Cytochrome P450 oxidase HPO and application thereof in synthesis of noottanone

Abstract

The invention discloses a cytochrome P450 oxidase HPO as well as a coding polynucleotide, an expression vector, a host cell and a production method of the cytochrome P450 oxidase HPO. The invention also discloses an application of the cytochrome P450 oxidase HPO in catalytic conversion of the valarene into the noottanone, and a method for converting the valarene into the noottanone. The invention provides the cytochrome P450 oxidase HPO mutant and the application of the cytochrome P450 oxidase HPO mutant in synthesis of noottanone in yeast by means of genetic engineering and enzyme engineering, the yield of noottanone is increased by providing the cytochrome P450 oxidase HPO mutant with improved catalytic activity, and conditions are created for industrial production of noottanone.

Description

Technical Field

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

[0002] Nootkatone can be derived from the oxidation of valencene, a sesquiterpene compound. It is a flavor-type sesquiterpene with a grapefruit aroma. It is also known as nootkatone and cedar ketone. Its chemical formula is C 15 H 22 O is a naturally occurring hydrophobic organic compound with high chemical stability, a melting point of 36-37°C, and is soluble in organic reagents such as ethanol and n-dodecane. In addition to its aromatic smell, oleanone has many biological properties, including antibacterial, insecticidal, antioxidant, anti-inflammatory and cardioprotective functions, so it is widely used in industries such as spices, cosmetics, food and medicine. Studies have found that oleanone exhibits significant inhibitory effects on a variety of Gram-positive bacteria, especially Staphylococcus aureus, Listeria monocytogenes and Corynebacterium diphtheriae. Among them, the inhibitory effect on Corynebacterium diphtheriae is the most significant, which makes oleanone a potential candidate for the development of new antibacterial agents. oleanone also has excellent insecticidal effects. It has been proven to be effective against ticks, cockroaches, mosquitoes, termites and lice. The U.S. Environmental Protection Agency (EPA) has officially approved oleanone for use as an insecticide. Related studies have shown that oleanone has excellent antioxidant activity, can inhibit the generation of reactive oxygen species (ROS), scavenge free radicals, and play an anti-inflammatory role. In addition, oleocanthal was shown to significantly reduce myocardial damage in mice, resulting in an increase in antioxidant proteins and a decrease in oxidative stress in the mouse hearts.

[0003] There are three common ways to synthesize oleanone: natural plant extraction, chemical synthesis and microbial synthesis. The traditional natural plant extraction method is costly, has a long cycle and a complicated extraction process. The oleanone produced by chemical synthesis has safety issues and cannot be added to food and medicine, which limits the use of oleanone. Microbial synthesis has become a strategy with great application potential due to its low cost, sustainability and short cycle.

[0004] The biosynthesis of oleanone can be generated by endogenous farnesyl pyrophosphate (FPP) through three steps of sequential catalysis. First, FPP is catalyzed by valencene synthase to generate valencene, then selectively hydroxylated by cytochrome P450 oxidase combined with P450 reductase (CPR) to generate nokadol, and finally oxidized by dehydrogenase to obtain oleanone. Ouyang et al. used resting cell experiments to screen out premnaspirodiene oxygenase (HPO) from Hyoscyamus muticus, cytochrome P450 reductase (AtCPR) from Arabidopsisthaliana, and alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae. These enzymes play a key role in the synthesis of valencene into oleanone. Through a combination of engineering strategies, the researchers increased the final total terpenoid production to 157 mg / L, a 56-fold increase over the initial value (Ouyang X et al. Stepwise engineering of Saccharomyces cerevisiae to produce (+)-valencene and its related sesquiterpenes [J]. RSC Advances, 2019, 9 (52): 30171-30181.). According to the study, the low catalytic efficiency of the cytochrome P450 oxidase-P450 reductase (HPO-CPR) system has always been the main challenge in the synthesis of valencene. Li Shuang's team obtained a mutant HPO with a catalytic performance 2.54 times that of the initial enzyme through a semi-rational design method. After metabolic engineering and endoplasmic reticulum engineering, 2.39 g / L of nootkatone was successfully obtained through batch fermentation (Liu T, Li W, Chen H, et al. Systematic optimization of HPO-CPR to boost (+)-nootkatone synthesis in engineered Saccharomyces cerevisiae [J]. Journal of Agricultural and Food Chemistry, 2022, 70 (49): 15548-15559.). Although the biosynthetic pathway of nootkatone has been established and the optimization strategy has been gradually updated, valencene is often not completely converted into nootkatone during the fermentation process, which indicates that the hydroxylation process between valencene and nootkatone is always the main rate-limiting step in the production of nootkatone. Therefore, there is still an unmet demand for HPO with stronger catalytic performance. Summary of the invention

[0005] The purpose of the present invention is to provide a mutant of cytochrome P450 oxidase HPO and its application in the biosynthesis of oleanone, so as to solve the problems of poor cytochrome P450 oxidase HPO activity, low valencene conversion rate and unsatisfactory oleanone yield in the prior art.

[0006] In one aspect, the present invention provides a cytochrome P450 oxidase HPO, wherein the cytochrome P450 oxidase HPO comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 12.

[0007] Preferably, the cytochrome P450 oxidase HPO has an amino acid sequence selected from any one of SEQ ID NOs: 1 to 12.

[0008] In one or more embodiments, the cytochrome P450 oxidase HPO comprises the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12.

[0009] In another aspect, the present invention provides a polynucleotide encoding the cytochrome P450 oxidase HPO as described in any embodiment herein.

[0010] In one or more embodiments, the polynucleotide has a nucleotide sequence as shown in any one of SEQ ID NOs: 13-24.

[0011] In one or more embodiments, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:24.

[0012] In another aspect, the present invention provides an expression vector comprising a polynucleotide as described in any of the embodiments herein.

[0013] In another aspect, the present invention provides a host cell comprising the cytochrome P450 oxidase HPO, polynucleotide, or expression vector as described in any embodiment herein.

[0014] Preferably, the host cell is Pichia pastoris.

[0015] In another aspect, the present invention provides a method for producing a cytochrome P450 oxidase HPO as described in any embodiment herein, the method comprising:

[0016] Introducing into cytochrome P450 oxidase HPO (V480A / V482A) (1) I205A mutation; (2) I205C mutation; (3) I205E mutation; (4) I205N mutation; (5) I205T mutation; (6) T483D mutation; (7) T483A mutation; (8) T483E mutation; (9) T483N mutation; (10) T483I mutation; (11) I205N mutation and T483D mutation; or (12) I205N mutation and T483I mutation; or

[0017] Using a polynucleotide as described in any of the embodiments herein for expression; or

[0018] Expressed using an expression vector as described in any of the embodiments herein; or

[0019] The expression is performed using a host cell as described in any of the embodiments herein.

[0020] In another aspect, the present invention provides the use of a cytochrome P450 oxidase HPO as described in any of the embodiments herein for catalyzing the conversion of valencene to nopalethone.

[0021] In another aspect, the present invention provides a method for converting valencene to nopalxone, the method comprising catalyzing using a cytochrome P450 oxidase HPO as described in any of the embodiments herein.

[0022] So far, only several cytochrome P450 oxidase mutants have been identified and tested for (+)-vinacetone biosynthesis, but these cytochrome P450 oxidases have low activity and cannot achieve high-yield preparation of vinacetone. The semi-rational transformation of cytochrome P450 oxidase HPO in current literature reports is usually only from the perspective of site-directed mutagenesis, single-site and multi-site mutations are performed on the improved HPO type to screen out mutants with the highest catalytic efficiency, but the final yield is mostly unsatisfactory. The key invention of the present invention is that by semi-rational transformation of cytochrome P450 oxidase HPO, the heterologous enzyme expression effect is optimized, the HPO activity is greatly improved, and the rate-limiting problem of the reaction process between valencene and vinacetone is solved.

[0023] The present invention obtains cytochrome P450 oxidase HPO mutant strains P106A, C110A, F118A, V204A, I205A, I205R, I205N, I205D, I205C, I205Q, I205E, I205G, I205H, I205L, I205K, I205M, I205F, I205P, I205S, I205T, I205W, I205Y, I205V, F296A, D297A, E304A, T483A through rational design and transformation and gene recombination. , T483R, T483N, T483D, T483C, T483Q, T483E, T483G, T483H, T483I, T483L, T483K, T483M, T483F, T483P, T483S, T483W, T483Y, T483V, I205N / T483D, and I205N / T483I. Compared with the improved strain, the shake flask fermentation yield of naringenone produced by the I205N / T483I mutant strain was significantly increased by 64%, and the yield in a 5L fermenter reached 2.47 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 pGAP-HPO I205N / T483I 's graph.

[0025] Figure 2 It is a graph of the production of naringenone by 10 recombinant Pichia pastoris strains.

[0026] Figure 3 It is a graph of the production of naringenone by some recombinant Pichia pastoris strains.

[0027] Figure 4 It is a graph of the production of naringenone by six recombinant Pichia pastoris strains.

[0028] Figure 5 The main component content and OD of the I205N / T483I mutant strain during fermentation 600 Change graph. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0030] The experimental methods in the following examples are marked with specific experimental conditions, which are usually carried out according to conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0031] The "HPO" in the present invention is a premnaspirodiene oxygenase derived from the plant Hyoscyamus muticus, which is a cytochrome P450 oxidase.

[0032] The sources or preparation methods of some materials involved in the present invention are as follows:

[0033] LB (Luria-Bertani) medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 2% (2 g / 100 mL) agar powder was added to the solid medium, and sterilized at 115°C for 20 min.

[0034] YPD (Yeast Extract Peptone Dextrose) medium: 20 g / L glucose (Dextrose), 10 g / L yeast extract (Yeast extract), 20 g / L peptone (Peptone), solid medium is additionally added with 2% (2 g / 100 mL) agar powder, and sterilized at high temperature and high pressure at 115°C for 20 min.

[0035] YPD-G418 solid culture medium: Slowly heat 100mL YPD solid culture medium in a microwave oven until it is completely melted, then rinse the bottle with room temperature tap water. When the culture medium temperature drops to an appropriate range, the antibiotic addition step needs to be performed. Take 100μL of 300μg / mL G418 solution, gently add it and mix it evenly, then quickly pour it into a clean culture dish in a clean bench, and wait for it to cool naturally to a solidified state before use.

[0036] 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.

[0037] Preparation of the standard solution of naringenone: Weigh 10 mg of naringenone standard (purchased from Sigma), dissolve it in n-dodecane solution, and make up to 1 mL. The concentration of the standard stock solution is 10 mg / mL.

[0038] Trace element solution: 0.086g CoCl2·6H2O, 1.91g EDTA, 0.512g FeSO4·H2O, 1.02g ZnSO4·H2O, 0.384g CaCl2·2H2O, 0.05g CuSO4 and 0.048g Na2MoO4. Weigh and add deionized water to 100mL, sterilize at 115℃ for 20min, and store at 4℃ for later use.

[0039] Vitamin solution: 0.1g pyridoxine, 0.005g biotin, 0.1g calcium pantothenate, 0.2g p-aminobenzoic acid, 0.1g thiamine hydrochloride, 0.1g nicotinic acid, 2.5g inositol. Weigh and dissolve in sufficient deionized water until the total volume reaches 100mL, filter and sterilize, and store at 4℃ for later use.

[0040] Preparation of 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 and make up to 1 L, sterilize with high-pressure steam at 115°C for 20 min, add 10 mL of trace element solution and 12 mL of vitamin solution after sterilization is completed and cooled.

[0041] The present invention adopts gas chromatography to carry out qualitative and quantitative analysis of grapefruit ketone, and the detection conditions are as follows:

[0042] Chromatographic column: HP-5 (30m×0.32mm, 0.25μm, Agilent, USA); injection port temperature 250℃; program temperature rise: column temperature starts at 100℃, maintained for 4min, then increases to 250℃ at a rate of 10℃ / min, maintained for 5min. Hydrogen flame ionization detector (FID) temperature: 320℃; split ratio = 5:1, split flow 6mL / min; air flow: 300mL / min; hydrogen flow: 30mL / min; tail gas flow (N2): 15mL / min; average linear velocity: 24.802cm / s; pressure: 7.1413psi; injection volume: 1μL.

[0043] The fermentation apparatus used in the present invention is a 5L fermentation tank produced by Shanghai Bailun Biotechnology Co., Ltd.

[0044] Example 1: Construction of valencene high-producing strain H53

[0045] In this example, a Pichia pastoris GS115 chassis strain was used to knock out part of its genome and knock in several expression cassettes to obtain a valencene high-producing strain H53, and the specific process is as follows. Pichia pastoris GS115 is commercially available.

[0046] Construction of EgVS expression cassette and tHMG1 expression cassette

[0047] The ΔADH900 gene cluster of the genome was knocked out using CRISPR / Cas9 technology, and the EgVS (valencene synthase from Eryngiumglaciale) gene expression cassette (pGAP promoter, EgVS gene, PMP20 terminator (PMP20TT)) and tHMG1 gene expression cassette (pGAP promoter, tHMG1 gene, Gcw14 terminator (PMP20TT)) were introduced into the ΔADH900 gene cluster position of the genome; the II-5 gene cluster of the genome was knocked out using CRISPR / Cas9 technology, and two copies of the EgVS (valencene synthase from Eryngiumglaciale) gene expression cassette (pGAP promoter, EgVS gene, PMP20 terminator (PMP20TT)) were introduced into the II-5 gene cluster position of the genome.

[0048] The sequence of the EgVS gene is shown in SEQ ID NO: 25, and the sequence of the tHMG1 gene is shown in SEQ ID NO: 26.

[0049] Construction of ERG10 expression cassette

[0050] The I-2 gene cluster of the genome was knocked out using CRISPR / Cas9 technology, and the ERG10 gene expression cassette (pGAP promoter, ERG10 gene, PMP20 terminator (PMP20TT)) was introduced into the I-2 gene cluster position of the genome. The sequence of the ERG10 gene is shown in SEQ ID NO: 27.

[0051] Construction of ERG20 expression cassette

[0052] The II-4 gene cluster of the genome was knocked out using CRISPR / Cas9 technology, and the ERG20 gene expression cassette (pGAP promoter, ERG20 gene, PMP20 terminator (PMP20TT)) was introduced into the II-4 gene cluster of the genome, and the endogenous promoter of ERG9 was replaced by promoter P HXT1 The sequence of the ERG20 gene is shown in SEQ ID NO: 28, and the promoter P HXT1 The sequence is shown in SEQ ID NO:33.

[0053] ScUTR1 expression cassette, zwf1 expression cassette, sol3 expression cassette construction and aprt expression cassette construction

[0054] The IV-9 gene cluster of the genome was knocked out using CRISPR / Cas9 technology, and the ScUTR1 (NAD+ kinase) gene expression cassette (pGAP promoter, ScUTR1 gene, PMP20 terminator (PMP20TT)), zwf1 (glucose-6-phosphate dehydrogenase) gene expression cassette (pGAP promoter, zwf1 gene, PMP20 terminator (PMP20TT)), sol3 (6-glucolactonase) gene expression cassette (pGAP promoter, sol3 gene, PMP20 terminator (PMP20TT)) and aprt (endogenous adenine phosphoribosyltransferase) gene expression cassette (pGAP promoter, aprt gene, PMP20 terminator (PMP20TT)) were introduced into the genome. The sequence of the ScUTR1 gene is shown in SEQ ID NO: 29, the sequence of the zwf1 gene is shown in SEQ ID NO: 30, the sequence of the sol3 gene is shown in SEQ ID NO: 31, and the sequence of the aprt gene is shown in SEQ ID NO: 32.

[0055] The tHMG1, ERG10, and ERG20 expression cassettes can achieve overexpression of the corresponding genes to optimize the MVA pathway; replacing the endogenous promoter of ERG9 with promoter P HXT1 , which can downregulate the synthesis of squalene in the product synthesis stage of cells and increase the production of valencene; there are 3 EgVS expression cassettes to better catalyze the accumulated upstream precursor compounds and promote the entire biosynthetic pathway; the introduction of zwf1 expression cassette, sol3 expression cassette, ScUTR1 expression cassette and aprt expression cassette can improve the supply of NADPH and ATP in Pichia pastoris strains.

[0056] The above expression cassette was constructed into the genome of Pichia pastoris GS115 to obtain the valencene high-producing strain H53.

[0057] Example 2: Construction of HPO gene expression cassette

[0058] The present invention is based on the mutant HPO reported by Liu et al. in 2022 V480A / V482A(The modified HPO in this article refers to Liu T, Li W, Chen H, et al. Systematic optimization of HPO-CPR to boost (+)-nootkatone synthesis in engineered Saccharomyces cerevisiae [J]. Journal of Agricultural and Food Chemistry, 2022, 70(49): 15548-15559.) as the basis for constructing a recombinant plasmid containing the target HPO gene expression cassette. HPO was obtained from the NCBI database V480A / V482A The coding gene sequence of (GenBank: EF569601.1) was obtained, and the codons were optimized according to Pichia pastoris as the expression host. The nucleotide sequence was fully synthesized, and the gene was cloned into a pGS plasmid vector containing a GAP promoter to obtain a pGAP-HPO plasmid. The pGS plasmid is commercially available.

[0059] Select pGAP-HPO plasmid or its mutant plasmid as template, and use the whole plasmid amplification method to complete the pGAP plasmid containing the HPO mutant gene according to the guidance of the rapid site-directed mutagenesis kit. Using PCR technology, design and synthesize the introduction of P106A, C110A, F118A, V204A, I205A, I205R, I205N, I205D, I205C, I205Q, I205E, I205G, I205H, I205L, I205K, I205M, I205F, I205P, I205S, I205T, I205W, I205Y, I205 The HPO gene was subjected to site-directed mutagenesis using the mutant primers of V, F296A, D297A, E304A, T483A, T483R, T483N, T483D, T483C, T483Q, T483E, T483G, T483H, T483I, T483L, T483K, T483M, T483F, T483P, T483S, T483W, T483Y, and T483V to obtain the HPO expression cassette. The primers used for PCR are shown in Table 2. Among them, pI205-R and pI205F-F, pI205L-F, pI205M-F, pI205V-F, pI205S-F, pI205P-F, pI205T-F, pI205Y-F, pI205H-F, pI205Q-F, pI205N-F, pI205K-F, pI205D-F, pI205E-F, pI205C-F, pI205W-F, pI205R-F or pI205G-F can respectively form a primer pair, and pT483-F and pT483F-R, pT483L-R, pT483 83I-R, pT483M-R, pT483V-R, pT483S-R, pT483P-R, pT483T-R, pT483Y-R, pT483H-R, pT483Q-R, pT483N-R, pT483K-R, pT483D-R, pT483E-R, pT483C-R, pT483W-R, pT483R-R or pT483G-R can respectively form a primer pair; the remaining mutation sites all have corresponding matching primer pairs, for example, pP106A-R and pP106A-F form a primer pair corresponding to the P106A mutation.

[0060] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; followed by 30 cycles (95°C for 15 s, 53°C for 5 s, and 72°C for 15 s); extension at 72°C for 5 min, and finally storage at 4°C.

[0061] The amplified product was recovered and purified after the size was verified by agarose gel electrophoresis, and then digested with restriction endonuclease DpnⅠ, and then connected with T4 ligase and introduced into DH5α competent cells through E. coli transformation. After the competent cells were spread on LB solid medium and cultured overnight, the monoclonal strains on LB solid medium were selected and cultured in LB liquid medium containing antibiotics for verification. The plasmids in the cultured bacterial solution were extracted, and primers were used to perform PCR sequencing verification on pUC19-R / pUC19-F. The E. coli strains with correct sequencing were maintained and plasmids were extracted to obtain the positive plasmid pGAP-HPO P106A , pGAP-HPO C110A , pGAP-HPO F118A , pGAP-HPO V204A , pGAP-HPO I205A , pGAP-HPO I205R , pGAP-HPO I205N , pGAP-HPO I205D , pGAP-HPO I205C , pGAP-HPO I205Q , pGAP-HPO I205E , pGAP-HPO I205G , pGAP-HPO I205H , pGAP-HPO I205L , pGAP-HPO I205K , pGAP-HPO I205M , pGAP-HPO I205F , pGAP-HPO I205P , pGAP-HPO I205S , pGAP-HPO I205T , pGAP-HPO I205W , pGAP-HPO I205Y , pGAP-HPO I205V , pGAP-HPO F296A , pGAP-HPO D297A , pGAP-HPO E304A , pGAP-HPO T483A , pGAP-HPO T483R , pGAP-HPO T483N , pGAP-HPO T483D , pGAP-HPO T483C , pGAP-HPO T483Q , pGAP-HPO T483E , pGAP-HPO T483G , pGAP-HPO T483H , pGAP-HPO T483I , pGAP-HPOT483L , pGAP-HPO T483K , pGAP-HPO T483M , pGAP-HPO T483F , pGAP-HPO T483P , pGAP-HPO T483S , pGAP-HPO T483W , pGAP-HPO T483Y and pGAP-HPO T483V , and then the HPO gene expression cassette (pGAP promoter, HPO target gene) is added to complete the construction of the recombinant plasmid. I205N / T483I Plasmid map Figure 1 shown.

[0062] Table 2: Primer sequence list

[0063]

[0064]

[0065] Example 3: Construction of recombinant Pichia pastoris expressing mutants

[0066] Using the Pichia transformation kit Hieff CloneTM One Step Cloning Kit, the improved and mutant plasmids constructed in Example 2 were used as cytochrome P450 oxidase expression vectors, and the expression cassette of the reduction chaperone protein CPR from Arabidopsisthaliana and the expression cassette of Pichia endogenous alcohol dehydrogenase ADH3 were cloned into the pGAP-HPO plasmid (expression vector of the improved HPO) and each positive plasmid, and then the cloned plasmids were transformed into the Pichia strain H53, and the plasmid addition amount was 20 to 200 ng. The recombinant Saccharomyces cerevisiae cells transformed with the plasmid were spread on YPD-G418 resistance plates (YPD-G418 solid culture medium) and cultured in a 30°C incubator for 2 to 3 days to obtain Pichia pastoris cytochrome P450 oxidase mutant expression strains P106A, C110A, F118A, V204A, I205A, I205R, I205N, I205D, I205C, I205Q, I205E, I205G, I205H, I205L, I205K, I205M, I205F, I205 P, I205S, I205T, I205W, I205Y, I205V, F296A, D297A, E304A, T483A, T483R, T483N, T483D, T483C, T483Q, T483E, T483G, T483H, T483I, T483L, T483K, T483M, T483F, T483P, T483S, T483W, T483Y, T483V and the HPO improved expression strain H-0 (expressing the mutant HPO in Example 2). V480A / V482A ).

[0067] Table 3: Recombinant HPO expression strains

[0068]

[0069]

[0070] Example 4: Production of naringenone by shake flask fermentation of recombinant Pichia pastoris

[0071] The recombinant strain and the HPO improved expression strain constructed in Example 2 were selected, transferred to a test tube containing 5 mL of YPD medium and cultured for 24 h, and 500 μL of the culture was transferred to a conical flask containing 50 mL of YPD medium and continued to be cultured for 12 h to obtain a seed solution. Subsequently, the absorbance value (OD) of the seed solution was measured at 600 nm. 600 ), take an appropriate amount of seed liquid and inoculate it into a 250 mL conical flask containing 50 mL YPD medium, so that the initial OD of the fermentation medium is 600The value was 0.15. Since oleanone is volatile and insoluble in water, 2% n-dodecane was added to the fermentation broth at the 24th hour of fermentation to reduce the volatility loss of oleanone. On the 7th day of fermentation, the OD value of the fermentation broth was detected. 600 ; Leave the shake flask to stand until the organic phase and the aqueous phase are separated, use a pipette to suck out the organic phase into an EP tube, centrifuge at 12000rpm for 10min, take out the upper organic phase, add an appropriate amount of anhydrous sodium sulfate, shake and mix thoroughly, centrifuge at 12000rpm for 10min, suck out the upper organic phase, filter through a membrane into a gas chromatography (GC) bottle, and use it as a GC sample to detect the yield of oleic acid ketone in the sample. The test results are as follows Figure 2 and Figure 3 As shown, the catalytic activities of strains I205A, I205C, I205E, I205N, I205T, T483D, T483A, T483E, T483N, and T483I were improved, and the production of naringenone was higher than that of the improved expression strain H-0; among them, the production of naringenone of I205N was significantly increased to 179.70 mg / L, and the increase ratio was 43%.

[0072] Referring to the methods of Examples 2 and 3, the mutation sites I205N, T483D and T483I with relatively high yields were randomly combined to construct recombinant strains I205N / T483D and I205N / T483I, and the strains were selected and shake flask fermented by the aforementioned method of this example. After 7 days, the yield of oleic acid and the OD value of the fermentation liquid were detected. 600 The test results are as follows: Figure 4 As shown, the naringenone production of the I205N / T483I strain was higher than that of the single point mutation strains of I205N, T483D and T483I and the I205N / T483D strain, and the naringenone production reached 206.13 mg / L.

[0073] Example 5: Production of naringenone by high-density fermentation of recombinant Pichia pastoris

[0074] The present embodiment is different from Liu et al. (Liu T, Li W, Chen H, et al. Systematic optimization of HPO-CPR to boost (+)-nootkatone synthesis in engineered Saccharomyces cerevisiae [J]. Journal of Agricultural and Food Chemistry, 2022, 70 (49): 15548-15559.) in the materials and processes used in fermentation. First, it is necessary to select and isolate the Pichia monoclonal from the YPD plate, and then inoculate it into a culture tube containing 5 mL YPD liquid culture medium. Under the conditions of a constant temperature of 30 ° C and a shaking table speed of 220 rpm, the culture was carried out for about 24 hours to ensure the full growth and reproduction of the monoclonal.

[0075] Next, 1.5 mL of bacterial liquid was taken from the initial culture tube and further inoculated into a 500 mL shake flask containing 150 mL YPD liquid culture medium. The culture was again carried out at a constant temperature of 30°C and a shaking speed of 220 rpm for about 24 hours to ensure further expansion of the bacterial number and improvement of activity. Through this series of steps, the Pichia seed liquid suitable for the fermentation tank can be obtained.

[0076] Weigh 1.25g magnesium sulfate, 25g yeast extract and 50g peptone, add deionized water to mix and dilute to 2L, put into the fermentation tank and sterilize at 115℃ high pressure steam for 20min for later use; prepare another 250mL conical flask, add 50g anhydrous glucose, dilute to 100mL with deionized water, sterilize at 115℃ high pressure steam for 20min, after sterilization and cooling, add 25mL trace element solution and 30mL vitamin solution. At this point, the basic culture medium is prepared and poured into the tank when the seed liquid is flame inoculated.

[0077] The fermentation process of the present invention is divided into two stages:

[0078] The first stage is the strain growth stage, in which the fermentation temperature is controlled at 30°C, the stirring speed is in the range of 300-850rpm, the pH is about 5.5-6.0, the dissolved oxygen (OD) is about 30%, and the strain grows in the basic culture medium for about 24 hours.

[0079] The second stage is the glucose feeding stage. The glucose content in the fermentation broth is measured using the glucose determination kit of Shanghai Rongsheng Biopharmaceutical Co., Ltd. When the glucose in the fermentation broth is exhausted, the glucose is replenished by adding an appropriate amount of feed medium to the fermenter to restore the glucose content in the fermentation broth to the initial concentration. Subsequently, glucose concentrate is added every 24 hours to ensure the continuous growth of the microbial cells. Once the cell growth enters the slow stage, that is, OD 600 When the glucose content reaches 350-400, the feeding is stopped and the fermentation stops after the strain consumes the remaining glucose. The fermentation conditions of this process are the same as above.

[0080] The two-stage fermentation process lasted for 7 days, during which the valencene, noetone, and OD in the fermenter were continuously monitored. 600 and glucose concentration, recorded as Figure 5 It can be seen that there was no obvious accumulation of valencene during the fermentation process. After 7 days, the concentration of valencene in the 5L fermenter reached 2.47 g / L, which was 11.9 times that of shake flask fermentation, indicating that the enzyme produced by the I205N / T483D strain had excellent catalytic performance.

[0081] In summary, the present invention provides a cytochrome P450 oxidase HPO mutant and its application in synthesizing annatto ketone in yeast by means of genetic engineering and enzyme engineering. By providing a cytochrome P450 oxidase HPO mutant with improved catalytic activity, the yield of annatto ketone is increased, thus creating conditions for the industrial production of annatto ketone.

[0082] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A cytochrome P450 oxidase HPO, characterized in that The cytochrome P450 oxidase HPO comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 12.

2. The cytochrome P450 oxidase HPO according to claim 1, characterized in that The cytochrome P450 oxidase HPO comprises an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:

12.

3. A polynucleotide, characterized in that The polynucleotide encodes the cytochrome P450 oxidase HPO as claimed in claim 1.

4. The polynucleotide according to claim 3, wherein The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:

24.

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

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 3, or the expression vector according to claim 5.

7. A method for producing the cytochrome P450 oxidase HPO according to claim 1, characterized in that: The method comprises: Introducing (1) an I205N mutation and a T483D mutation into cytochrome P450 oxidase HPO(V480A / V482A); (2) an I205N mutation and a T483I mutation; or Using the polynucleotide of claim 3 for expression; or Using the expression vector of claim 5 for expression; or The host cell according to claim 6 is used 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.

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