Cytochrome P450 oxidase HPO and application thereof in synthesis of noottanone

Abstract

The invention discloses a cytochrome P450 oxidase HPO and application thereof in synthesis of noottanone, and by combining modification of a target gene of the cytochrome P450 oxidase HPO and metabolic engineering optimization modification of an expression host pichia pastoris genome, the shake flask yield of the noottanone is remarkably increased to 356.93 mg / L; compared with the prior art, the high-yield strain is improved by 185%; furthermore, recombinant pichia pastoris is used for high-density fermentation, so that the yield of noottanone reaches 3365.36 mg / L, and a foundation is laid for industrialization of biosynthesis of noottanone.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology, and in particular relates to a cytochrome P450 oxidase HPO and its application in the synthesis of grapefruit ketone. Background Art

[0002] Nootkatone, also known as nootkatone, is a natural sesquiterpenoid compound with a unique citrus aroma and multiple biological activities. Its chemical formula is C 15 H 22 O, whose core structure consists of three isoprene units, contains a unique bicyclic backbone and a keto functional group. This complex structure confers unique biological activities, including anti-carcinogenic, antioxidant, anti-inflammatory, and antiviral effects.

[0003] In the food industry, oleic acid ketone is widely used as a natural flavoring, particularly in beverages and candies, due to its strong citrus aroma. In the pharmaceutical field, its antioxidant and anti-inflammatory properties are being used to develop functional drugs, such as those that inhibit tumor cell proliferation and alleviate inflammatory responses. Furthermore, oleic acid ketone has been shown to inhibit a variety of pathogens, such as malaria parasites and dengue virus, demonstrating potential anti-infective applications. The antioxidant properties of oleic acid ketone make it an active ingredient in cosmetics for anti-aging and skin care products. Its natural origin and safety further enhance its potential for application in high-end cosmetics.

[0004] There are various methods for extracting naringone, including extraction, distillation, and chemical synthesis. Due to the low vapor pressure of naringone at atmospheric pressure, extraction and distillation methods are ineffective, resulting in low oil yields and naringone content. Since valencene is a precursor to naringone, naringone can also be obtained through oxidative synthesis using valencene as a raw material. However, chemical synthesis has three drawbacks: first, the raw material valencene is extremely scarce and expensive; second, the synthetic product yields a racemic mixture of naringone, requiring an additional resolution step to obtain the natural dextrorotatory naringone, significantly reducing yield and increasing costs; and third, the chemical synthesis process involves hazardous reagents and costly processes. To overcome the limitations of natural extraction and chemical synthesis, biosynthesis of naringone has become a research hotspot. Biosynthesis, typically catalyzed by microorganisms, avoids the toxic reagents and heavy metal catalysts used in traditional chemical synthesis, reducing environmental pollution. Biosynthesis also offers advantages such as environmental friendliness, mild reaction conditions, a wide range of raw material sources, and sustainability, making it an attractive alternative to traditional production methods. Lu Wenyu and Guo Xiaoyan from Tianjin University used recombinant Yarrowia lipolytica to achieve a 0.5 mg / L yield of tautomerone (CN107723252A); the team of Li Shuang and Liu Tong from South China University of Technology optimized a mutant of valencene oxidase to achieve a 322.62 mg / L yield of tautomerone (CN115927488A). Although the biosynthetic pathway of tautomerone has been established, its production in commonly used yeast hosts is still very low. In addition, the semi-rational modification strategies for cytochrome P450 oxidase HPO reported so far are mainly site-directed mutagenesis, that is, single-site or multi-site mutations are performed on the wild type of HPO, and mutants with improved catalytic efficiency are obtained through screening. Although these mutants have improved catalytic activity, the yield of their final products often still fails to reach the ideal level. Therefore, there is still an unmet demand for HPO with stronger catalytic performance. Summary of the Invention

[0005] The present invention aims to provide a mutant of cytochrome P450 oxidase HPO and its application in the biosynthesis of oleic acid ketone, thereby solving the problems of poor cytochrome P450 oxidase HPO activity and unsatisfactory oleic acid ketone production in the prior art.

[0006] In one aspect, the present invention provides a cytochrome P450 oxidase HPO, wherein the cytochrome P450 oxidase HPO comprises the amino acid sequence shown in SEQ ID NO: 2.

[0007] In another aspect, the present invention provides a polynucleotide encoding the cytochrome P450 oxidase HPO according to any embodiment herein.

[0008] Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:4.

[0009] In another aspect, the present invention provides an expression vector comprising the polynucleotide according to any embodiment herein.

[0010] In one or more embodiments, the expression vector comprises an EgVS gene expression cassette.

[0011] Preferably, the expression vector comprises an EgVS gene expression cassette and a tHMG1 gene expression cassette, and the ΔADH900 and II-5 gene clusters are knocked out. More preferably, the EgVS gene expression cassette is located at the sites where the knocked-out ΔADH900 and II-5 gene clusters were originally located, respectively, with the number of copies of the EgVS gene expression cassette at the site where the knocked-out II-5 gene cluster was originally located being two; and the tHMG1 gene expression cassette is located at the site where the knocked-out ΔADH900 gene cluster was originally located. More preferably, the EgVS gene expression cassette and the tHMG1 gene expression cassette are located at the site where the knocked-out ΔADH900 gene cluster was originally located, and the EgVS gene expression cassette and the tHMG1 gene expression cassette are linked sequentially.

[0012] Preferably, the EgVS gene expression cassette consists of a pGAP promoter, an EgVS gene, and a PMP20 terminator. More preferably, the sequence of the EgVS gene is shown in SEQ ID NO:5.

[0013] Preferably, the tHMG1 gene expression cassette consists of a pGAP promoter, a tHMG1 gene, and a Gcw14 terminator. More preferably, the sequence of the tHMG1 gene is shown in SEQ ID NO: 6.

[0014] In one or more embodiments, the expression vector comprises one or more selected from the group consisting of an ERG10 gene expression cassette, an ERG20 gene expression cassette, a PHXT1 promoter, a ScUTR1 gene expression cassette, a zwf1 gene expression cassette, a sol3 gene expression cassette, aprt gene expression cassette, an ICE2p gene expression cassette, a RAD52 gene expression cassette, and a GCS gene expression cassette.

[0015] Preferably, the expression vector comprises an ERG10 gene expression cassette and an ERG20 gene expression cassette, and the I-2 gene cluster and the II-4 gene cluster are knocked out. More preferably, the ERG10 gene expression cassette is located at the site where the knocked-out I-2 gene cluster was originally located, and the ERG20 gene expression cassette is located at the site where the knocked-out II-4 gene cluster was originally located.

[0016] Preferably, the expression vector comprises the PHXT1 promoter, and the endogenous promoter of the ERG9 gene is knocked out. More preferably, the PHXT1 promoter is located at the site where the endogenous promoter of the knocked-out ERG9 gene was originally located.

[0017] Preferably, the expression vector comprises a ScUTR1 gene expression cassette, a zwf1 gene expression cassette, a sol3 gene expression cassette, and an aprt gene expression cassette, and the IV-9 gene cluster is knocked out. More preferably, the ScUTR1 gene expression cassette, the zwf1 gene expression cassette, the sol3 gene expression cassette, and the aprt gene expression cassette are located at the site where the knocked-out IV-9 gene cluster was originally located. More preferably, the ScUTR1 gene expression cassette, the zwf1 gene expression cassette, the sol3 gene expression cassette, and the aprt gene expression cassette are linked sequentially.

[0018] Preferably, the expression vector comprises an ICE2p gene expression cassette, a RAD52 gene expression cassette, and a GCS gene expression cassette, and the II-9 gene cluster is knocked out. More preferably, the ICE2p gene expression cassette, the RAD52 gene expression cassette, and the GCS gene expression cassette are located at the site where the knocked-out II-9 gene cluster was originally located. More preferably, the ICE2p gene expression cassette, the RAD52 gene expression cassette, and the GCS gene expression cassette are linked sequentially.

[0019] Preferably, the ERG10 gene expression cassette consists of a pGAP promoter, an ERG10 gene, and a PMP20 terminator. More preferably, the sequence of the ERG10 gene is shown in SEQ ID NO: 7.

[0020] Preferably, the ERG20 gene expression cassette consists of a pGAP promoter, an ERG20 gene, and a PMP20 terminator. More preferably, the sequence of the ERG20 gene is shown in SEQ ID NO:8.

[0021] Preferably, the sequence of the PHXT1 promoter is shown in SEQ ID NO:9.

[0022] Preferably, the ScUTR1 gene expression cassette consists of a pGAP promoter, a ScUTR1 gene, and a PMP20 terminator. More preferably, the sequence of the ScUTR1 gene is shown in SEQ ID NO: 10.

[0023] Preferably, the zwf1 gene expression cassette consists of a pGAP promoter, a zwf1 gene, and a PMP20 terminator. More preferably, the sequence of the zwf1 gene is shown in SEQ ID NO: 11.

[0024] Preferably, the sol3 gene expression cassette consists of a pGAP promoter, a sol3 gene, and a PMP20 terminator. More preferably, the sequence of the sol3 gene is shown in SEQ ID NO: 12.

[0025] Preferably, the aprt gene expression cassette consists of a pGAP promoter, an aprt gene and a PMP20 terminator. More preferably, the sequence of the aprt gene is shown in SEQ ID NO: 13.

[0026] Preferably, the ICE2p gene expression cassette consists of a pGAP promoter, an ICE2p gene, and a PMP20 terminator. More preferably, the sequence of the ICE2p gene is shown in SEQ ID NO: 14.

[0027] Preferably, the RAD52 gene expression cassette consists of a pGAP promoter, a RAD52 gene, and a PMP20 terminator. More preferably, the sequence of the RAD52 gene is shown in SEQ ID NO: 15.

[0028] Preferably, the GCS gene expression cassette consists of a pGAP promoter, a GCS gene and a PMP20 terminator. More preferably, the sequence of the GCS gene is shown in SEQ ID NO: 16.

[0029] More preferably, the sequence of the pGAP promoter is shown as SEQ ID NO: 47; more preferably, the sequence of the PMP20 terminator is shown as SEQ ID NO: 48.

[0030] In another aspect, the present invention provides a host cell comprising the cytochrome P450 oxidase HPO, polynucleotide or expression vector according to any embodiment of the present invention.

[0031] Preferably, the host cell is yeast. More preferably, the host cell is Pichia pastoris.

[0032] In another aspect, the present invention provides a method for producing the cytochrome P450 oxidase HPO as described in any embodiment herein, the method comprising: using the polynucleotide described in any embodiment herein for expression; using the expression vector described in any embodiment herein for expression; or, using the host cell described in any embodiment herein for expression.

[0033] In another aspect, the present invention provides use of the cytochrome P450 oxidase HPO according to any embodiment herein in the synthesis of naringenone.

[0034] In another aspect, the present invention provides a method for preparing naringenone, the method comprising catalyzing the cytochrome P450 oxidase HPO according to any embodiment of the present invention.

[0035] In another aspect, the present invention provides a method for screening a highly active cytochrome P450 oxidase HPO mutant, the method comprising the following steps:

[0036] S1: A homologous structural model of cytochrome P450 oxidase HPO was constructed using Alphafold software, and valencene molecules were docked into the model;

[0037] S2: Generate sequence reconstruction pathway for cytochrome P450 oxidase HPO by ancestral sequence reconstruction (ASR);

[0038] S3: Creation of a cytochrome P450 oxidase HPO mutant library by ancestral combinatorial library directed evolution (CLADE);

[0039] S4: The sequences of the cytochrome P450 oxidase HPO mutants in the library and the molecular structure of valencene were input into the system, and the cytochrome P450 oxidase HPO was subjected to virtual saturation mutagenesis using the DLKcat software, and the turnover number Kcat value was predicted;

[0040] S5: Experimental verification of the activity of cytochrome P450 oxidase HPO mutants with Kcat values ​​higher than the control.

[0041] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0042] 1. The present invention combines the modification of the cytochrome P450 oxidase HPO target gene with the metabolic engineering optimization modification of the expression host Pichia pastoris genome to significantly increase the shake flask yield of naringenone to 356.93 mg / L, which is 185% higher than the high-yielding strain in the prior art. Furthermore, high-density fermentation using the recombinant Pichia pastoris has increased the naringenone yield to 3365.36 mg / L, laying the foundation for the industrialization of the biosynthesis of naringenone.

[0043] 2. The present invention utilizes a molecular docking model to obtain a mutagenesis library through ancestral sequence reconstruction and directed evolution of an ancestral combinatorial library. This is combined with virtual saturation mutagenesis and the prediction of the Kcat value of HPO during mutagenesis to preliminarily screen for HPO mutants with high Kcat and potential high catalytic activity. This provides a new approach for screening HPO mutants and increasing the yield of oleic acid ketone. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the plasmid pGAP-HPO H54A's map.

[0045] Figure 2 It is a graph showing the production of nootkatone by the mutant expression strain.

[0046] Figure 3 This is a graph showing the production of nootkatone by the metabolically engineered strain.

[0047] Figure 4 The correlation between the content of nootkatone, valencene and glucose and OD during the high-density fermentation of recombinant Pichia pastoris 600 Change graph. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0049] The following examples contain experimental methods with specific experimental conditions. Conventional experimental conditions or those recommended by the manufacturer were generally followed. Materials and reagents used were commercially available unless otherwise specified.

[0050] The "HPO" in the present invention is a premnaspirodiene oxygenase derived from the plant Hyoscyamus muticus, which is a cytochrome P450 oxidase.

[0051] The sources or preparation methods of some materials involved in the present invention are as follows:

[0052] LB (Luria-Bertani) medium: Weigh 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. For solid medium, add 2% (2 g / 100 mL) agar powder, dilute to volume with water, and sterilize at 115°C for 20 min.

[0053] YPD (Yeast Extract Peptone Dextrose) medium: Weigh 20 g / L glucose (Dextrose), 10 g / L yeast extract (Yeast extract), and 20 g / L peptone (Peptone). For solid medium, add 2% (2 g / 100 mL) agar powder, dilute to volume with water, and sterilize at 115°C for 20 min.

[0054] YPD-G418 solid medium: Slowly heat 100 mL of YPD solid medium in a microwave oven until completely melted. Rinse the bottle with room-temperature tap water. When the medium temperature drops to the appropriate range, add antibiotics. Gently add 100 μL of 300 μg / mL G418 solution and mix thoroughly. Pour the mixture into a clean culture dish in a laminar flow hood and allow it to cool naturally until solidified before use.

[0055] The 2× PrimeSTAR MAX kit used in PCR was purchased from Takara; the Hieff CloneTM One Step Cloning Kit was purchased from YEASEN; the ExTaq enzyme was purchased from Thermo Scientific; the HieffCloneTM One Step Cloning Kit was purchased from YEASEN; and the Hieff MutTM Site-Directed Mutagenesis Kit (rapid site-directed mutagenesis kit) was purchased from Shanghai Yishen Biotechnology Co., Ltd.

[0056] Trace element solution: Weigh 0.086 g CoCl2·6H2O, 1.91 g EDTA, 0.512 g FeSO4·H2O, 1.02 g ZnSO4·H2O, 0.384 g CaCl2·2H2O, 0.05 g CuSO4, and 0.048 g Na2MoO4, add deionized water, mix, and dilute to 100 mL. Sterilize at 115°C for 20 min and store at 4°C until used.

[0057] Vitamin solution: Weigh 0.1 g pyridoxine, 0.005 g biotin, 0.1 g calcium pantothenate, 0.2 g p-aminobenzoic acid, 0.1 g thiamine hydrochloride, 0.1 g niacin, and 2.5 g inositol, add deionized water and dilute to 100 mL. Sterilize by filtration and store at 4°C until use.

[0058] Fermentation medium:

[0059] Component 1: Weigh 1.25 g of magnesium sulfate, 25 g of yeast extract, and 50 g of peptone, add deionized water, mix, and dilute to 2 L. Sterilize in a fermenter with high-pressure steam at 115°C for 20 min.

[0060] Component 2: Take 50g of anhydrous glucose, add deionized water and mix, and dilute to 100mL. Sterilize with high-pressure steam at 115℃ for 20min. After sterilization is completed and cooled, add 25mL of trace element solution and 30mL of vitamin solution, and pour into the tank during flame inoculation of seed liquid.

[0061] Feed medium: Weigh 400 g of glucose, 10 g of yeast extract, 3.5 g of potassium phosphate, 9 g of potassium dihydrogen phosphate, 0.28 g of sodium sulfate, and 2.5 g of magnesium sulfate, add deionized water, mix, and dilute to 1 L. Sterilize by high-pressure steam at 115°C for 20 min. After sterilization is completed and cooled, add 10 mL of trace element solution and 12 mL of vitamin solution.

[0062] In the present invention, the method for extracting grapefruit ketone from the culture medium is preferably as follows: after the fermentation is completed, the shake flask is allowed to stand, and the organic phase and the aqueous phase are separated, the organic phase is aspirated with a pipette tip and transferred to an EP tube, centrifuged at 1,2000 rpm for 10 minutes, the upper organic phase is taken out, an appropriate amount of anhydrous sodium sulfate is added, and the mixture is thoroughly shaken and mixed, centrifuged at 1,2000 rpm for 10 minutes, and the upper organic phase is aspirated and filtered through a membrane into a gas chromatography bottle as a GC sample to be detected.

[0063] In the present invention, the method for extracting grapefruit ketone from the fermentation culture broth is preferably as follows: after the start of fermentation, sampling is performed from the sample outlet of the fermentation tank every 6 hours; after the fermentation is completed, the fermentation product is allowed to stand, and the organic phase and the aqueous phase are separated, the organic phase is aspirated and transferred to an EP tube, centrifuged at 1,2000 rpm for 10 minutes, the upper organic phase is taken out, an appropriate amount of anhydrous sodium sulfate is added, and the mixture is thoroughly shaken and mixed, centrifuged at 1,2000 rpm for 10 minutes, and the upper organic phase is aspirated and filtered through a membrane into a gas chromatography bottle as a GC sample to be detected.

[0064] Preparation of grapefruit ketone standard solution: Weigh 10 mg of grapefruit ketone standard (purchased from Sigma), dissolve it in n-dodecane solution, and dilute to 1 mL to obtain a grapefruit ketone standard stock solution with a concentration of 10 mg / mL.

[0065] The present invention adopts gas chromatography to carry out qualitative and quantitative analysis of grapefruit ketone, and the preferred detection conditions are as follows:

[0066] Chromatographic column: HP-5 (30 m × 0.32 mm, 0.25 μm, Agilent Technologies, USA); inlet temperature: 250°C; temperature program: starting at 100°C, hold for 4 min, then increasing to 250°C at a rate of 10°C / min, hold for 5 min. Flame ionization detector (FID) temperature: 320°C; split ratio = 5:1, split flow rate: 6 mL / min; air flow rate: 300 mL / min; hydrogen flow rate: 30 mL / min; makeup gas flow rate (N2): 15 mL / min; average linear velocity: 24.802 cm / s; pressure: 7.1413 psi; injection volume: 1 μL.

[0067] In the present invention, the construction of a gene expression cassette or the knockout of a gene cluster can be achieved by conventional CRISPR / Cas9 technology, such as constructing an sgRNA expression vector and a Cas9 protein expression vector, and introducing them into host cells.

[0068] Example 1: Construction of a valencene-producing strain

[0069] In this example, Pichia pastoris GS115 was used as the base strain to construct a valencene-producing strain by optimizing the expression of key enzymes in the MVA pathway, downregulating the competitiveness of the bypass branch, increasing the copy number of the valencene synthase gene (EgVS), and optimizing the supply of NADPH and ATP. The specific process is as follows. Pichia pastoris GS115 is commercially available.

[0070] Construction of EgVS and tHMG1 gene expression cassettes (optimization of valencene synthase expression)

[0071] The ΔADH900 gene cluster in the genome was knocked out using CRISPR / Cas9 technology, and the EgVS (valencene synthase from Eryngium glaciale) gene expression cassette and the tHMG1 gene expression cassette were introduced into the site of the original ΔADH900 gene cluster in the genome. The EgVS gene expression cassette and the tHMG1 gene expression cassette were connected in sequence; wherein, the EgVS gene expression cassette consists of the pGAP promoter, the EgVS gene and the PMP20 terminator (PMP20TT); the tHMG1 gene expression cassette consists of the pGAP promoter, the tHMG1 gene and the Gcw14 terminator (Gcw14TT).

[0072] The II-5 gene cluster in the genome was knocked out using CRISPR / Cas9 technology, and two copies of the EgVS gene expression cassette were introduced into the site where the II-5 gene cluster was originally located in the genome.

[0073] The sequence of the EgVS gene is shown in SEQ ID NO: 5, and the sequence of the tHMG1 gene is shown in SEQ ID NO: 6.

[0074] Construction of ERG10 and ERG20 gene expression cassettes (MVA pathway optimization)

[0075] The I-2 gene cluster was knocked out using CRISPR / Cas9 technology. An ERG10 gene expression cassette was introduced into the genome at the site where the I-2 gene cluster originally resided. The ERG10 gene expression cassette consisted of the pGAP promoter, the ERG10 gene, and the PMP20 terminator (PMP20TT).

[0076] The II-4 gene cluster of the genome was knocked out using CRISPR / Cas9 technology. An ERG20 gene expression cassette was introduced into the genome at the site where the II-4 gene cluster was originally located. The ERG20 gene expression cassette consists of the pGAP promoter, the ERG20 gene, and the PMP20 terminator (PMP20TT).

[0077] The sequence of the ERG10 gene is shown in SEQ ID NO: 7, and the sequence of the ERG20 gene is shown in SEQ ID NO: 8.

[0078] Promoter replacement (competitive downregulation of bypass branches)

[0079] The endogenous promoter of the ERG9 gene in the genome was replaced with the PHXT1 promoter using CRISPR / Cas9 technology. The sequence of the PHXT1 promoter is shown in SEQ ID NO: 9.

[0080] Construction of ScUTR1, zwf1, sol3, and aprt gene expression cassettes (NADPH and ATP expression optimization)

[0081] Using CRISPR / Cas9 technology, the IV-9 gene cluster was knocked out of the genome. The ScUTR1, zwf1, sol3, and aprt gene expression cassettes were then introduced into the genome at the site where the IV-9 gene cluster originally resided. The ScUTR1, zwf1, sol3, and aprt gene expression cassettes were sequentially linked. Each expression cassette consists of the pGAP promoter, the ScUTR1, zwf1, sol3, or aprt gene, and the PMP20 terminator (PMP20TT).

[0082] The sequence of the ScUTR1 gene is shown in SEQ ID NO: 10, the sequence of the zwf1 gene is shown in SEQ ID NO: 11, the sequence of the sol3 gene is shown in SEQ ID NO: 12, and the sequence of the aprt gene is shown in SEQ ID NO: 13.

[0083] The above expression cassettes were constructed into the genome of Pichia pastoris GS115 to obtain the valencene-producing strain H53.

[0084] Example 2: Preparation of cytochrome P450 oxidase HPO mutants

[0085] Liu et al. reported an HPO mutant HPO in 2022 G302G / V480A / V482A / A484A(hereinafter referred to as HPO prototype). In this example, the coding sequence of the mutant (GenBank: EF569601.1) was codon-optimized using Pichia pastoris as the host to obtain the optimized coding sequence, the nucleotide sequence of which is shown in SEQ ID NO: 1. This coding sequence was then cloned into a pGS plasmid vector containing the GAP promoter and the PMP20 terminator to obtain the pGAP-HPO plasmid. The pGS plasmid is commercially available.

[0086] Then, mutants were screened based on the HPO prototype. The valencene molecule was docked into the HPO homology structure model obtained by AlphaFold v2.0 software. First, the most likely sequence reconstruction path was generated for the ancestral protein (HPO) by ancestral sequence reconstruction (ASR), that is, the probability distribution of amino acids at each position on the path was calculated. Then, the ancestral combinatorial library for directed evolution (CLADE) strategy was adopted. CLADE uses the uncertainty of this probability distribution to create an HPO mutant library (mutagenized library). The CLADE results showed that there is a main pathway in the ASR process of HPO, and the relevant mutagenesis library contains HPO. H54X 、HPO D212X 、HPO D115X 、HPO D297X 、HPO K390X etc. mutants (X represents any natural amino acid).

[0087] The sequences of all the aforementioned mutants and the simplified valencene molecular structure were input, and the machine learning function of the DLKcat software was used to perform virtual saturation mutagenesis on the amino acid residues of HPO. The turnover number (Kcat) values ​​of HPO were predicted, and the relative activity of those predicted Kcat values ​​exceeding that of the HPO prototype was experimentally verified. The predicted Kcat values ​​of the mutants are shown in Table 1.

[0088] Table 1: Kcat values ​​of various HPO mutants predicted by DLKcat

[0089]

[0090]

[0091]

[0092] Using the pGAP-HPO plasmid as a template, according to the turnover number Kcat value predicted by DLKcat, mutant HPO with a large change in Kcat value compared with the HPO prototype was selected. H54A 、HPO H54L 、HPO H54F 、HPO D115A 、HPO D115L 、HPO D115F、HPO D212A 、HPO D212L 、HPO D212F 、HPO D297A 、HPO D297L 、HPO D297F 、HPO E304A 、HPO E304L 、HPO E304F 、HPO T305A 、HPO K390A 、HPO K390L and HPO K390F , respectively designed mutation primers, and used PCR technology to perform site-directed mutagenesis on the cytochrome P450 oxidase HPO gene to obtain the expression plasmids of each mutant.

[0093] The primers used for PCR are shown in Table 2. PCR amplification conditions were: initial denaturation at 95°C for 3 minutes, followed by 30 cycles (95°C for 15 seconds, 53°C for 5 seconds, and 72°C for 15 seconds), extension at 72°C for 5 minutes, and storage at 4°C. PCR products were verified by agarose gel electrophoresis and then purified.

[0094] The reduced chaperone protein CPR (AtCPR) gene expression cassette from Arabidopsis thaliana (pTEF1 promoter, AtCPR gene, TP11 terminator (TP11TT)) and the Pichia pastoris endogenous alcohol dehydrogenase ADH3 gene expression cassette (pGAP promoter, ADH3 gene, PMP2 terminator (PMP2TT)) were cloned into the pGAP-HPO plasmid from which the HPO gene expression cassette (GAP promoter, HPO gene and PMP20 terminator) was removed to obtain a helper plasmid; wherein, the AtCPR gene expression cassette and the ADH3 gene expression cassette were sequentially connected.

[0095] The mutant expression plasmid and auxiliary plasmid were digested with Dpn I and ligated with T4 ligase, and then transformed into Escherichia coli DH5α competent cells. After the competent cells were cultured overnight in LB solid medium, clones were selected and cultured in LB liquid medium containing antibiotics. After the plasmid was extracted, the pUC19-R / pUC19-F primers were used for PCR sequencing verification. After the sequencing was correct, the positive mutant plasmid pGAP-HPO was obtained. H54A , pGAP-HPO H54L , pGAP-HPO H54F , pGAP-HPO D115A , pGAP-HPO D115L , pGAP-HPO D115F , pGAP-HPO D212A , pGAP-HPO D212L , pGAP-HPOD212F , pGAP-HPO D297A , pGAP-HPO D297L , pGAP-HPO D297F , pGAP-HPO E304A , pGAP-HPO E304L , pGAP-HPO E304F , pGAP-HPO T305A , pGAP-HPO K390A , pGAP-HPO K390L , pGAP-HPO K390F And auxiliary plasmid pGAP-AtCPR-ADH3. Among them, the exemplary expression vector pGAP-HPO H54A The plasmid map of Figure 1 shown.

[0096] Table 2: PCR primer sequences

[0097]

[0098]

[0099]

[0100] Example 3: Construction of recombinant Pichia pastoris expressing cytochrome P450 oxidase mutants

[0101] The plasmids of the HPO prototype or the plasmids of each mutant and the helper plasmid constructed in Example 2 were respectively introduced into Pichia pastoris strains using the Hieff Clone™ One Step Cloning Kit. The plasmids were then spread on G418-YPD resistant plates and cultured in a 30°C incubator for 2-3 days to obtain Pichia pastoris cytochrome P450 oxidase mutant expression strains H54A, H54L, H54F, D115A, D115L, D115F, D212A, D212L, D212F, D297A, D297L, D297F, E304A, E304L, E304F, T305A, K390A, K390L, K390F, and the HPO prototype expression strain H-0, respectively.

[0102] Table 3: Recombinant HPO expression strains

[0103]

[0104]

[0105] Example 4: Metabolic Engineering Optimization of Cytochrome P450 Oxidase

[0106] Overexpression of ICE2p in yeast can lead to proliferation of the endoplasmic reticulum membrane and the formation of staggered layers, and its overexpression can stabilize the level of intracellular membrane-anchored cytochrome P450 enzymes; the DNA repair protein RAD52 is believed to promote the hydroxylation reaction of P450 enzymes in Pichia pastoris, and overexpression of its gene can improve the biosynthesis efficiency of oleic acid ketone; overexpression of the ceramide synthase gene (GCS) can improve the lipid environment of yeast, promote the correct folding and stability of P450 enzymes, and play a positive role in promoting the biosynthesis of oleic acid ketone.

[0107] In this example, the expression capacity and stability of cytochrome P450 enzymes in an engineered Pichia pastoris strain were optimized based on the above principles. Pichia pastoris H54A constructed in Example 3 was used as the starting strain, part of its genome was knocked out, and several expression cassettes were knocked in to construct a tamarindone-producing strain H60. The specific process is as follows:

[0108] Construction of ICE2p, RAD52 and GCS gene expression cassettes

[0109] CRISPR / Cas9 technology was used to knock out the II-9 gene cluster in the genome, and ICE2p, RAD52, and GCS gene expression cassettes were introduced into the genome at the site where the II-9 gene cluster originally resided. Each expression cassette consists of the pGAP promoter, ICE2p, RAD52, or GCS gene, and the PMP20 terminator (PMP20TT).

[0110] The sequence of the ICE2p gene is shown in SEQ ID NO: 14, the sequence of the RAD52 gene is shown in SEQ ID NO: 15, and the sequence of the GCS gene is shown in SEQ ID NO: 16.

[0111] The above expression cassette was constructed into the genome of the H54A strain to obtain the oleic acid ketone-producing strain H60.

[0112] Example 5: Production of naringenone by shake flask fermentation of recombinant Pichia pastoris

[0113] Using H-0 as a control, mutant expression strains constructed in Examples 3 and 4 were cultured in a test tube containing 5 mL of YPD medium for 24 h. Afterwards, 500 μL of the culture solution was inoculated into a conical flask containing 50 mL of YPD medium and cultured for a further 12 h to obtain a seed solution. The OD600 of the seed solution was measured, and an appropriate amount was inoculated into a 250 mL conical flask containing 50 mL of YPD medium for fermentation, resulting in an initial OD600 value of 0.15 for the fermentation broth. Because oleic acid is volatile and insoluble in water, 2% n-dodecane was added to the fermentation broth 24 h after fermentation to conduct bidirectional fermentation to reduce volatile losses of oleic acid. The oleic acid yield was measured on the 7th day of fermentation.

[0114] like Figure 2 and Figure 3 As shown, the naringenone production of strain H54A was significantly improved compared with the wild-type expression strain, with a yield of 287.67 mg / L ( Figure 2 ), the increase ratio was 130%; and the naringone production of strain H60 (overexpressing ICE2p, RAD52 and GCS genes) reached 356.93 mg / L ( Figure 3 ), which is 1.24 times the yield of the H54A mutant strain (287.67 mg / L), and the yield is increased by 185%.

[0115] Example 6: Production of naringenone by high-density fermentation of recombinant Pichia pastoris

[0116] Seed culture: The mutant expression strain H60 constructed in Example 4 was selected and inoculated into a culture tube containing 5 mL of YPD liquid medium, and cultured in a shaking incubator at 30°C and 220 rpm for about 24 h. 1.5 mL of the culture solution was taken out and inoculated into a 500 mL shake flask containing 150 mL of YPD liquid medium and cultured in a bed at 30°C and 220 rpm for about 24 h to obtain the seed solution.

[0117] Fermentation: All seed liquid was inoculated into a 5 L fermenter containing 3 L fermentation medium and fermented for 7 days (168 h). The fermentation temperature was controlled at 30°C, the stirring speed was controlled in the range of 300-850 rpm, the pH was maintained at 5.5-6.0, and the dissolved oxygen (OD) was controlled at 30%.

[0118] Intermittent feeding: The fermenter is equipped with a glucose concentration monitoring device. When the glucose in the fermentation broth is completely consumed, the glucose can be replenished by adding an appropriate amount of feed medium to the fermenter. After that, 60% (w / v) glucose solution can be added every 24 hours.

[0119] The results of intermittent fed-batch fermentation are as follows Figure 4 As shown, the naringenone production of strain H60 reached 3365.36 mg / L, which was 9.4 times that of shake flask fermentation.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention is conventional technology.

Claims

1. A cytochrome P450 oxidase HPO, characterized in that The cytochrome P450 oxidase HPO comprises the amino acid sequence shown in SEQ ID NO:

2.

2. A polynucleotide, characterized in that The polynucleotide encodes the cytochrome P450 oxidase HPO according to claim 1.

3. An expression vector, characterized in that The expression vector comprises the polynucleotide according to claim 2.

4. The expression vector according to claim 3, wherein The expression vector includes an EgVS gene expression cassette.

5. The expression vector according to claim 4, wherein The expression vector comprises one or more selected from the group consisting of an ERG10 gene expression cassette, an ERG20 gene expression cassette, a ScUTR1 gene expression cassette, a zwf1 gene expression cassette, a sol3 gene expression cassette, aprt gene expression cassette, an ICE2p gene expression cassette, a RAD52 gene expression cassette and a GCS gene expression cassette.

6. A host cell, characterized in that The host cell comprises the cytochrome P450 oxidase HPO according to claim 1, the polynucleotide according to claim 2, or the expression vector according to claim 3.

7. A method for producing the cytochrome P450 oxidase HPO according to claim 1, characterized in that: The method comprises: using the polynucleotide of claim 2 for expression; using the expression vector of any one of claims 3 to 5 for expression; or using the host cell of claim 6 for expression.

8. Use of the cytochrome P450 oxidase HPO as claimed in claim 1 in the synthesis of grapefruit ketone.

9. A method for preparing grapefruit ketone, characterized in that: The method comprises using the cytochrome P450 oxidase HPO as claimed in claim 1 for catalysis.

10. A method for screening highly active cytochrome P450 oxidase HPO mutants, characterized in that: The method comprises the following steps: S1: A homologous structural model of cytochrome P450 oxidase HPO was constructed using Alphafold software, and valencene molecules were docked into the model; S2: Generate sequence reconstruction pathway for cytochrome P450 oxidase HPO by ancestral sequence reconstruction (ASR); S3: Creation of a cytochrome P450 oxidase HPO mutant library by ancestral combinatorial library directed evolution (CLADE); S4: The sequences of the cytochrome P450 oxidase HPO mutants in the library and the molecular structure of valencene were input into the system, and the cytochrome P450 oxidase HPO was subjected to virtual saturation mutagenesis using the DLKcat software, and the turnover number Kcat value was predicted; S5: Experimental verification of the activity of cytochrome P450 oxidase HPO mutants with Kcat values ​​higher than the control.

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