Cytochrome p450 enzyme cyp153c3 and use thereof

Abstract

The present application belongs to the field of enzyme catalysis and bioengineering technology, and relates to a cytochrome P450 enzyme CYP153C3 and application thereof. The amino acid sequence of the enzyme is shown in SEQ ID NO. 1 of the sequence listing. The nucleotide sequence of the DNA molecule encoding the enzyme is shown in SEQ ID NO. 2 of the sequence listing. The cytochrome P450 enzyme CYP153C3 provided by the present application has catalytic activity on C8-C14 chain length saturated fatty acids, various unsaturated fatty acids and short-chain alkanes (octane, decane); meanwhile, the enzyme shows excellent ω-1 hydroxylation capacity on saturated fatty acids, unsaturated fatty acids and alkane substrates, has high regioselectivity, and can be used for green bio-manufacturing of high-value hydroxyl fatty acids and hydroxyl alkane compounds.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis and bioengineering technology, and relates to a cytochrome P450 enzyme CYP153C3 and its applications. Background Technology

[0002] Hydroxylated hydrocarbons have significant industrial applications, but the selective oxidation of alkanes remains a considerable challenge due to the high C–H bond dissociation energy and large oxidation potential. Therefore, developing efficient catalytic systems with excellent selectivity is a crucial issue that urgently needs to be addressed in this field. Cytochrome P450 is one of the most broadly catalytic and selective oxidase families in nature, capable of achieving specific oxidative modification of various substrates, including alkanes and fatty acids.

[0003] The CYP153 family, a class of soluble and highly regioselective terminal alkane hydroxylases, overcomes the limitations of traditional membrane-bound terminal hydroxylases, such as difficulties in heterologous expression and purification, and poor selectivity. It is not only an ideal model for studying the mechanism of alkane terminal hydroxylation but also shows significant industrial application potential in biomanufacturing and environmental remediation. Hydroxylated hydrocarbons have important industrial applications, but the selective oxidation of alkanes has always faced considerable challenges due to the high C–H bond dissociation energy and large oxidation potential. Therefore, developing efficient catalytic systems with excellent selectivity has become a key problem urgently needing to be solved in this field. Cytochrome P450 is one of the most broadly catalytic and selective oxidase families in nature, capable of specific oxidative modification of various substrates such as alkanes and fatty acids. The CYP153 family, as a class of soluble and highly regioselective alkane hydroxylases, overcomes the limitations of traditional membrane-bound hydroxylases, such as difficulties in heterologous expression and purification, and poor selectivity, showing significant industrial application potential in biomanufacturing. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a cytochrome P450 enzyme CYP153C3 and its applications. This enzyme is derived from... Novosphingobium percolationis C1, this enzyme can stably catalyze the hydroxylation of fatty acids and alkane substrates under suitable temperature and reaction conditions, and exhibits unique site selectivity.

[0005] The technical solution provided by this invention is as follows: a cytochrome P450 enzyme CYP153C3, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0006] Furthermore, the present invention provides a DNA molecule encoding the cytochrome P450 enzyme CYP153C3, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] Furthermore, the present invention provides a recombinant expression vector comprising the aforementioned DNA molecule.

[0008] Furthermore, the present invention provides an engineered strain comprising the recombinant expression vector described above.

[0009] Furthermore, the present invention provides the application of the cytochrome P450 enzyme CYP153C3 in the preparation of subterminal hydroxylated aliphatic compounds.

[0010] Furthermore, the present invention provides a method for preparing subterminal hydroxylated aliphatic compounds, which uses the cytochrome P450 enzyme CYP153C3 to catalyze the subterminal hydroxylation of saturated fatty acids, unsaturated fatty acids or alkane compounds to obtain the corresponding compounds.

[0011] The cytochrome P450 enzyme CYP153C3 provided by this invention exhibits catalytic activity for C8-C14 chain saturated fatty acids and various unsaturated fatty acids, and also shows detectable conversion activity for short-chain alkanes (octane, decane). Unlike most reported CYP153 family enzymes that specifically catalyze terminal (ω-position) hydroxylation, the cytochrome P450 enzyme CYP153C3 of this invention demonstrates highly specific subterminal (ω-1 position) hydroxylation catalysis for saturated fatty acids, unsaturated fatty acids, and alkane substrates, exhibiting strong regioselectivity. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the construction of the CYP153C3 recombinant expression vector in an embodiment of the present invention; Figure 2 This is a schematic diagram of SDS-PAGE detection of CYP153C3; where M: protein marker; 1: CYP153C3; Figure 3 The CO differential spectrum of CYP153C3; Figure 4 GC detection of the reaction of CYP153C3 with alkanes (octane, decane and dodecane); Figure 5 The results of GC-MS identification of the products of the reaction of CYP153C3 with octane; Figure 6 The catalytic activity of CYP153C3 for saturated fatty acids (octanoic acid, capric acid, dodecanoic acid, and tetradecanoic acid); Figure 7 The results of GC-MS identification of the reaction products of CYP153C3 with decanoic acid; Figure 8 The catalytic activity of CYP153C3 for partially unsaturated fatty acids; Figure 9The results of GC-MS identification of the reaction product of CYP153C3 with trans-2-decenoic acid are shown. Detailed Implementation

[0013] To facilitate understanding of the present invention, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. The following examples or drawings are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0014] I. Origin and Acquisition of CYP153C3 This invention is from Novosphingobium percolationis A novel cytochrome P450 enzyme, CYP153C3 (abbreviated as CYP153C3), was discovered and identified in the C1 genome. It belongs to the CYP153C3 subfamily of the CYP153 family. The amino acid sequence of this enzyme is shown in SEQ ID NO.1 in the sequence listing.

[0015] II. Cloning of the CYP153C3 gene The entire gene was synthesized using the CYP153C3 amino acid sequence, and codon optimization was performed for expression in *E. coli*. Using the primers listed in Table 1 and pET30a as the vector, the gene was expressed... Novosphingobium percolationis C1 The genome was linearized and amplified. PCR amplification conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, Tm-2-5℃ annealing for 15 s, 72℃ extension for the corresponding duration, for 35 cycles; final extension at 72℃ for 10 min, followed by storage at 4℃. The obtained CYP153C3 DNA fragment (SEQ ID NO.2) was ligated to the linearized pET30a vector fragment using homologous recombinase C115 (Novazia, Nanjing) to form the expression vector pET30a- cyp153c3 and transformed Escherichia coli expression strains Escherichia coli BL21(DE3) was used to obtain the expression strain BL21 / pET30a- cyp153c3 BL21 / pET30a- cyp153c3 The expressed CYP153C3 protein was purified by nickel column affinity chromatography combined with ultrafiltration desalting. The purified protein was then subjected to activity assays such as absorption spectroscopy and in vitro enzymatic reactions.

[0016] Table 1 Primers used to clone CYP153C3 .

[0017] III. Expression and Purification of CYP153C3 Select BL21 / pET30a- cyp153C3Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate resistance and cultured at 37°C and 220 rpm for 8–10 h to prepare seed culture. For scale-up culture, the seed culture was inoculated into 500 mL LB medium at a ratio of 1:100, and 1 M vitamin B1 (VB1) and 50 μg / mL kanamycin sulfate were added at a ratio of 1:1000. The culture was then incubated at 37°C and 220 rpm for 4–5 h. When the OD600 reached 0.8–1, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 200 μM and 5-aminolevulinic acid (5-Ala) at a final concentration of 0.5 mM were added. The culture conditions were adjusted to 18°C ​​and 180 rpm for 24 h of induction. After induction, the bacterial culture was centrifuged at 4°C and 8000 g for 10 min, and the bacterial cells were collected and stored at -80°C for later use.

[0018] Remove the frozen bacterial cells, thaw at room temperature, and then add 3-4 volumes of pre-cooled Lysis buffer. Vortex resuspend until no sterile clumps aggregate. Place the bacterial suspension in an ice-water mixture and sonicate the cells (25% power, 2 s sonication, 3 s interval, total duration 20-30 min) until the bacterial solution is clear. Centrifuge the mixture at 4℃ and 10000 × g for 60 min and collect the supernatant.

[0019] Load an appropriate amount of nickel resin into the chromatography column. Equilibrate the resin with 5 column volumes of Lysis buffer. Add the collected supernatant to the chromatography column for column loading. Recover the effluent and repeat the column loading process once. Then wash the nickel column with 5 volume of Wash buffer twice to remove impurities. Elute the target protein with 10 mL of Elution buffer and collect the colored elution fraction.

[0020] Select an appropriate ultrafiltration tube based on the target protein molecular weight, and concentrate the protein by centrifugation at 4℃ and 5000 × g for 30-50 min. Add the concentrated protein to a PD-10 desalting column pre-equilibrated with desalting buffer, elute with 3.5 mL of desalting buffer, and collect the colored eluent. Concentrate the desalted protein appropriately using an ultrafiltration tube (avoiding over-concentration which could lead to protein aggregation), and take 10 μL for SDS-PAGE analysis. Figure 2 The remaining protein was flash-frozen in liquid nitrogen and stored at -80°C.

[0021] The formulations of the above protein purification buffers are as follows: Lysis buffer (1 L): 7.8 g NaH2PO4·2H2O, 17.532 g NaCl, 100 g glycerol, 0.6808 g imidazole, pH 8.0; Wash buffer (1 L): 7.8 g NaH2PO4·2H2O, 17.532 g NaCl, 100 g glycerol, 1.3616 g imidazole, pH 8.0; Elution buffer (1 L): 7.8 g NaH2PO4·2H2O, 17.532 g NaCl, 100 g glycerol, 17.02 g imidazole, pH 8.0; Desalting buffer (1 L): 7.8 g NaH2PO4·2H2O, 100 g glycerol, pH 7.4.

[0022] IV. Determination of CYP153C3 Concentration Determination of CO differential absorption spectrum of CYP153C3 ( Figure 3 ), using the characteristic extinction coefficient (ε) 450-490 =91,000 M -1 •cm -1 Concentration calculations are performed.

[0023] (1) Take out CYP153C3 stored at -80℃ and thaw it on ice; (2) Dilute the protein to a suitable concentration using desalting buffer, with a final volume of 100-200 μL; (3) The diluted CYP153C3 was purged with CO for 1 min in a fume hood; (4) Transfer the protein dilution to a 100 μL cuvette and perform characteristic spectral scanning using a multi-functional microplate reader. Scan once every 2 seconds, with a scanning wavelength range of 350-500 nm. (5) Add an appropriate amount of sodium hydrosulfite, mix well, and then perform a spectral scan again; (6) Record the spectral data of the two scans, calculate the difference between the absorbance value after sodium hydrosulfite reduction and the absorbance value before addition, and plot the characteristic absorption spectrum of CYP153C3 with this value as the ordinate and time as the abscissa. Figure 3 ); (7) Calculate the concentration of CYP153C3 using the following formula: ; in, C Protein concentration (unit: mM); A Absorbance at a specific wavelength; ΔA: The difference in absorbance (reduced state - oxidized state) before and after adding sodium hydrosulfite at the same wavelength; ε The molar extinction coefficient of heme b (91,000 M) -1 •cm -1 ); L : Optical path of cuvette (fixed at 1 cm).

[0024] Multiply by the corresponding dilution factor to get the concentration of the protein being measured.

[0025] V. Determination of the activity of CYP153C3 against alkanes In this example, octane, decane, and dodecane were used as reaction substrates to detect the activity of CYP153C3. In the system shown in Table 2, CYP153C3 was incubated with 3 mM alkane substrates at pH 7.4 and 30 °C for 12 h. The reaction was then terminated by adding 10 μL of concentrated hydrochloric acid. After extraction with ethyl acetate, BSTFA derivatization, and incubation at 70 °C for 30 min, the mixture was cooled to room temperature, and the amount of product generated was determined by gas chromatography.

[0026] Table 2. In vitro enzymatic reaction system of CYP153C3 with alkanes .

[0027] The reaction results are as follows Figure 4 CYP153C3 can react with short-chain alkanes (octane and decane), but has almost no catalytic activity with long-chain alkanes such as dodecane.

[0028] Taking octane substrates as an example, product 1c was identified by gas chromatography-mass spectrometry (GC-MS). m / z 275.19 is the [M-15] fragment peak, suggesting a molecular weight of approximately 290 ( Figure 5 ), calculations confirmed that product 1c is octanediol. Further analysis revealed that in the spectrum m / z 117 is a fragment resulting from the cleavage of the subterminal ω-1 hydroxylation. Combined with the above evidence, the 1c product is confirmed to be 2,7-octanediol. Therefore, CYP153C3 can specifically catalyze the ω-1 hydroxylation of alkanes.

[0029] VI. Determination of the activity of CYP153C3 against saturated fatty acids This example uses octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, and hexadecanoic acid as reaction substrates to compare CYP153C3 with the positive control CYP153A. M.aqThe activity was determined by incubating the system according to Table 3 at 30°C for 90 min, then adding 10 μL of concentrated hydrochloric acid to terminate the reaction. After extraction with ethyl acetate, derivatization with BSTFA, and heating at 70°C for 30 min, the reaction was cooled to room temperature, and the substrate consumption and product formation were determined by gas chromatography.

[0030] Table 3. In vitro enzymatic reaction systems of CYP153C3 with saturated and unsaturated fatty acids .

[0031] The reaction results are as follows Figure 6 As shown, the conversion rates of CYP153C3 for medium-chain saturated fatty acids octanoic acid, decanoic acid, dodecanoic acid, and tetradecanoic acid were 16.82 ± 5.27%, 31.66 ± 5.92%, 76.25 ± 4.67%, and 48.49 ± 17.54%, respectively. This demonstrates its great application potential in the high-value utilization of fatty acids and the synthesis of bio-based chemicals. Furthermore, CYP153C3 enhances the conversion of saturated fatty acid products with ω-hydroxylase CYP153A. M.aq The difference indicates that the catalytic site for fatty acids is not the ω-site.

[0032] Taking decanoic acid as an example, CYP153C3 exhibits unique regioselectivity in catalyzing saturated fatty acids. This invention identified it using GC-MS technology and compared it with a database. Figure 7 Mass spectrometry analysis confirmed that the product was a subterminal (ω-1) hydroxylated product, 9-hydroxydecanoic acid. This regioselectivity is consistent with its catalytic behavior on alkane substrates (catalyzing the sequential oxidation of octane to 2,7-octanediol), confirming that this recombinase possesses unique ω-1 regioselectivity.

[0033] VII. Determination of the activity of CYP153C3 against unsaturated fatty acids This example uses oleic acid, trans-2-decenoic acid, arachidonic acid (ARA), α-linolenic acid (ALA), and ricinoleic acid as reaction substrates to compare CYP153C3 with the positive control CYP153A. M.aq The activity was determined. The system in Table 3 was incubated at 30°C for 4 h. The reaction termination and sample preparation procedures were the same as in Experiment 6.

[0034] The reaction results are as follows Figure 8As shown, CYP153C3 exhibited near 100% conversion rates for arachidonic acid and ricinoleic acid substrates, and conversion rates for oleic acid, trans-2-decenoic acid, and α-linolenic acid were 3.18 ± 0.17%, 21.31 ± 1.11%, and 22.08 ± 1.46%, respectively. GC-MS analysis and database comparison revealed that the catalytic product of trans-2-decenoic acid from CYP153C3 was 9-hydroxy-2-decenoic acid, i.e., the catalytic site was ω-1 (…). Figure 9 ).

Claims

1. A cytochrome P450 enzyme CYP153C3, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

2. A DNA molecule encoding the cytochrome P450 enzyme CYP153C3 of claim 1, characterized in that, The nucleotide sequence is shown in the sequence listing SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: It includes the DNA molecule as described in claim 2.

4. An engineered bacterial strain, characterized in that: It includes the recombinant expression vector as described in claim 3.

5. The use of the cytochrome P450 enzyme CYP153C3 according to claim 1 in the synthesis of subterminal hydroxylated aliphatic compounds.

6. A method for synthesizing aliphatic compounds with terminal hydroxylation, characterized in that: The cytochrome P450 enzyme CYP153C3 described in claim 1 is used to catalyze the subterminal hydroxylation of alkanes, saturated fatty acids, and some unsaturated fatty acid compounds to obtain the corresponding compounds.

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