Method for biosynthesizing omega-hydroxy fatty acid and genetically engineered bacterium

Abstract

The invention provides a method for biosynthesizing omega-hydroxy fatty acid and a genetically engineered bacterium. The method comprises the following steps: carrying out catalytic reaction on fatty acid by adopting P450 hydroxylase to obtain the omega-hydroxy fatty acid, wherein the P450 hydroxylase has any one of the following amino acid sequences: SEQ ID NO: 6, SEQ ID NO: 12 to 14, SEQ ID NO: 22, SEQ ID NO: 26 to 27, SEQ ID NO: 30 to 32 or SEQ ID NO: 37. The method has the advantages of high catalytic efficiency and high regioselectivity, also has the characteristics of greenness and environmental protection, meets the requirements of sustainable development, has a wide application prospect, and has a relatively high economic value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for biosynthesizing ω-hydroxy fatty acids and genetically engineered bacteria. Background Technology

[0002] Rising energy costs and environmental concerns have highlighted the necessity of producing sustainable fuels and chemicals. Biorefining, which utilizes renewable biomass feedstocks to produce oil-based chemicals, is transitioning from a "laboratory technology" to a "commercial product" due to its high efficiency and environmental friendliness, and is expected to replace some petroleum-based products in the energy, chemical, and materials industries.

[0003] Fatty acids and their derivatives, as useful petrochemicals, are widely used in cosmetics, lubricants, surfactants, coatings, and biofuels. Notably, ω-hydroxy fatty acids are important chemicals used in adhesives, lubricants, cosmetic intermediates, and as potential anticancer agents. Furthermore, due to the presence of both hydroxyl and carboxyl groups at both ends of the molecule, they can be further oxidized to produce fatty aldehydes and dicarboxylic acids, serving as effective precursors for polymers, lactones, and pseudoceramides. They can also be used in the preparation of coatings, fragrances, preservatives, and polyketide antibiotics.

[0004] Due to the inertness of long fatty acyl chains, ω-hydroxy fatty acids are difficult to synthesize chemically. Furthermore, chemical synthesis methods are limited by cumbersome steps, poor stereoselectivity, and harsh reaction conditions. In addition, fatty acids are structurally diverse compounds, controlled by chain length, saturation, branching, unsaturated bond position, and cis-trans conformation; therefore, chemical catalysts for specific hydroxylation reactions of particular fatty acids are also limited. These difficulties restrict the commercial application of ω-hydroxy fatty acids.

[0005] Biological methods are considered promising synthetic approaches for synthesizing structurally diverse compounds due to their high efficiency, high stereoselectivity, high substrate selectivity, and high regioselectivity. Microorganisms and plants and animals can naturally synthesize ω-hydroxy fatty acids, thus forming a natural enzyme library from which highly specific catalytic enzymes can be discovered. Currently, various P450 enzymes derived from bacteria, yeast, and plants and animals have been reported to synthesize ω-hydroxy fatty acids. Although the variety is extensive, problems such as poor protein expression, low catalytic efficiency, and difficulties in enzyme purification exist. Therefore, screening for a highly efficient P450 enzyme for the synthesis of ω-hydroxy fatty acids and developing an efficient method for their production are of great significance. Summary of the Invention

[0006] The main objective of this invention is to provide a method for biosynthesizing ω-hydroxy fatty acids and a genetically engineered bacterium to solve the problem of low efficiency in the biosynthesis of ω-hydroxy fatty acids in the prior art.

[0007] To achieve the above objective, according to a first aspect of the present invention, a method for biosynthesizing ω-hydroxy fatty acids is provided, the method comprising: catalyzing a fatty acid reaction with a P450 hydroxylase to obtain the aforementioned ω-hydroxy fatty acid; wherein the aforementioned P450 hydroxylase has any one of the following amino acid sequences: 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.

[0008] Furthermore, the above-mentioned catalytic reactions also include redox chaperones.

[0009] Furthermore, the fatty acids mentioned above are selected from any one of the following: C12 saturated fatty acids, C14 saturated fatty acids, C16 saturated fatty acids, C16 monounsaturated fatty acids, or C18 monounsaturated fatty acids.

[0010] Furthermore, the aforementioned redox chaperones are selected from any one of the following:

[0011] The reductase domain BMR of the enzyme CYP102A1 from Bacillus megaterium P450;

[0012] The reductase domain RhFRed is derived from the P450 enzyme CYP116B2 of Rhodococcus spp.;

[0013] The redox synergies CamA and CamB derived from *Pseudomonas putida*;

[0014] The redox chaperones PetH and PetF are derived from Synechocystis.

[0015] Or it may be derived from the reductase AtATR2 of type II P450 in the plant Arabidopsis thaliana.

[0016] Further, the amino acid sequence of the above-mentioned BMR is shown in SEQ ID NO: 44; the amino acid sequence of the above-mentioned RhFRed is shown in SEQ ID NO: 45; the amino acid sequence of the above-mentioned CamA is shown in SEQ ID NO: 46; the amino acid sequence of the above-mentioned CamB is shown in SEQ ID NO: 47; the amino acid sequence of the above-mentioned PetH is shown in SEQ ID NO: 48; the amino acid sequence of the above-mentioned PetF is shown in SEQ ID NO: 49; and the amino acid sequence of the above-mentioned AtATR2 is shown in SEQ ID NO: 50.

[0017] Furthermore, the above methods include:

[0018] The bacterial cells, the above-mentioned fatty acids, and the carbon source were mixed and subjected to a hydroxylation reaction to obtain the above-mentioned ω-hydroxy fatty acids;

[0019] The aforementioned bacterial cells are able to express the aforementioned P450 hydroxylase and the aforementioned redox chaperone.

[0020] Furthermore, the method for preparing the above-mentioned bacterial cells includes:

[0021] The first recombinant cells, cultured overnight, were inoculated into the first culture medium for the first expansion culture. The culture was carried out until the OD600 reached 0.6-0.8. An inducer and cofactor were added to induce expression for 24-48 hours. The precipitate was collected by centrifugation to obtain the above-mentioned bacterial cells. The above-mentioned first recombinant cells were able to express the above-mentioned P450 hydroxylase and the above-mentioned redox chaperone.

[0022] Furthermore, the carbon source includes glucose and glycerol; the final concentration of glucose is 0.2 g / L-0.5 g / L; and the final concentration of glycerol is 5 g / L-10 g / L.

[0023] Furthermore, the final concentration of the above fatty acids is 0.5 mM-1 mM.

[0024] Furthermore, the first culture medium mentioned above is LB medium.

[0025] Furthermore, the conditions for the first expanded culture were 30℃-37℃ and 200rpm-220rpm.

[0026] Furthermore, the conditions for inducing expression were 28℃-30℃ and 200rpm-220rpm.

[0027] Furthermore, the above methods include:

[0028] The second recombinant cells, cultured overnight, were seeded into a second culture medium for a second expansion culture. The culture was carried out until the OD600 reached 0.6-0.8. Inducers and cofactors were added, and fermentation was induced for 48-72 hours to obtain the above-mentioned ω-hydroxy fatty acids. Among them, the above-mentioned second recombinant cells were able to express the above-mentioned P450 hydroxylase, the above-mentioned redox chaperone, and thioesterase.

[0029] Furthermore, the formulation of the second culture medium includes: ammonium chloride 4-6 g / L, potassium dihydrogen phosphate 5-10 g / L, citric acid 0.3-0.5 g / L, yeast extract 3-5 g / L, glycerol 15-30 g / 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.

[0030] Furthermore, the formulation of the above-mentioned metal trace element storage solution includes: 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.

[0031] Furthermore, the aforementioned thioesterases are derived from Escherichia coli.

[0032] Furthermore, the amino acid sequence of the thioesterase derived from the above-mentioned Escherichia coli is shown in SEQ ID NO: 9.

[0033] Furthermore, the conditions for the second expanded culture were 30℃-37℃ and 200rpm-220rpm.

[0034] Furthermore, the conditions for the above-mentioned induced fermentation are 28℃-30℃ and 200rpm-220rpm.

[0035] Furthermore, the inducer is IPTG; the final concentration of IPTG is 0.1 mM-1 mM.

[0036] Furthermore, the aforementioned cofactors include δ-aminolevulinic acid and FeSO4; wherein the final concentration of the aforementioned δ-aminolevulinic acid is 0.3-0.5 mM; and the final concentration of the aforementioned FeSO4 is 0.05-0.1 mM.

[0037] To achieve the above objectives, according to a second aspect of the present invention, a genetically engineered bacterium is provided, comprising a gene encoding a P450 hydroxylase; wherein the P450 hydroxylase has any one of the following amino acid sequences: 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.

[0038] Furthermore, the aforementioned genetically engineered bacteria also include genes encoding redox chaperones.

[0039] Furthermore, the aforementioned redox chaperones are selected from any of the following: the reductase domain BMR of the Bacillus megaterium P450 enzyme CYP102A1; the reductase domain RhFRed of the Rhodococcus P450 enzyme CYP116B2; the redox chaperones CamA and CamB of Pseudomonas putida; the redox chaperones PetH and PetF of Synechocystis; or the reductase AtATR2 of type II P450 from the plant Arabidopsis thaliana.

[0040] Further, the amino acid sequence of the above-mentioned BMR is shown in SEQ ID NO: 44; the amino acid sequence of the above-mentioned RhFRed is shown in SEQ ID NO: 45; the amino acid sequence of the above-mentioned CamA is shown in SEQ ID NO: 46; the amino acid sequence of the above-mentioned CamB is shown in SEQ ID NO: 47; the amino acid sequence of the above-mentioned PetH is shown in SEQ ID NO: 48; the amino acid sequence of the above-mentioned PetF is shown in SEQ ID NO: 49; and the amino acid sequence of the above-mentioned AtATR2 is shown in SEQ ID NO: 50.

[0041] Furthermore, the aforementioned genetically engineered bacteria also include genes encoding thioesterases.

[0042] Furthermore, the aforementioned thioesterases are derived from Escherichia coli.

[0043] Furthermore, the amino acid sequence of the thioesterase derived from the above-mentioned Escherichia coli is shown in SEQ ID NO: 9.

[0044] To achieve the above objectives, according to a third aspect of the present invention, a method for the above-described biosynthesis of ω-hydroxy fatty acids or the application of the above-described genetically engineered bacteria in the synthesis of ω-hydroxy fatty acids is provided.

[0045] By applying the technical solution of this invention, the screened P450 hydroxylase (having any one of the following amino acid sequences: 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) can convert fatty acids into ω-hydroxy fatty acids with high catalytic efficiency. This not only accelerates the chemical reaction but also helps to increase the yield of the target product and reduce production costs. Compared with chemical synthesis methods, the biocatalytic pathway for synthesizing ω-hydroxy fatty acids has higher regioselectivity, reduces the generation of byproducts, and improves product purity. It is also green and environmentally friendly, meeting the requirements of sustainable development, and has broad application prospects and high economic value. Attached Figure Description

[0046] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0047] Figure 1 The present invention illustrates a synthetic route for the synthesis of 16-hydroxy-9Z-hexadecenoic acid from a 16-carbon, Δ9 monounsaturated fatty acid using the P450 enzyme P450-31 according to Example 6.

[0048] Figure 2 The liquid chromatography-mass spectra of 14-hydroxy-C14 saturated fatty acid, 16-hydroxy-C16 saturated fatty acid and 18-hydroxy-C18-Δ9-monounsaturated fatty acid synthesized by using P450 hydroxylase P450-31 to catalyze C14 saturated fatty acid, C16 saturated fatty acid and C18,Δ9, monounsaturated fatty acid, respectively, are shown in Example 5 of the present invention; wherein (1) is 14-hydroxy-C14 saturated fatty acid; (2) is 16-hydroxy-C16 saturated fatty acid; and (3) is 18-hydroxy-C18-Δ9-monounsaturated fatty acid.

[0049] Figure 3 The liquid chromatogram of 16-hydroxy-9Z-hexadecenoic acid synthesized using P450 hydroxylase according to Example 4 of the present invention is shown, wherein (1) is the 16-hydroxy-9Z-hexadecenoic acid product synthesized using P450 hydroxylase CYP153A33, and (2) is the 16-hydroxy-9Z-hexadecenoic acid product synthesized using P450-31 of the present invention.

[0050] Figure 4 The liquid chromatography-mass spectrum of 16-hydroxy-9Z-hexadecenoic acid synthesized using P450 hydroxylase according to Example 4 of the present invention is shown, wherein (1) is the 16-hydroxy-9Z-hexadecenoic acid product synthesized using P450 hydroxylase CYP153A33, and (2) is the 16-hydroxy-9Z-hexadecenoic acid product synthesized using P450-31 of the present invention.

[0051] Figure 5 The gas chromatography-mass spectrum of 16-hydroxy-9Z-hexadecenoic acid synthesized using P450 hydroxylase P450-31 according to Example 4 of the present invention is shown.

[0052] Figure 6 The optimal fit material obtained by searching the NIST database using gas chromatography-mass spectra of 16-hydroxy-9Z-hexadecenoic acid synthesized using P450 hydroxylase P450-31, according to Example 4 of the present invention, is shown.

[0053] Figure 7 A roadmap for the biosynthesis of ω-hydroxy fatty acids according to the present invention is shown. Detailed Implementation

[0054] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0055] Terminology Explanation:

[0056] Regioselectivity: In organic chemical reactions, if a molecule has multiple possible reaction sites, the reactants may react at different sites. However, when enzymes catalyze these reactions, they typically preferentially act on one or a few specific sites. This site-specific selectivity is called regioselectivity. For example, in fatty acid hydroxylation, P450 hydroxylases can selectively introduce a hydroxyl group at the ω position (the terminal carbon atom) of the fatty acid chain, rather than at other positions in the chain. This property allows enzyme-catalyzed reactions to generate specific, high-value-added products, thus having important applications in pharmaceutical synthesis, fine chemicals, and biomaterials.

[0057] Redox chaperones are proteins that work synergistically with redox enzymes (such as P450 hydroxylases) in biocatalytic reactions to help transfer electrons and achieve the catalytic reaction. Redox chaperones play a crucial role in connecting the electron donor (NADH / NADPH) and the electron acceptor (O2).

[0058] As mentioned in the background section, ω-hydroxy fatty acids have important applications in adhesives, lubricants, cosmetic intermediates, and potential anticancer agents. In addition, ω-hydroxy fatty acids have two functional groups, hydroxyl and carboxyl groups, at both ends of the molecule, so they can be further oxidized to produce fatty aldehydes and dicarboxylic acids, which can serve as effective precursors for polymers, lactones, and pseudoceramides. Therefore, they can be widely used in the preparation of coatings, fragrances, preservatives, and polyketide antibiotics.

[0059] While various P450 hydroxylases derived from bacteria, yeast, and plants and animals can synthesize ω-hydroxy fatty acids in the prior art, they face problems such as difficulty in protein expression and low catalytic efficiency. In this application, the inventors attempted to screen P450 hydroxylases with high catalytic efficiency from 5000 P450 hydroxylases in an existing database for the efficient production of ω-hydroxy fatty acids, and thus proposed the technical solution of this application.

[0060] In a first typical embodiment of the present invention, a method for biosynthesizing ω-hydroxy fatty acids is provided, the method comprising: catalyzing a fatty acid reaction with a P450 hydroxylase to obtain the aforementioned ω-hydroxy fatty acid; wherein the aforementioned P450 hydroxylase has any one of the following amino acid sequences: 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.

[0061] P450 hydroxylase is a multifunctional oxidase capable of hydroxylating fatty acids at specific positions to generate high-value-added ω-hydroxy fatty acids. P450 hydroxylases with amino acid sequences shown in any 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 exhibit highly efficient conversion capabilities for fatty acids of different chain lengths and saturation levels during catalysis. This not only accelerates the chemical reaction but also helps increase the yield of the target product and reduce production costs. Compared to chemical synthesis methods, the biocatalytic synthesis of ω-hydroxy fatty acids offers higher regioselectivity, reduces byproduct formation, improves product purity, and is environmentally friendly, meeting the requirements of sustainable development. It has broad application prospects and high economic value.

[0062] The catalytic activity of P450 hydroxylase depends on a stable electron supply, a role primarily undertaken by redox chaperones. Redox chaperones act as electron transfer agents in the catalytic reaction, working synergistically with P450 hydroxylase and serving as cofactors in the synthesis of ω-hydroxy fatty acids. Therefore, in a preferred embodiment of the present invention, the above-mentioned catalytic reaction further includes a redox chaperone.

[0063] Redox chaperones from different sources exhibit different activities. To further increase the yield of ω-hydroxy fatty acids, in a preferred embodiment of the present invention, the redox chaperones are selected from any of the following: the reductase domain BMR of Bacillus megaterium P450 hydroxylase CYP102A1; the reductase domain RhFRed of Rhodococcus P450 hydroxylase CYP116B2; redox chaperones CamA and CamB from Pseudomonas putida; redox chaperones PetH and PetF from Synechocystis; or the reductase AtATR2 of type II P450 from Arabidopsis thaliana.

[0064] In a preferred embodiment of the present invention, the amino acid sequence of the above-mentioned BMR is shown in SEQ ID NO: 44; the amino acid sequence of the above-mentioned RhFRed is shown in SEQ ID NO: 45; the amino acid sequence of the above-mentioned CamA is shown in SEQ ID NO: 46; the amino acid sequence of the above-mentioned CamB is shown in SEQ ID NO: 47; the amino acid sequence of the above-mentioned PetH is shown in SEQ ID NO: 48; the amino acid sequence of the above-mentioned PetF is shown in SEQ ID NO: 49; and the amino acid sequence of the above-mentioned AtATR2 is shown in SEQ ID NO: 50.

[0065] Fatty acids, as substrates for the catalytic reaction of this invention, are selected from, but are not limited to, C12 saturated fatty acids, C14 saturated fatty acids, C16 saturated fatty acids, C16 monounsaturated fatty acids, or C18 monounsaturated fatty acids. This broad substrate adaptability stems from the high compatibility and adaptability of the aforementioned P450 hydroxylase to substrates, enabling precise hydroxylation at the ω-terminus of the fatty acid chain to generate the corresponding ω-hydroxy fatty acid. This not only demonstrates the high regioselectivity of the enzyme but also provides flexibility and diversity for the biosynthesis of ω-hydroxy fatty acids. In a preferred embodiment of this invention, the fatty acid is selected from any one of the following: C12 saturated fatty acids, C14 saturated fatty acids, C16 saturated fatty acids, C16 monounsaturated fatty acids, or C18 monounsaturated fatty acids.

[0066] The methods for biosynthesizing ω-hydroxy fatty acids described above include, but are not limited to, using the aforementioned P450 hydroxylase for in vitro catalytic reactions or using cells capable of expressing P450 hydroxylase for in vivo catalytic reactions. When using P450 hydroxylase for in vitro catalytic reactions, the catalytic reaction system should also contain the aforementioned redox chaperone and NADH / NADPH; wherein, the aforementioned P450 hydroxylase can be either a crude enzyme solution obtained after heterologous expression or a purified enzyme. This application allows for the selection of either a crude enzyme solution or a purified enzyme according to actual production needs.

[0067] When using P450 hydroxylase for in vivo catalytic reactions, the methods for biosynthesizing ω-hydroxy fatty acids include, but are not limited to, de novo synthesis and salvage synthesis. De novo synthesis refers to recombinant cells expressing P450 hydroxylase, the aforementioned redox chaperones, and thioesterases (which participate in fatty acid metabolism within cells, releasing free fatty acids through the hydrolysis of thioester bonds) being able to synthesize fatty acids from a simple carbon source (such as glucose) through their own metabolic pathways, followed by further catalysis to generate ω-hydroxy fatty acids. Salvage synthesis refers to recombinant cells expressing P450 hydroxylase and the aforementioned redox chaperones being able to synthesize ω-hydroxy fatty acids through whole-cell catalytic reactions using fatty acids as substrates (see [link to relevant documentation]). Figure 1 ).

[0068] It should be noted that when constructing recombinant cells containing the genes encoding P450 hydroxylase, redox chaperones, and thioesterase, these three genes can be independently linked to three separate plasmids, simultaneously linked to the same plasmid, or any two genes can be linked to one plasmid while the third gene is linked to a separate plasmid. Similarly, when constructing recombinant cells containing the genes encoding P450 hydroxylase and redox chaperones, these two genes can be independently constructed to two separate plasmids, or simultaneously constructed to the same plasmid.

[0069] In a preferred embodiment of the present invention, the above-mentioned ω-hydroxy fatty acid is synthesized using a salvage synthesis method. In a preferred embodiment of the present invention, the above-mentioned method for biosynthesizing ω-hydroxy fatty acid includes: mixing bacterial cells, the above-mentioned fatty acid and a carbon source and performing a hydroxylation reaction to obtain the above-mentioned ω-hydroxy fatty acid; wherein the above-mentioned bacterial cells are capable of expressing the above-mentioned P450 hydroxylase and the above-mentioned redox chaperone.

[0070] In a more preferred embodiment of the present invention, the method for preparing the above-mentioned bacterial cells includes: inoculating overnight recombinant cells into a first culture medium for a first expansion culture, culturing until the OD600 reaches 0.6-0.8, adding an inducer and cofactor, inducing expression for 24-48 hours, centrifuging to collect the precipitate, and obtaining the above-mentioned bacterial cells; wherein the above-mentioned first recombinant cells can express the above-mentioned P450 hydroxylase and the above-mentioned redox chaperone. The induction time is limited, and the enzyme content produced by the bacteria is not high. Therefore, after induction, the bacteria need to be concentrated, and then the concentrated bacteria are used for catalytic reactions. The above-mentioned method for producing ω-hydroxy fatty acids has the advantages of simplicity and speed, which is helpful for the large-scale screening of enzyme types.

[0071] In the biosynthesis of ω-hydroxy fatty acids, carbon sources such as glucose and glycerol not only support the growth and metabolism of microorganisms but also promote the expression and activity of specific enzymes by providing necessary energy, metabolites, and electron donors, ultimately improving catalytic efficiency and product yield. In a preferred embodiment of the present invention, the carbon sources include glucose and glycerol; the final concentration of glucose is 0.2 g / L-0.5 g / L; and the final concentration of glycerol is 5 g / L-10 g / L.

[0072] Fatty acids, as substrates for P450 hydroxylases, allow those skilled in the art to select appropriate concentrations for biosynthesis to match enzyme catalytic efficiency with product selectivity. A suitable final fatty acid concentration ensures sufficient substrate at the enzyme's active site to maintain a high catalytic rate, while avoiding enzyme inhibition or non-specific reactions caused by excessively high substrate concentrations, thus resulting in higher product purity and yield. Furthermore, regulating fatty acid concentration helps maintain the stability of the bioreaction system, minimizing or even avoiding toxic effects caused by substrate or product accumulation, thereby promoting healthy host strain growth and more stable enzyme expression. In a preferred embodiment of the present invention, the final concentration of the fatty acid is 0.5 mM - 1 mM. Using this concentration of fatty acid promotes efficient catalytic reaction.

[0073] In a preferred embodiment of the present invention, the first culture medium is LB medium. LB medium provides sufficient nutrients for the growth and metabolism of the strain, supports rapid microbial growth, and provides the necessary biomass for subsequent catalytic reactions.

[0074] In a preferred embodiment of the present invention, the conditions for the first expansion culture are 30℃-37℃ and 200rpm-220rpm. In a preferred embodiment of the present invention, the conditions for the induction of expression are 28℃-30℃ and 200rpm-220rpm. Regulating the above expansion culture and induction of expression conditions facilitates the expression of P450 hydroxylase and redox chaperone.

[0075] To further simplify the production process and improve production efficiency, in another preferred embodiment of the present invention, the above-mentioned ω-hydroxy fatty acids are synthesized using a de novo synthesis method. In a preferred embodiment of the present invention, the above-mentioned method for biosynthesizing ω-hydroxy fatty acids includes: inoculating overnight recombinant cells into a second culture medium for a second expansion culture, culturing until the OD600 reaches 0.6-0.8, adding an inducer and cofactor, and inducing fermentation for 48-72 hours to obtain the above-mentioned ω-hydroxy fatty acids; wherein the above-mentioned second recombinant cells are able to express the above-mentioned P450 hydroxylase, the above-mentioned redox chaperone, and thioesterase.

[0076] In this preferred embodiment, recombinant cells containing P450 hydroxylase, the aforementioned redox chaperone, and thioesterase are used for biosynthesis. The thioesterase participates in fatty acid metabolism within the cell, releasing free fatty acids through the hydrolysis of thioester bonds. This not only increases the supply of fatty acid substrates for the enzymatic reaction but also improves the dynamic equilibrium of the catalytic reaction, leading to the conversion of more fatty acids into ω-hydroxy fatty acids. In other words, the co-expression of thioesterase within the cell promotes the release and circulation of free fatty acids, thereby enhancing the synthesis efficiency of ω-hydroxy fatty acids. Therefore, the method described above offers the advantage of high yield in the production of ω-hydroxy fatty acids.

[0077] In a preferred embodiment of the present invention, the formulation of the second culture medium includes: ammonium chloride 4-6 g / L, potassium dihydrogen phosphate 5-10 g / L, citric acid 0.3-0.5 g / L, yeast extract 3-5 g / L, glycerol 15-30 g / 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 antibiotic 50 mg / L-100 mg / L. In a preferred embodiment of the present invention, the formulation of the metal trace element stock solution includes: ferric chloride 15-18 g / L, zinc chloride 1-2 g / L, sodium molybdate 1-2 g / L, copper sulfate 1-1.5 g / L, and boric acid 0.3-0.5 g / L. Using the above-mentioned culture medium helps to increase the biomass of recombinant cells and the expression levels of various proteins (e.g., P450 hydroxylase, redox chaperone, and thioesterase), thereby further increasing the production of ω-hydroxy fatty acids.

[0078] It is important to note that the type of antibiotic used depends on the resistance gene on the plasmid transferred into the recombinant cells (either the first or second recombinant cell). For example, when two plasmids containing different resistance genes (e.g., chloramphenicol resistance gene and kanamycin resistance gene) are transferred into the recombinant cells, the culture medium (either the first or second culture medium) contains 50 mg / L–100 mg / L chloramphenicol and 50 mg / L–100 mg / L kanamycin. When only one plasmid (e.g., chloramphenicol resistance plasmid or kanamycin resistance plasmid) is transferred into the recombinant cells, the fermentation medium contains only 50 mg / L–100 mg / L chloramphenicol or 50 mg / L–100 mg / L kanamycin.

[0079] As mentioned above, thioesterases can promote the release and flow of fatty acids, thereby enhancing the synthesis efficiency of ω-hydroxy fatty acids. Therefore, any thioesterase with this function is applicable to this application. In a preferred embodiment of the present invention, the above-mentioned thioesterase is derived from *Escherichia coli*. In a preferred embodiment of the present invention, the amino acid sequence of the above-mentioned thioesterase derived from *Escherichia coli* is shown in SEQ ID NO: 9. The thioesterase derived from the above-mentioned source has the advantage of high catalytic activity.

[0080] In a preferred embodiment of the present invention, the conditions for the second scale-up culture are 30℃-37℃ and 200rpm-220rpm. In another preferred embodiment of the present invention, the conditions for the induced fermentation are 28℃-30℃ and 200rpm-220rpm. Fermentation under these conditions helps to increase the yield of ω-hydroxy fatty acids.

[0081] In a preferred embodiment of the present invention, the inducer is IPTG; the final concentration of IPTG is 0.1 mM-1 mM. IPTG can induce the expression of heterologous genes (e.g., genes encoding P450 hydroxylase, genes encoding redox chaperones, or genes encoding thioesterases) in recombinant cells, and its final concentration is preferably 0.1 mM-1 mM, which can promote the efficient expression of enzymes without affecting the health of host cells.

[0082] The cofactors δ-aminolevulinic acid (ALA) and FeSO4 help maintain the activity and stability of P450 hydroxylase. ALA is the starting material for heme biosynthesis, and heme plays a crucial role in the structural integrity and catalytic activity of P450 hydroxylase. By providing ALA, P450 hydroxylase can have sufficient heme cofactors to perform its monooxygenase catalytic function, thereby efficiently converting fatty acids into ω-hydroxy fatty acids.

[0083] FeSO4 (ferrous sulfate) is an important electron transport component in the P450 hydroxylase catalytic cycle. P450 hydroxylase relies on the redox state transition of iron atoms in the hydroxylation reaction, and the addition of FeSO4 provides the required iron ions. In a preferred embodiment of the present invention, the cofactors include δ-aminolevulinic acid and FeSO4; wherein the final concentration of δ-aminolevulinic acid is 0.3 mM - 0.5 mM; and the final concentration of FeSO4 is 0.05 mM - 0.1 mM.

[0084] In a second typical embodiment of the present invention, a genetically engineered bacterium is provided, comprising a gene encoding a P450 hydroxylase; wherein the P450 hydroxylase has any one of the following amino acid sequences: 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 genetically engineered bacterium containing the gene encoding the P450 hydroxylase exhibits highly efficient catalytic activity and substrate selectivity for saturated and unsaturated fatty acids of different chain lengths. This characteristic enables the genetically engineered bacterium to efficiently synthesize ω-hydroxy fatty acids over a wide substrate range, providing a powerful tool for biorefining and green chemistry.

[0085] In a preferred embodiment of the present invention, the genetically engineered bacteria further includes a gene encoding a redox chaperone. By introducing redox chaperones derived from different microorganisms and plants (such as BMR, RhFRed, CamA / CamB, PetH / PetF, and AtATR2), the genetically engineered bacteria of this application can achieve more efficient electron transfer, which directly improves the catalytic efficiency and stability of P450 hydroxylase. These redox chaperones not only support the activity of P450 hydroxylase, but also, through their specific amino acid sequences (as shown in SEQ ID NOs: 44-50), enable electron donors (NADH or NADPH) to reach P450 hydroxylase rapidly and efficiently, thereby facilitating the smooth progress of the hydroxylation reaction.

[0086] In a preferred embodiment of the present invention, the redox chaperones are selected from any of the following: the reductase domain BMR of Bacillus megaterium P450 hydroxylase CYP102A1; the reductase domain RhFRed of Rhodococcus spp. P450 hydroxylase CYP116B2; redox chaperones CamA and CamB of Pseudomonas putida; redox chaperones PetH and PetF of Synechocystis; or the reductase AtATR2 of type II P450 from Arabidopsis thaliana.

[0087] In a preferred embodiment of the present invention, the amino acid sequence of the above-mentioned BMR is shown in SEQ ID NO: 44; the amino acid sequence of the above-mentioned RhFRed is shown in SEQ ID NO: 45; the amino acid sequence of the above-mentioned CamA is shown in SEQ ID NO: 46; the amino acid sequence of the above-mentioned CamB is shown in SEQ ID NO: 47; the amino acid sequence of the above-mentioned PetH is shown in SEQ ID NO: 48; the amino acid sequence of the above-mentioned PetF is shown in SEQ ID NO: 49; and the amino acid sequence of the above-mentioned AtATR2 is shown in SEQ ID NO: 50.

[0088] The addition of thioesterase enhances the mobility of fatty acids within host cells, thereby increasing the synthesis efficiency of ω-hydroxy fatty acids by promoting the release of free fatty acids. In a preferred embodiment of the present invention, the genetically engineered bacteria further includes a gene encoding a thioesterase. In a preferred embodiment of the present invention, the thioesterase is derived from *Escherichia coli*. In a preferred embodiment of the present invention, the amino acid sequence of the thioesterase derived from *E. coli* is shown in SEQ ID NO: 9. The genetically engineered bacteria containing the gene encoding a thioesterase (particularly the gene having the amino acid sequence shown in SEQ ID NO: 9) can promote the efficient synthesis of ω-hydroxy fatty acids, reducing the genetically engineered bacteria's need for additional fatty acid substrates.

[0089] In a third typical embodiment of the present invention, a method for biosynthesizing ω-hydroxy fatty acids as described above or the application of the above-described genetically engineered bacteria in the synthesis of ω-hydroxy fatty acids is provided.

[0090] The aforementioned methods for biosynthesizing ω-hydroxy fatty acids, or the aforementioned genetically engineered bacteria, improve production efficiency and selectivity while reducing environmental impact. By regulating enzyme expression and reaction conditions, the direct and efficient conversion from simple carbon sources to high-value ω-hydroxy fatty acids is achieved, providing strong technical support for the industrial production of green chemistry and sustainable bioproducts, demonstrating significant economic and environmental benefits. This not only promotes the widespread application of ω-hydroxy fatty acids in multiple industries but also opens up new pathways for cutting-edge exploration of biocatalysis technology.

[0091] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0092] Example 1 Construction of recombinant Escherichia coli BL21(DE3) / pRSF-P450

[0093] Considering the structural diversity of fatty acids due to chain length, saturation, branching, unsaturated bond position, and cis-trans configuration, and the resulting differences in substrate-enzyme compatibility, P450 hydroxylases that efficiently catalyze short-chain fatty acids (less than 6 carbons) or medium-chain fatty acids (6-12 carbons) may not be able to catalyze long-chain fatty acids (greater than 12 carbons), and P450 hydroxylases that efficiently catalyze saturated fatty acids may not be able to catalyze unsaturated fatty acids. Therefore, obtaining highly efficient P450 hydroxylases for fatty acid catalysis requires analysis and screening of multiple P450 hydroxylases.

[0094] Therefore, the inventors screened P450s from various databases (approximately 5000 in total) and performed docking analysis with substrates, initially identifying 43 P450 hydroxylases that may synthesize ω-hydroxy fatty acids. Based on the codon usage preferences of *E. coli*, the codons of these 43 P450 hydroxylases were optimized and synthesized in the pRSF-Duet-1 vector (Sigma-Aldrich, catalog number 71341), resulting in 43 pRSF-P450 plasmids (Table 1). All nucleic acids used in this invention were synthesized by Genewiz Biotechnology Co., Ltd.

[0095] Table 1 P450 hydroxylase expression vectors

[0096]

[0097] To obtain a positive control, the P450 hydroxylase CYP153A33, which is reported to catalyze the synthesis of 16-hydroxy-9Z-hexadecenoic acid from 16-carbon,Δ9 monounsaturated fatty acids, was also optimized according to E. coli codon usage preferences and synthesized in the pRSF-Duet-1 vector (Table 2).

[0098] Table 2 CYP153A33 expression vector

[0099]

[0100] The 44 recombinant plasmids shown in Tables 1-2 above were transformed into Escherichia coli BL21(DE3) (Full Gold, catalog number CD601-03) to obtain 44 recombinant Escherichia coli BL21(DE3) / pRSF-P450 strains.

[0101] The specific transformation method is as follows: Thaw BL21(DE3) competent cells (50 μL of competent cells in 1.5 ml EP tube) on ice, add 50 ng of plasmid to the competent cells, mix well, incubate on ice for 30 min, heat shock in a 42℃ water bath for 90 s, add 600 μL of recovery solution (LB medium), incubate at 37℃ for 50 min, after incubation, spread all the bacterial culture on plates containing 50 mg / L kanamycin (LB medium), and incubate overnight at 37℃.

[0102] Example 2: Verification of protein expression of P450 hydroxylase

[0103] To determine whether the synthesized P450 hydroxylase is expressed normally, protein expression verification is required. The specific method is as follows: Forty-four engineered strains expressing P450 hydroxylase from the BL21(DE3) chassis strain were inoculated at 0.2% (v / v) into test tubes containing 5 mL LB liquid medium (with 50 mg / L kanamycin). After overnight incubation at 37°C, 1% (v / v) was inoculated into 250 mL shake flasks containing 50 mL LB liquid medium (with 50 mg / L kanamycin). The flasks were shaken at 37°C until the OD600 reached 0.8. IPTG was added to a final concentration of 0.1 mM, and the mixture was induced to mature on a shaker at 30°C for 20 hours. Then, 3 mL of the bacterial culture was collected, centrifuged at 12000 rpm at 4°C, and the cells were resuspended in 1 mL of PBS buffer. The cells were then sonicated for 5 min using an ultrasonic homogenizer. Centrifuge at rpm for 10 minutes to separate the supernatant protein and the precipitated protein, and perform SDS-PAGE analysis separately. The results showed that the P450 hydroxylases used in this application were expressed normally.

[0104] Example 3 Construction of recombinant Escherichia coli BL21(DE3) / pRSF-P450, pCDF-CPR

[0105] Functional expression of P450 requires the assistance of a P450 redox chaperone (see...) Figure 7 Therefore, it is necessary to construct a CPR expression plasmid. Different P450 hydroxylases have different spatial positions and electron transfer efficiencies with different redox chaperones. Therefore, to avoid the possibility of failing to screen for P450 hydroxylases due to mismatch, five redox chaperones were used, including:

[0106] 1) A redox chaperone for type VIII P450, namely the reductase domain (BMR) of Bacillus megaterium P450 hydroxylase CYP102A1, the amino acid sequence of which is SEQ ID NO: 44;

[0107] 2) A redox chaperone for type VII P450, namely the reductase domain (RhFRed) of the P450 hydroxylase CYP116B2 from Rhodococcus spp., SEQ ID NO: 45;

[0108] 3) Two redox chaperones for type I P450, namely redox chaperones (CamA and CamB) derived from *Pseudomonas putida* and redox chaperones (PetH and PetF) derived from *Syntrophus spp.*, wherein the amino acid sequence of CamA is SEQ ID NO: 46, the amino acid sequence of CamB is SEQ ID NO: 47, the amino acid sequence of PetH is SEQ ID NO: 48, and the amino acid sequence of PetF is SEQ ID NO: 49;

[0109] 4) A redox chaperone (AtATR2) derived from type II P450 of the plant Arabidopsis thaliana, with the amino acid sequence SEQ ID NO: 50.

[0110] Based on the codon usage preferences of *E. coli*, these redox chaperones were codon optimized and synthesized in the pCDF-Duet-1 (Merck, catalog number 71340) vector, resulting in a total of 5 pCDF-CPR plasmids (Table 3). All nucleic acids used in this invention were synthesized by Genewiz Biotechnology Co., Ltd.

[0111] Table 3 CPR expression vectors

[0112]

[0113] The five recombinant plasmids expressing CPR shown in Table 3 above were transformed into Escherichia coli BL21(DE3) / pRSF-P450 to obtain 220 recombinant Escherichia coli BL21(DE3) / pRSF-P450, pCDF-CPR strains.

[0114] The specific transformation method is as follows: Take BL21(DE3) / pRSF-P450 competent cells (50 μL of competent cells in 1.5 ml EP tube) and thaw them on ice. Add 50 ng of plasmid to the competent cells, mix well, incubate on ice for 30 min, heat shock in a 42℃ water bath for 90 s, add 600 μL of recovery solution (LB medium), and incubate at 37℃ for 50 min. After incubation, spread all the bacterial culture on plates containing 50 mg / L kanamycin and 50 mg / L streptomycin (LB medium) and incubate overnight at 37℃.

[0115] Example 4: Whole-cell catalysis of 16-carbon,Δ9 monounsaturated fatty acid synthesis of 16-hydroxy-9Z-hexadecenoic acid

[0116] Whole-cell catalytic reactions were performed using 220 recombinant Escherichia coli strains BL21(DE3) / pRSF-P450 and pCDF-CPR constructed in Example 3.

[0117] The specific method is as follows: 220 strains of recombinant Escherichia coli BL21(DE3) / pRSF-P450, pCDF-CPR glycerol bacteria were inoculated at a volume of 0.2% into test tubes containing 5 mL of LB liquid medium (with 50 mg / L kanamycin and 50 mg / L streptomycin). After overnight incubation at 37°C, the bacteria were inoculated at a volume of 1% into 250 mL shake flasks containing 50 mL of LB liquid medium (with 50 mg / L kanamycin and 50 mg / L streptomycin). The culture was shaken at 37°C until the OD600 reached 0.8. IPTG, δ-aminolevulinic acid (δ-aminolevulinic acid), and FeSO4 were added to a final concentration of 0.1 mM. After induction culture at 30°C for 20 hours, the bacterial cells were collected by centrifugation at 12,000 rpm. The bacterial cells were controlled to OD600 = 30 and resuspended in 0.1 mM KPB buffer.

[0118] The following reaction system was prepared using the cell slurry: 2 mL cell slurry, 100 µL 16-carbon, Δ9 monounsaturated fatty acid (the mother liquor was 20 mM 16-carbon, Δ9 monounsaturated fatty acid stored in DMSO), 26.6 µL 60% glycerol, and 132 µL 6 g / L glucose. The system was reacted at 30 °C and 220 rpm for 4 h. Then, 200 µL 6 M hydrochloric acid and 2 mL ethyl acetate were added, and the mixture was shaken for 10 min. After centrifugation, the supernatant was collected, and the amount of 16-hydroxy-9Z-hexadecenoic acid synthesized in the reaction system was determined by HPLC.

[0119] The chromatographic conditions were as follows: column: ZORBAX SB-C8 4.6 x 150 mm, P / N: 863953-906; mobile phase: H2O + 0.1% TFA and ACN + 0.1% TFA; flow rate: 1.0 ml / min; column temperature: 40℃; ELSD detector; evaporator temperature: 40℃; nebulizer temperature: 40℃; nitrogen flow rate: 1.6 SLM; detection time: 25 min.

[0120] As shown in Tables 4-1, 4-2, 4-3, 4-4, and 4-5 below, after the whole-cell catalytic reaction, significant synthesis of 16-hydroxy-9Z-hexadecenoic acid was detected in the control strains CYP153A33, P450-6, P450-12, P450-13, P450-14, P450-21, P450-22, P450-26, P450-27, P450-30, P450-31, P450-32, P450-37, and P450-38 among the 220 engineered strains (see Tables 4-1, 4-2, 4-3, 4-4, and 4-5 for details).

[0121] The liquid phase diagram of 16-hydroxy-9Z-hexadecenoic acid synthesized using P450-31 is shown below. Figure 3 As shown. To further confirm the synthesis of 16-hydroxy-9Z-hexadecenoic acid, the liquid phase sample was analyzed by mass spectrometry. The LC-MS spectrum is shown below. Figure 4 As shown, using anion exchange mode, the extracted molecular weight was 269, therefore the molecular weight of this substance is 270, consistent with 16-hydroxy-9Z-hexadecenoic acid. To further determine the hydroxylation position, the mass spectrometry sample was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (containing trimethylchlorosilane) and subjected to gas chromatography-mass spectrometry (GC-MS). The GC-MS chromatogram is shown below. Figure 5 As shown, the main peak of the gaseous fragment is 73, and the gaseous results can be compared with those obtained by searching the NIST database. Figure 6 The hydroxylation products shown matched, thus further confirming the synthesis of 16-hydroxy-9Z-hexadecenoic acid.

[0122] Therefore, it can be determined that the P450 hydroxylase P450-31 obtained by screening in this invention can catalyze the synthesis of 16-hydroxy-9Z-hexadecenoic acid from 16-carbon,Δ9 monounsaturated fatty acids, and that the P450-31 screened in this invention has higher catalytic efficiency than the reported P450 hydroxylases.

[0123] Table 4-1 Ability of P450 and BMR from different sources to synthesize 16-hydroxy-9Z-hexadecenoic acid

[0124]

[0125] Note: - represents no synthesized product, + represents a yield of 1-10 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 1 mg / L but excluding the endpoint value of 10 mg / L), ++ represents a yield of 10-30 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 10 mg / L but excluding the endpoint value of 30 mg / L), +++ represents a yield of 30-50 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 30 mg / L but excluding the endpoint value of 50 mg / L), ++++ represents a yield of 50-100 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 50 mg / L but excluding the endpoint value of 100 mg / L), and +++++ represents a yield of 100 mg / L or more of 16-hydroxy-9Z-hexadecenoic acid.

[0126] Table 4-2 Ability of P450 and RhFRed from different sources to synthesize 16-hydroxy-9Z-hexadecenoic acid

[0127]

[0128] Note: - represents no synthesized product, + represents a yield of 1-10 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 1 mg / L but excluding the endpoint value of 10 mg / L), ++ represents a yield of 10-30 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 10 mg / L but excluding the endpoint value of 30 mg / L), +++ represents a yield of 30-50 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 30 mg / L but excluding the endpoint value of 50 mg / L), ++++ represents a yield of 50-100 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 50 mg / L but excluding the endpoint value of 100 mg / L), and +++++ represents a yield of 100 mg / L or more of 16-hydroxy-9Z-hexadecenoic acid.

[0129] Table 4-3 Ability of P450 from different sources to synthesize 16-hydroxy-9Z-hexadecenoic acid with camB-camA

[0130]

[0131] Note: - represents no synthesized product, + represents a yield of 1-10 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 1 mg / L but excluding the endpoint value of 10 mg / L), ++ represents a yield of 10-30 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 10 mg / L but excluding the endpoint value of 30 mg / L), +++ represents a yield of 30-50 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 30 mg / L but excluding the endpoint value of 50 mg / L), ++++ represents a yield of 50-100 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 50 mg / L but excluding the endpoint value of 100 mg / L), and +++++ represents a yield of 100 mg / L or more of 16-hydroxy-9Z-hexadecenoic acid.

[0132] Table 4-4 Ability of P450 from different sources to synthesize 16-hydroxy-9Z-hexadecenoic acid with petH-petF

[0133]

[0134] Note: - represents no synthesized product, + represents a yield of 1-10 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 1 mg / L but excluding the endpoint value of 10 mg / L), ++ represents a yield of 10-30 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 10 mg / L but excluding the endpoint value of 30 mg / L), +++ represents a yield of 30-50 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 30 mg / L but excluding the endpoint value of 50 mg / L), ++++ represents a yield of 50-100 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 50 mg / L but excluding the endpoint value of 100 mg / L), and +++++ represents a yield of 100 mg / L or more of 16-hydroxy-9Z-hexadecenoic acid.

[0135] Table 4-5 Ability of P450 from different sources to synthesize 16-hydroxy-9Z-hexadecenoic acid with AtATR2

[0136]

[0137] Note: - represents no synthesized product, + represents a yield of 1-10 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 1 mg / L but excluding the endpoint value of 10 mg / L), ++ represents a yield of 10-30 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 10 mg / L but excluding the endpoint value of 30 mg / L), +++ represents a yield of 30-50 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 30 mg / L but excluding the endpoint value of 50 mg / L), ++++ represents a yield of 50-100 mg / L of 16-hydroxy-9Z-hexadecenoic acid (including the endpoint value of 50 mg / L but excluding the endpoint value of 100 mg / L), and +++++ represents a yield of 100 mg / L or more of 16-hydroxy-9Z-hexadecenoic acid.

[0138] Example 5 Whole-cell catalytic synthesis of ω-hydroxy fatty acids

[0139] To further expand the practicality of the screened P450 hydroxylase P450-31, the engineered strain FAEc119 and the control strain FAEc132 were subjected to whole-cell catalytic reactions using various fatty acids as substrates. The specific method is as follows: Recombinant Escherichia coli FAEc119 and control strain FAEc132 glycerol bacteria were inoculated at a volume of 0.2% into test tubes containing 5 mL LB liquid medium (with 50 mg / L kanamycin and 50 mg / L streptomycin). After overnight incubation at 37°C, the bacteria were inoculated at a volume of 1% into 250 mL shake flasks containing 50 mL LB liquid medium (with 50 mg / L kanamycin and 50 mg / L streptomycin). The culture was shaken at 37°C until the OD600 reached 0.8. IPTG, ALA (δ-aminolevulinic acid), and FeSO4 were added to a final concentration of 0.1 mM, 0.5 mM, and 0.1 mM. After induction culture in a shaker at 30°C for 20 hours, the bacterial cells were collected by centrifugation at 12000 rpm. The bacterial cells were controlled to OD600=30 and resuspended in 0.1 mM KPB buffer.

[0140] The following reaction system was prepared using the cell slurry: 2 mL cell slurry, 100 µL fatty acid (the mother liquor was 20 mM fatty acid stored in DMSO), 26.6 µL 60% glycerol, and 132 µL 6 g / L glucose. The system was reacted at 30 °C and 220 rpm for 4 h. Then, 200 µL 6 M hydrochloric acid and 2 mL ethyl acetate were added, and the mixture was shaken for 10 min. After centrifugation, the supernatant was collected, and the amount of ω-hydroxy fatty acid synthesized in the reaction system was determined by HPLC.

[0141] The whole-cell catalysis results are shown in Tables 5 and 6. The results indicate that P450-31, screened in this patent, also shows a significant advantage over the reported CYP153A33 in catalyzing the synthesis of ω-hydroxy products from C14 saturated fatty acids and C18,Δ9, monounsaturated fatty acids. This suggests that P450-31 screened in this patent has certain substrate universality. To further determine the 14-hydroxy-C14 saturated fatty acids, 16-hydroxy-C16 saturated fatty acids, and 18-hydroxy-C18-Δ9-monounsaturated fatty acids synthesized from C14 saturated fatty acids, C16 saturated fatty acids, and C18,Δ9, monounsaturated fatty acids by P450-31, mass spectrometry was performed on the liquid phase samples. The LC-MS spectra are shown below. Figure 2 As shown.

[0142] Table 5. FAEc119-catalyzed fatty acid synthesis of ω-hydroxy fatty acids

[0143]

[0144] Table 6. FAEc132-catalyzed fatty acid synthesis of ω-hydroxy fatty acids

[0145]

[0146] Example 6 Construction of recombinant Escherichia coli producing ω-hydroxy fatty acids

[0147] To enable Escherichia coli to directly synthesize ω-hydroxy fatty acids from a simple carbon source, TesA' (nucleotide sequence SEQ ID NO: 9), obtained by removing the signal peptide region of the thioesterase TesA from E. coli, was constructed into the vectors pRSF-P450-31 and pRSF-CYP153A33, respectively, to obtain pRSF-P450-31-tesA' and pRSF-CYP153A33-tesA'. These were then co-introduced into E. coli BL21(DE3) with the well-selected CPR plasmid pCDF-camB-camA, resulting in engineered strains HFA01 and HFA02 that directly produce ω-hydroxy fatty acids from a simple carbon source.

[0148] Example 7: Synthesis of ω-hydroxy fatty acids from a simple carbon source by recombinant Escherichia coli

[0149] Engineered strains HFA01 and HFA02 were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain seed culture. The seed culture was then inoculated into fresh fermentation medium at a volume of 1% and cultured at 37°C and 220 rpm for 2 h. After that, 0.1 mM IPTG, 0.5 mM ALA (δ-aminolevulinic acid), and 0.1 mM FeSO4 were added to induce fermentation at 30°C and 220 rpm for 70 h. The yield of ω-hydroxy fatty acids was detected by high performance liquid chromatography.

[0150] The fermentation medium formulation includes: ammonium chloride 6 g / L, potassium dihydrogen phosphate 8.5 g / L, citric acid 0.5 g / L, yeast extract 5 g / L, glycerol 30 g / L, magnesium sulfate heptahydrate 1 g / L, calcium chloride dihydrate 0.07 g / L, trace element stock solution 4 mL / L, thiamine 100 mg / L, MOPS 50 g / L, kanamycin 50 mg / L, and streptomycin 50 mg / L. The trace element stock solution formulation includes: ferric chloride hexahydrate 27 g / L, zinc chloride 2 g / L, sodium molybdate dihydrate 2 g / L, copper sulfate pentahydrate 1.9 g / L, and boric acid 0.5 g / L.

[0151] The experimental results are shown in Table 7. The results indicate that after 72 hours of fermentation, the engineered strain HFA01 containing CYP153A33 could synthesize 68.14 mg / L of 16-hydroxy-9Z-hexadecenoic acid, while the engineered strain HFA02 containing P450-31 obtained through screening in this patent could synthesize 177.27 mg / L of 16-hydroxy-9Z-hexadecenoic acid. This demonstrates that P450-31 screened in this invention can more efficiently catalyze the synthesis of 16-hydroxy-9Z-hexadecenoic acid from 16-carbon,Δ9 monounsaturated fatty acids.

[0152] In addition to 16-hydroxy-9Z-hexadecenoic acid, after 72 h of fermentation, the engineered strain HFA02 can also synthesize 12-hydroxy-dodecanoic acid, 14-hydroxy-tetradecanoic acid, 16-hydroxy-hexadecanoic acid, and 18-hydroxy-11Z-octadecenoic acid, and the corresponding peak areas are significantly higher than those of the engineered strain HFA01 (Table 8).

[0153] Table 7. Yield of 16-hydroxy-9Z-hexadecenoic acid synthesized in vivo by recombinant engineered strains

[0154]

[0155] Table 8 Peak area of ​​ω-hydroxy fatty acid synthesis in recombinant engineered strains

[0156]

[0157] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: The present invention is the first to discover that cytochrome P450 hydroxylases with amino acid sequences such as 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 can efficiently catalyze the synthesis of ω-hydroxy fatty acids from fatty acids.

[0158] In particular, the cytochrome P450 hydroxylase P450-31 (with the amino acid sequence shown in SEQ ID NO: 31) catalyzes the synthesis of 16-hydroxy-9Z-hexadecenoic acid from 16-carbon,Δ9 monounsaturated fatty acids and the synthesis of ω-hydroxy products from C14 saturated fatty acids and C18,Δ9 monounsaturated fatty acids with significantly higher efficiency than currently reported P450 hydroxylases.

[0159] Simultaneously, an in vivo fatty acid synthesis strain was constructed, and P450 hydroxylase P450-31 and reductase were co-expressed. Through fermentation, ω-hydroxy fatty acids were synthesized from a simple carbon source. The yields of synthesized 16-hydroxy-9Z-hexadecanoic acid, 12-hydroxy-dodecanoic acid, 14-hydroxy-tetradecanoic acid, 16-hydroxy-hexadecanoic acid, and 18-hydroxy-11Z-octadecanoic acid were significantly higher than those reported using currently reported P450 hydroxylases. Compared to other P450 hydroxylases in existing technologies, this enzyme exhibits high activity and high yield of ω-hydroxy fatty acids, possessing unique advantages and promising applications in the microbial industrial production of ω-hydroxy fatty acids.

[0160] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for biosynthesizing ω-hydroxy fatty acids, characterized in that, The method includes: The ω-hydroxy fatty acid was obtained by catalyzing the reaction of fatty acids with P450 hydroxylase. The P450 hydroxylase has any one of the following amino acid sequences: 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. The method according to claim 1, characterized in that, The catalytic reaction also includes a redox partner.

3. The method according to claim 2, characterized in that, The fatty acid is selected from any one of the following: C12 saturated fatty acid, C14 saturated fatty acid, C16 saturated fatty acid, C16 monounsaturated fatty acid, or C18 monounsaturated fatty acid.

4. The method according to claim 3, characterized in that, The redox chaperone is selected from any one of the following: The reductase domain BMR of the enzyme CYP102A1 from Bacillus megaterium P450; The reductase domain RhFRed is derived from the P450 enzyme CYP116B2 of Rhodococcus spp.; The redox synergies CamA and CamB derived from *Pseudomonas putida*; The redox chaperones PetH and PetF are derived from Synechocystis. Or it may be derived from the reductase AtATR2 of type II P450 in the plant Arabidopsis thaliana.

5. The method according to claim 4, characterized in that, The amino acid sequence of BMR is shown in SEQ ID NO: 44; the amino acid sequence of RhFRed is shown in SEQ ID NO: 45; the amino acid sequence of CamA is shown in SEQ ID NO: 46; the amino acid sequence of CamB is shown in SEQ ID NO: 47; the amino acid sequence of PetH is shown in SEQ ID NO: 48; the amino acid sequence of PetF is shown in SEQ ID NO: 49; and the amino acid sequence of AtATR2 is shown in SEQ ID NO:

50.

6. The method according to claim 5, characterized in that, The method includes: The bacterial cells, the fatty acid, and the carbon source were mixed and subjected to a hydroxylation reaction to obtain the ω-hydroxy fatty acid; The bacterial cells are capable of expressing the P450 hydroxylase and the redox chaperone.

7. The method according to claim 6, characterized in that, The method for preparing the bacterial cells includes: The first recombinant cells, cultured overnight, were inoculated into the first culture medium for the first expansion culture. The culture was carried out until the OD600 reached 0.6-0.

8. An inducer and cofactor were added to induce expression for 24-48 hours. The precipitate was collected by centrifugation to obtain the bacterial cells. The first recombinant cell is able to express the P450 hydroxylase and the redox chaperone.

8. The method according to claim 7, characterized in that, The carbon source includes glucose and glycerol; the final concentration of glucose is 0.2 g / L-0.5 g / L; and the final concentration of glycerol is 5 g / L-10 g / L.

9. The method according to claim 8, characterized in that, The final concentration of the fatty acid is 0.5 mM-1 mM.

10. The method according to claim 8, characterized in that, The first culture medium is LB medium.

11. The method according to claim 10, characterized in that, The conditions for the first expansion culture were 30℃-37℃ and 200rpm-220rpm.

12. The method according to claim 11, characterized in that, The conditions for inducing expression are 28℃-30℃ and 200rpm-220rpm.

13. The method according to claim 5, characterized in that, The method includes: The second recombinant cells, cultured overnight, were seeded into a second culture medium for a second expansion culture. The culture was carried out until the OD600 reached 0.6-0.

8. Inducing agents and cofactors were added, and fermentation was induced for 48-72 hours to obtain the ω-hydroxy fatty acids. The second recombinant cell is capable of expressing the P450 hydroxylase, the redox chaperone, and the thioesterase.

14. The method according to claim 13, characterized in that, The second culture medium consists of: ammonium chloride 4-6 g / L, potassium dihydrogen phosphate 5-10 g / L, citric acid 0.3-0.5 g / L, yeast extract 3-5 g / L, glycerol 15-30 g / 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. The method according to claim 14, characterized in that, The formulation of the metal trace element storage solution includes: 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. The method according to claim 15, characterized in that, The thioesterase is derived from Escherichia coli.

17. The method according to claim 16, characterized in that, The amino acid sequence of the thioesterase derived from the *Escherichia coli* is shown in SEQ ID NO:

9.

18. The method according to claim 17, characterized in that, The conditions for the second expansion culture were 30℃-37℃ and 200rpm-220rpm.

19. The method according to claim 18, characterized in that, The conditions for induced fermentation are 28℃-30℃ and 200rpm-220rpm.

20. The method according to any one of claims 7 to 19, characterized in that, The inducing agent is IPTG; the final concentration of IPTG is 0.1 mM-1 mM.

21. The method according to claim 20, characterized in that, The cofactors include δ-aminolevulinic acid and FeSO4; wherein the final concentration of δ-aminolevulinic acid is 0.3-0.5 mM; and the final concentration of FeSO4 is 0.05-0.1 mM.

22. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria include a gene encoding P450 hydroxylase; The P450 hydroxylase has any one of the following amino acid sequences: 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.

23. The genetically engineered bacterium according to claim 22, characterized in that, The genetically engineered bacteria also include genes encoding redox chaperones.

24. The genetically engineered bacterium according to claim 23, characterized in that, The redox chaperone is selected from any one of the following: The reductase domain BMR of the enzyme CYP102A1 from Bacillus megaterium P450; The reductase domain RhFRed is derived from the P450 enzyme CYP116B2 of Rhodococcus spp.; The redox synergies CamA and CamB derived from *Pseudomonas putida*; The redox chaperones PetH and PetF are derived from Synechocystis. Or it may be derived from the reductase AtATR2 of type II P450 in the plant Arabidopsis thaliana.

25. The genetically engineered bacteria according to claim 24, characterized in that, The amino acid sequence of BMR is shown in SEQ ID NO: 44; the amino acid sequence of RhFRed is shown in SEQ ID NO: 45; the amino acid sequence of CamA is shown in SEQ ID NO: 46; the amino acid sequence of CamB is shown in SEQ ID NO: 47; the amino acid sequence of PetH is shown in SEQ ID NO: 48; the amino acid sequence of PetF is shown in SEQ ID NO: 49; and the amino acid sequence of AtATR2 is shown in SEQ ID NO:

50.

26. The genetically engineered bacteria according to claim 25, characterized in that, The genetically engineered bacteria also include genes encoding thioesterases.

27. The genetically engineered bacterium according to claim 26, characterized in that, The thioesterase is derived from Escherichia coli.

28. The genetically engineered bacteria according to claim 27, characterized in that, The amino acid sequence of the thioesterase derived from the *Escherichia coli* is shown in SEQ ID NO:

9.

29. The method for biosynthesizing ω-hydroxy fatty acids according to any one of claims 1 to 21, or the use of genetically engineered bacteria according to any one of claims 22 to 28 in the synthesis of ω-hydroxy fatty acids.

Images