hydroxylase, the redox partner, and a thioesterase.
  14. 14. The method according to claim 13, wherein the second medium is formulated of ammonium chloride 4-6g/L, potassium dihydrogen phosphate 5-10g/L, citric acid 0.3-0.5g/L, yeast extract 3-5g/L, glycerol 15-30g/L, magnesium sulfate 0.5-1 g/L, calcium chloride 0.05-0.07 g/L, metal trace element stock solution 4 mL/L, thiamine 50-100 mg/L, MOPS 40-50 g/L, and antibiotics 50 mg/L-100 mg/L.
  15. 15. The method of claim 14, wherein the formulation of the stock solution of metal trace elements comprises 15-18 g/L ferric chloride, 1-2 g/L zinc chloride, 1-2 g/L sodium molybdate, 1-1.5 g/L copper sulfate, and 0.3-0.5 g/L boric acid.
  16. 16. The method of claim 15, wherein the thioesterase is derived from escherichia coli.
  17. 17. The method according to claim 16, wherein the thioesterase derived from the E.coli has the amino acid sequence shown in SEQ ID NO. 9.
  18. 18. The method of claim 17, wherein the conditions of the second expansion culture are 30 ℃ to 37 ℃ and 200rpm to 220rpm.
  19. 19. The method of claim 18, wherein the conditions for inducing fermentation are 28 ℃ to 30 ℃ and 200rpm to 220rpm.
  20. 20. A method according to any one of claims 7 to 19, wherein the inducer is IPTG and the final concentration of IPTG is 0.1mM to 1mM.

Description

Method for biosynthesis of omega-hydroxy fatty acid and genetically engineered bacterium Technical Field The invention relates to the technical field of biology, in particular to a method for biosynthesizing omega-hydroxy fatty acid and genetically engineered bacteria. Background The increase in energy costs and environmental concerns highlight the necessity of producing sustainable fuels and chemicals. The utilization of renewable biomass raw materials for producing oily chemicals, namely biorefinery, is changing from laboratory technology to commercial products due to its high efficiency and green environmental protection, and is expected to replace part of petroleum-based products in the fields of energy, chemical industry, materials and the like. Fatty acids and their derivatives are widely used in cosmetics, lubricants, surfactants, paints and biofuels as one of the useful oleaginous chemicals. Notably, omega-hydroxy fatty acids are important chemicals for adhesives, lubricants, cosmetic intermediates, and potential anticancer agents, and further, due to the presence of both hydroxyl and carboxyl functional groups at both ends of the molecule, can be further oxidized to form fatty aldehydes and dicarboxylic acids, which can be useful precursors for polymers, lactones and pseudoceramides, and can be used in the preparation of coatings, fragrances, preservatives, and polyketide antibiotics. Omega-hydroxy fatty acids are more difficult to chemically synthesize due to the inertness of the long chain fatty acyl chains, and furthermore chemical synthesis methods are limited by cumbersome steps, poor stereoselectivity and harsh reaction conditions. In addition, chemical catalysts for specific hydroxylation reactions of specific fatty acids are limited because of the diversity of fatty acid structures, which are controlled by chain length, saturation, whether there are branches, unsaturated bond positions, and cis-trans conformations. These difficulties limit the commercial use of omega-hydroxy fatty acids. Biological methods are considered potential synthetic methods in the synthesis of structurally diverse compounds due to their high efficiency, high stereoselectivity, high substrate selectivity and high regioselectivity. Microorganisms and animals and plants can naturally synthesize omega-hydroxy fatty acids and are therefore natural enzyme libraries from which highly specific catalytic enzymes can be extracted. At present, a plurality of P450 enzymes derived from bacteria, yeast and animals and plants are reported to be capable of synthesizing omega-hydroxy fatty acid, and the problems of difficult expression of protein, low catalytic efficiency, difficult enzyme purification and the like are faced although the variety is wide. Therefore, the method has important significance in screening out P450 enzyme for efficiently synthesizing omega-hydroxy fatty acid and developing a method for efficiently producing omega-hydroxy fatty acid. Disclosure of Invention The invention mainly aims to provide a method for biosynthesizing omega-hydroxy fatty acid and genetically engineered bacteria, which are used for solving the problem of low efficiency of biosynthesizing omega-hydroxy fatty acid in the prior art. In order to achieve the aim, according to a first aspect of the invention, a method for biosynthesizing omega-hydroxy fatty acid is provided, which comprises the step of carrying out catalytic reaction on fatty acid by using P450 hydroxylase to obtain the omega-hydroxy fatty acid, wherein the P450 hydroxylase has any one of the following amino acid sequences of SEQ ID NO. 6, SEQ ID NOs 12-14, SEQ ID NO. 22, SEQ ID NOs 26-27, SEQ ID NOs 30-32 or SEQ ID NO. 37. Further, the catalytic reaction also includes a redox partner. Further, the fatty acid is selected from any one of C12 saturated fatty acid, C14 saturated fatty acid, C16 monounsaturated fatty acid and C18 monounsaturated fatty acid. Further, the redox partner is selected from any one of the following: A reductase domain BMR derived from bacillus megatherium P450 enzyme CYP102 A1; a reductase domain RhFRed derived from rhodococcus P450 enzyme CYP116B 2; Redox partners CamA and CamB derived from pseudomonas putida; redox partners PetH and PetF derived from synechocystis; Or reductase AtATR2 derived from type II P450 of Arabidopsis thaliana. Further, the amino acid sequence of BMR is shown as SEQ ID NO. 44, rhFRed as SEQ ID NO. 45, camA as SEQ ID NO. 46, camB as SEQ ID NO. 47, petH as SEQ ID NO. 48, petF as SEQ ID NO. 49 and AtATR2 as SEQ ID NO. 50. Further, the method comprises the steps of: Mixing thalli, the fatty acid and a carbon source and carrying out hydroxylation reaction to obtain omega-hydroxy fatty acid; wherein the bacterial strain expresses the P450 hydroxylase and the redox partner. Further, the preparation method of the thalli comprises the following steps: And inoculating the first recombinant cells cultured overnight into a first culture medi