Cytochrome p450 enzyme cyp153d40 and use thereof

Abstract

This invention belongs to the field of enzyme catalysis and bioengineering technology, and relates to a cytochrome P450 enzyme CYP153D40 and its applications. The amino acid sequence of the enzyme is shown in SEQ ID NO.1. The nucleotide sequence of the DNA molecule encoding the enzyme is shown in SEQ ID NO.2. The cytochrome P450 enzyme CYP153D40 provided by this invention exhibits catalytic activity for C8-C14 chain saturated fatty acids, various unsaturated fatty acids, and short-chain alkanes (octane, decane); simultaneously, this enzyme shows excellent terminal (ω-position) hydroxylation ability for saturated fatty acids, unsaturated fatty acids, and alkane substrates, with high regioselectivity, and can be used for the green biomanufacturing of high-value-added hydroxy fatty acids and hydroxyalkane compounds.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis and bioengineering technology, and relates to a cytochrome P450 enzyme CYP153D40 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 terminal alkane hydroxylases with high regioselectivity, overcomes the limitations of traditional membrane-bound terminal hydroxylases, such as difficulties in heterologous expression and purification, and poor selectivity. They are not only ideal models for studying the mechanism of alkane terminal hydroxylation but also demonstrate significant industrial application potential in biomanufacturing and environmental remediation. Hydroxylated hydrocarbons have important industrial applications, but the selective oxidation of alkanes remains a significant challenge 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 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 specific oxidative modification of various substrates such as alkanes and fatty acids. The CYP153 family, as a class of soluble terminal alkane hydroxylases with high regioselectivity, 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 important 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 CYP153D40 and its applications. This enzyme is derived from... Novosphingobium aquiterrae This enzyme exhibits extremely high terminal hydroxylation activity and regioselectivity for saturated or unsaturated fatty acids, and also has catalytic ability for short-chain alkanes.

[0005] The technical solution provided by the present invention is as follows: a cytochrome P450 enzyme CYP153D40, 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 CYP153D40, the nucleotide sequence of which is shown in SEQ ID NO.2 of the sequence listing.

[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 CYP153D40 in the preparation of terminally hydroxylated aliphatic compounds.

[0010] Furthermore, the present invention provides a method for synthesizing terminally hydroxylated aliphatic compounds, which uses the cytochrome P450 enzyme CYP153D40 to catalyze the terminal hydroxylation of saturated fatty acids, unsaturated fatty acids or short-chain alkanes to obtain the corresponding compounds.

[0011] The cytochrome P450 enzyme CYP153D40 provided by this invention exhibits extremely high terminal hydroxylation activity and regioselectivity for saturated or unsaturated fatty acids, and also has catalytic ability for short-chain alkanes. It can meet the industrial biocatalysis demand for efficient and highly selective hydroxylases, and make up for the shortcomings of previously reported CYP153 family enzymes in terms of substrate coverage, catalytic efficiency and stability. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the construction of the CYP153D40 expression vector in an embodiment of the present invention; Figure 2 The results of SDS-PAGE analysis of CYP153D40 are shown; where M: protein marker; 1: CYP153D40. Figure 3 The concentration of CYP153D40 was determined by the pyridine heme proto-method. Figure 4 The catalytic activity of CYP15340 for alkanes (octane, decane, dodecane); Figure 5 The catalytic activity of CYP15340 for saturated fatty acids (octanoic acid, capric acid, dodecanoic acid, tetradecanoic acid, and hexadecanoic acid); Figure 6 This refers to the ω-hydroxylation reaction of saturated fatty acids catalyzed by CYP153D40. Figure 7 The catalytic activity of CYP153D40 for partially unsaturated fatty acids; Figure 8 A comparison diagram of the oxidation of different long-chain alkanes to dicarboxylic acids catalyzed by CYP153D40. 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] one, Origin and Acquisition of CYP153D40 This invention is from Novosphingobium aquiterrae A novel cytochrome P450 enzyme, CYP153D40 (abbreviated as CYP153D40), was discovered and identified in the 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 of the sequence listing.

[0015] II. Cloning of the CYP153D40 gene The entire gene was synthesized using the CYP153D40 amino acid sequence, and codon optimization was performed for expression in *E. coli*. Following the system described in Table 1, pET30a was used as the vector, and specific primer pairs were employed. Novosphingobium aquiterra The genome was 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, 35 cycles; 72℃ final extension for 10 min, then cooled to 4℃ for storage. The obtained CYP153D40 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- cyp153d40 and transformed Escherichia coli expression strains Escherichia coli BL21(DE3) was used to obtain the expression strain BL21 / pET30a- cyp153d40 BL21 / pET30a- cyp153d40 The expressed CYP153D40 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 CYP153D40 .

[0017] III. Expression and Purification of CYP153D40 Select BL21 / pET30a- cyp153d40Single 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 using an ultrasonic homogenizer (25% power, 2 seconds sonication, 3-second intervals, total duration 20-30 min) until the bacterial solution is clear. Centrifuge the homogenized mixture at 4°C 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 CYP153D40 Concentration by Pyridine Hemochromatin Method (1) Preparation of reagents Solution I (reaction solution): In a fume hood, mix "2 / 5 volume 0.5 M NaOH + 2 / 5 volume pyridine + 1 / 200 volume Solution II + balance deionized water" to achieve a final concentration of 0.2 M NaOH, 40% (v / v) pyridine, and 500 µM potassium ferricyanide. This solution is used to bind with heme and oxidize Fe(III). Solution II (potassium ferricyanide stock solution): Prepare a 0.1 M K3[Fe(CN)6] aqueous solution, which must be used within 24 hours. Solution III (reducing agent): Dissolve sodium dithionite in 0.5 M NaOH to prepare a 0.5 M concentration solution, and use immediately after preparation. If the P450 sample contains reducing agents such as DTT, it must be removed beforehand (to avoid interfering with the oxidation state spectrum). (2) Turn on the spectrophotometer, set the scanning range to 500-600 nm, the data interval to 1 nm, and maintain room temperature. (3) Take an equal volume (e.g., 0.5 mL) of solution I and mix it with the desalting buffer of the CYP153D40 sample, pour it into a 100 μL cuvette, and place it in a spectrophotometer for blank calibration. (4) Empty the cuvette, add equal amounts of solution I and CYP153D40 sample to it, and gently blow to mix evenly; (5) Place the mixture in a spectrophotometer and record the spectrum in the range of 500-600 nm. This is the oxidation state spectrum. Figure 3 ); (6) Keeping the oxidized mixture in the cuvette unchanged, add 10 µL of solution III and mix quickly. The sample will turn red, indicating that Fe(III) has begun to be reduced to Fe(II). (7) Start the scan immediately, and repeat the scan once every 1 min until the absorbance at 557 nm (Hemeb characteristic peak) in two consecutive scans no longer increases. Take the spectrum with the highest absorbance as the reduced state spectrum. Figure 3 ); (8) Based on Beer's Law, calculate the basic formula for heme concentration: ; C Heme concentration (unit: mM); A Absorbance at 557 nm in the reduced state - absorbance at 540 nm in the oxidized state; ε The molar extinction coefficient differential spectrum of heme b is 23.98 mM. -1 •cm -1 ; L : Optical path length of cuvette (fixed at 1 cm); Dilution factor correction: The calculated value needs to be adjusted. C Multiply by the dilution factor to obtain the actual concentration of heme in the CYP153D40 sample.

[0023] VI. Determination of the activity of CYP153D40 against alkanes In this example, octane, decane, and dodecane were used as reaction substrates to detect the activity of CYP153D40. In the system shown in Table 2, the purified CYP153D40 enzyme was incubated with 3 mM alkane substrate 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.

[0024] Table 2. In vitro enzymatic reaction system of CYP153D40 against alkanes .

[0025] CYP153D40 showed substrate conversions of 17.96 ± 0.93% and 2.82 ± 0.26% for short-chain alkanes (octane and decane), but almost no catalytic activity for long-chain alkanes such as dodecane. Figure 4 This demonstrates its application potential in the fields of petroleum hydrocarbon degradation, biomass resource utilization, and green synthesis of high-value-added compounds. Furthermore, the reaction product of CYP153D40 with short-chain alkanes (octane and decane) is an α,ω-diol, meaning that hydroxylation occurs at both ends. Figure 6 ).

[0026] VII. Determination of the activity of CYP153D40 against saturated fatty acids This example uses octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, and hexadecanoic acid as reaction substrates to compare CYP153D40 with the positive control CYP153A. M.aq The 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.

[0027] Table 3. In vitro enzymatic reaction systems of CYP153D40 with saturated and unsaturated fatty acids .

[0028] The reaction results are as follows Figure 5 As shown, CYP153D40 achieved nearly 100% conversion of medium-chain saturated fatty acid (decanoic acid and dodecanoic acid) substrates, while also exhibiting certain catalytic activity towards octanoic acid and tetradecanoic acid, with conversion rates of 61.63 ± 5.77% and 63.85 ± 4.77%, respectively, demonstrating its significant application potential in the high-value utilization of fatty acids and the synthesis of bio-based chemicals. Analysis revealed that the catalytic site for saturated fatty acids by CYP153D40 is the ω-position (…). Figure 6 ).

[0029] VIII. Determination of the activity of CYP153D40 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 CYP153D40 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 VI.

[0030] The reaction results are as follows Figure 7 As shown, CYP153D40 exhibits nearly 100% conversion of selected unsaturated fatty acid (arachidonic acid, α-linolenic acid, ricinoleic acid) substrates, demonstrating its significant application potential in the green preparation of functional hydroxy fatty acids, pharmaceutical intermediates, and health product raw materials. Figure 8 As shown, when the substrate is decane, the yield of dicarboxylic acid product is much higher than that of octane; indicating that the enzyme's catalytic conversion efficiency for 10-carbon decane is significantly better than that for 8-carbon octane.

Claims

1. A cytochrome P450 enzyme CYP153D40, 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 CYP153D40 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 CYP153D40 according to claim 1 in the synthesis of terminally hydroxylated aliphatic compounds.

6. A method for synthesizing terminally hydroxylated aliphatic compounds, characterized in that: The cytochrome P450 enzyme CYP153D40 described in claim 1 is used to catalyze the terminal hydroxylation of saturated fatty acids, unsaturated fatty acids, or short-chain alkane compounds to obtain the corresponding compounds.

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