Recombinant bacteria for expressing SCOX gene and PMCAT gene and application of recombinant bacteria in preparation of L-xylose

Abstract

The invention discloses a recombinant bacterium for expressing an SCOX gene and a PMCAT gene and application of the recombinant bacterium in preparation of L-xylose. The present invention provides a recombinant microorganism having a SCOX gene and a PMCAT gene. The invention also provides a method for preparing the recombinant microorganism for producing the L-xylose, which comprises the following steps: introducing the SCOX gene and the PMCAT gene into the microorganism to obtain the recombinant microorganism with the SCOX gene and the PMCAT gene, namely the recombinant microorganism for producing the L-xylose. The invention also provides an application of the recombinant microorganism in preparation of L-xylose. The invention also provides a method for preparing L-xylose, which comprises the following step: preparing L-xylose by taking xylitol as a raw material and the recombinant microorganism as a whole-cell catalyst. The method can generate huge economic benefits for production of the L-xylose, and has great popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a recombinant bacterium expressing a SCOX gene and a PMCAT gene and an application thereof in preparing L-xylose. Background Art

[0002] L-xylose is a pentose and an L-type rare sugar. L-xylose is a non-caloric sweetener and is widely used in medicine, food and other fields. The main uses of L-xylose are as follows: (1) As a pharmaceutical intermediate, L-xylose plays an important role in anti-cancer, anti-inflammatory, anti-viral, and anti-diabetic effects; (2) As a raw material, L-xylose can be used to synthesize a healthy sweetener, xylitol; (3) As a sweetener, it can improve the microbial environment of the human body, enhance immunity, and can be used as an auxiliary therapeutic agent for diabetics.

[0003] Different from D-xylose naturally occurring in nature, L-xylose is not a naturally occurring substance. At present, the production method of L-xylose is mainly through chemical synthesis and biotransformation. The chemical synthesis method has the problems of complicated steps, harsh reaction conditions, low yield, many by-products, insufficient stereoselectivity, etc. There are fewer reports on biotransformation methods, and the process is immature. It is limited to the laboratory research stage and is difficult to obtain large-scale production and application in industry. In addition, L-xylulose can also obtain L-xylose by isomerization reaction, but L-xylulose also belongs to rare sugar and is relatively expensive.

[0004] In summary, there is an urgent need in the art to develop a new and efficient method for synthesizing L-xylose. Summary of the invention

[0005] The technical problem solved by the present invention is how to efficiently synthesize L-xylose. Therefore, the present invention provides an engineered bacterium for preparing L-xylose.

[0006] The invention discloses a recombinant bacterium expressing SCOX gene and PMCAT gene and application of the recombinant bacterium in preparing L-xylose.

[0007] The present invention provides a recombinant microorganism having a SCOX gene and a PMCAT gene. The present invention also provides a method for preparing a recombinant microorganism for producing L-xylose, comprising the following steps: introducing SCOX gene and PMCAT gene into the microorganism to obtain a recombinant microorganism having SCOX gene and PMCAT gene, that is, the recombinant microorganism for producing L-xylose.

[0008] The present invention also provides the use of the recombinant microorganism in preparing L-xylose.

[0009] In the application, the recombinant microorganism serves as a whole-cell catalyst.

[0010] In the application, xylitol is used as a raw material to prepare L-xylose.

[0011] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0012] The present invention also provides a method for preparing L-xylose, comprising the following steps: using xylitol as a raw material and the recombinant microorganism as a whole-cell catalyst to prepare L-xylose.

[0013] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0014] The present invention also provides the use of biological material I in preparing recombinant microorganisms for producing L-xylose; The biomaterial I includes SCOX gene-related biomaterials and PMCAT gene-related biomaterials; the SCOX gene-related biomaterials are SCOX genes, expression cassettes with SCOX genes or recombinant vectors with SCOX genes; the PMCAT gene-related biomaterials are PMCAT genes, expression cassettes with PMCAT genes or recombinant vectors with PMCAT genes.

[0015] The biological material I also includes microorganisms (microorganisms as receptor materials).

[0016] In the application, xylitol is used as a raw material to prepare L-xylose.

[0017] The present invention also provides the use of biological material II in preparing recombinant microorganisms for producing L-xylose; The biological material II includes double-gene related biological materials; the double genes are SCOX gene and PMCAT gene; the double-gene related biological materials are the double genes, expression boxes with the double genes or recombinant vectors with the double genes.

[0018] The biological material II also includes microorganisms (microorganisms as receptor materials).

[0019] In the application, xylitol is used as a raw material to prepare L-xylose.

[0020] The present invention also provides the use of biological material III in the preparation of L-xylose; The biomaterial III includes SCOX gene-related biomaterials and PMCAT gene-related biomaterials; the SCOX gene-related biomaterials are SCOX genes, expression cassettes with SCOX genes, recombinant vectors with SCOX genes or recombinant microorganisms with SCOX genes; the PMCAT gene-related biomaterials are PMCAT genes, expression cassettes with PMCAT genes, recombinant vectors with PMCAT genes or recombinant microorganisms with PMCAT genes.

[0021] The biological material III also includes microorganisms (microorganisms as receptor materials).

[0022] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0023] In the application, xylitol is used as a raw material to prepare L-xylose.

[0024] The present invention also provides the use of biological material IV in the preparation of L-xylose; The biological material IV includes double-gene related biological materials; the double genes are SCOX gene and PMCAT gene; the double-gene related biological materials are the double genes, expression boxes with the double genes, recombinant vectors with the double genes or recombinant microorganisms with the double genes.

[0025] The biological material IV also includes microorganisms (microorganisms as receptor materials).

[0026] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0027] In the application, xylitol is used as a raw material to prepare L-xylose.

[0028] Any of the above SCOX genes encodes a SCOX protein.

[0029] Specifically, the function of SCOX protein is: xylitol oxidase.

[0030] Specifically, the SCOX protein is derived from Streptomyces coelicolor ( Streptomyces coelicolor ).

[0031] The SCOX protein is the protein shown in SEQ ID NO:1.

[0032] The SCOX protein is as follows (a1) or (a2) or (a3) ​​or (a4): (a1) the protein shown in SEQ ID NO: 1; (a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in (a1) and having the same function as (a1); (a4) A protein that is 98% or more identical to (a1) and has the same function as (a1).

[0033] Any of the above PMCAT genes encodes PMCAT protein.

[0034] Specifically, the function of PMCAT protein is: catalase.

[0035] Specifically, the PMCAT protein is derived from Proteus mirabilis ( Proteus mirabilis ).

[0036] The PMCAT protein is the protein shown in SEQ ID NO:3.

[0037] The PMCAT protein is as follows (b1) or (b2) or (b3) or (b4): (b1) the protein shown in SEQ ID NO: 3; (b2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (b1); (b3) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in (b1) and having the same function as (b1); (b4) A protein that is 98% or more identical to (b1) and has the same function as (b1).

[0038] Specifically, the SCOX gene is derived from Streptomyces coelicolor ( Streptomyces coelicolor ).

[0039] Specifically, the SCOX gene is a DNA molecule whose coding region is shown in SEQ ID NO: 2.

[0040] Specifically, the SCOX gene is as follows (c1) or (c2) or (c3): (c1) a DNA molecule having a coding region as shown in SEQ ID NO: 2; (c2) a DNA molecule having more than 90% identity with (c1) and encoding the protein; (c3) A DNA molecule that hybridizes to the nucleotide sequence defined in (c1) under stringent conditions and encodes the protein.

[0041] Specifically, the PMCAT gene is derived from Proteus mirabilis ( Proteus mirabilis ).

[0042] Specifically, the PMCAT gene is a DNA molecule whose coding region is shown in SEQ ID NO:4.

[0043] Specifically, the PMCAT gene is as follows (d1) or (d2) or (d3): (d1) a DNA molecule having a coding region as shown in SEQ ID NO: 4; (d2) a DNA molecule having more than 90% identity with (d1) and encoding the protein; (d3) A DNA molecule that hybridizes to the nucleotide sequence defined in (d1) under stringent conditions and encodes the protein.

[0044] Specifically, the recombinant vector with the SCOX gene can be a recombinant plasmid obtained by inserting the SCOX gene into an expression vector. Specifically, the expression vector can be a vector pBAD / HisB. Specifically, the recombinant vector with the SCOX gene can be: using the vector pBAD / HisB as a starting plasmid, replacing the SCOX gene shown in SEQ ID NO: 2 with XOt I and Pst The recombinant plasmid was obtained by digesting the small fragment between the recognition sequences with I.

[0045] Specifically, the recombinant microorganism having the SCOX gene may be a recombinant microorganism obtained by introducing the SCOX gene into a starting microorganism. Specifically, the recombinant microorganism having the SCOX gene may be a recombinant microorganism obtained by introducing a recombinant vector having the SCOX gene into a starting microorganism. Specifically, the microorganism may be a bacterium. Specifically, the microorganism may be Escherichia coli. Specifically, the microorganism may be Escherichia coli BW25113.

[0046] Specifically, the recombinant vector with PMCAT gene can be a recombinant plasmid obtained by inserting PMCAT gene into an expression vector. Specifically, the expression vector can be vector pBAD / HisB. Specifically, the recombinant vector with PMCAT gene can be: vector pBAD / HisB is used as a starting plasmid, and the PMCAT gene shown in SEQ ID NO: 4 is replaced Eco RI and Hind The recombinant plasmid was obtained by digesting the small fragment between the recognition sequences by III restriction enzyme. Specifically, the recombinant microorganism having the PMCAT gene may be a recombinant microorganism obtained by introducing the PMCAT gene into a starting microorganism. Specifically, the recombinant microorganism having the PMCAT gene may be a recombinant microorganism obtained by introducing a recombinant vector having the PMCAT gene into a starting microorganism. Specifically, the microorganism may be a bacterium. Specifically, the microorganism may be Escherichia coli. Specifically, the microorganism may be Escherichia coli BW25113.

[0047] Specifically, the recombinant vector with the double genes may be a recombinant plasmid obtained by inserting the SCOX gene and the PMCAT gene into an expression vector. Specifically, the expression vector may be a vector pBAD / HisB. Specifically, the recombinant vector with the double genes may be: using the vector pBAD / HisB as a starting plasmid, replacing the SCOX gene shown in SEQ ID NO: 2 with XOt I and Pst I enzyme cuts the small fragment between the recognition sequences and replaces it with the PMCAT gene shown in SEQ ID NO: 4 Eco RI and Hind III enzyme cuts the small fragment between the recognition sequences to obtain the recombinant plasmid.

[0048] Specifically, the recombinant microorganism with the double gene may be a recombinant microorganism obtained by introducing the SCOX gene and the PMCAT gene into a starting microorganism. Specifically, the recombinant microorganism with the double gene may be a recombinant microorganism obtained by introducing a recombinant vector with the SCOX gene and the PMCAT gene into a starting microorganism. Specifically, the microorganism may be a bacterium. Specifically, the microorganism may be Escherichia coli. Specifically, the microorganism may be Escherichia coli BW25113.

[0049] Specifically, the microorganism is a bacterium, and the microorganism is a recombinant bacterium.

[0050] Specifically, the microorganism is Escherichia coli, and the microorganism is recombinant Escherichia coli.

[0051] Specifically, the Escherichia coli is Escherichia coli BW25113.

[0052] Specifically, the recombinant microorganism after induced culture is obtained by induced culture of the recombinant microorganism.

[0053] Exemplarily, the induction culture is: L-arabinose induction culture. Exemplarily, the induction culture is: using L-arabinose induction culture at a concentration of 0.1-0.3g / 100mL. Exemplarily, the induction culture is: using L-arabinose induction culture at a concentration of 0.2g / 100mL. Exemplarily, the temperature of the induction culture is: 30-40°C. Exemplarily, the temperature of the induction culture is: 37°C. Exemplarily, the time of the induction culture is: 10-16 hours. Exemplarily, the time of the induction culture is: 12-16 hours. Exemplarily, the time of the induction culture is: 12 hours. Exemplarily, the induction culture is shaking culture. Exemplarily, the rotation speed of the shaking culture is 100-300rpm. Exemplarily, the rotation speed of the shaking culture is 200rpm. Exemplarily, the conditions of the induction culture are shaking culture at 37°C and 200rpm for 12 hours.

[0054] Exemplarily, the method for preparing the recombinant microorganism after induced culture comprises the following steps: ① Inoculate the recombinant microorganism into liquid culture medium and culture until OD 600nm The value is 0.6~0.8; ②After completing step ①, add L-arabinose to the culture system for induction culture.

[0055] ③ After completing step ②, collect the bacterial precipitate, which is the recombinant microorganism after induced culture.

[0056] In the step ①, the liquid culture medium may be liquid LB culture medium.

[0057] In the step ①, the liquid culture medium may be a liquid LB culture medium containing ampicillin.

[0058] In the step ①, the liquid culture medium may be a liquid LB culture medium containing 50 μg / mL ampicillin.

[0059] In step ①, the culture conditions are: 37° C., 220 rpm shaking culture.

[0060] In the step ②, the concentration of L-arabinose in the system is 0.1-0.3 g / 100 mL.

[0061] In step ②, the concentration of L-arabinose in the system is 0.2 g / 100 mL.

[0062] In step ②, the culture time is 10-16 hours.

[0063] In step ②, the culture time is: 12-16 hours.

[0064] In the step ②, the culture time is: 12 hours.

[0065] In the step ②, the culture temperature is 30-40°C.

[0066] In step ②, the culture temperature is: 37°C.

[0067] Exemplarily, the induction culture is a shaking culture. Exemplarily, the rotation speed of the shaking culture is 100-300 rpm. Exemplarily, the rotation speed of the shaking culture is 200 rpm.

[0068] In step ②, the culture conditions are: 37° C., 200 rpm shaking culture for 12 hours.

[0069] In the step ③, the bacterial precipitate is collected by centrifugation.

[0070] Exemplarily, the centrifugation parameters are: 6000 rpm for 15 min.

[0071] L-xylose is prepared by taking xylitol as raw material and the recombinant microorganism as whole-cell catalyst.

[0072] In the method for preparing L-xylose, the reaction temperature is 30°C-60°C.

[0073] In the method for preparing L-xylose, the reaction temperature is 30°C-50°C.

[0074] In the method for preparing L-xylose, the reaction temperature is 37°C.

[0075] In the method for preparing L-xylose, the reaction pH is 6.0-8.0.

[0076] In the method for preparing L-xylose, the reaction pH is 7.0.

[0077] In the method for preparing L-xylose, the reaction time is 10-16 hours.

[0078] In the method for preparing L-xylose, the reaction time is 12 hours.

[0079] In the method for preparing L-xylose, the content of xylitol in the reaction system is 0.5M-0.7M.

[0080] In the method for preparing L-xylose, the content of xylitol in the reaction system is 0.7M.

[0081] In the method for preparing L-xylose, the content of the recombinant microorganism in the reaction system is 20-40 g (wet weight) / L.

[0082] In the method for preparing L-xylose, the content of the recombinant microorganism in the reaction system is 30 g (wet weight) / L.

[0083] In the method for preparing L-xylose, the reaction system is composed of the recombinant microorganism, xylitol and phosphate buffer. In the reaction system, the cell content of the recombinant microorganism is 30g (wet weight) / L, and the xylitol content is 0.7M.

[0084] In the method for preparing L-xylose, shaking culture is used. Exemplarily, the rotation speed of the shaking culture is 100-300 rpm. Exemplarily, the rotation speed of the shaking culture is 220 rpm.

[0085] In the method for preparing L-xylose, the reaction conditions are: 37° C., 220 rpm shaking reaction for 12 hours.

[0086] The invention will generate huge economic benefits for the production of L-xylose and has great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Schematic diagram of the pathway for converting xylitol to L-xylose by SCOX and PMCA.

[0088] Figure 2 HPLC spectra of xylitol standard and L-xylose standard. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0090] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. Unless otherwise specified, the quantitative tests in the following examples are all repeated three times, and the results are averaged. The vector pBAD / HisB is provided by the pBAD / His kit, which is a product of Invitrogen, and the product catalog number is V43001. Escherichia coli BW25113: NBRP E.coli strain, the product catalog number is ME9062. Xylitol (CAS number: 87-99-0; molecular formula: C5H 12O5; molecular weight: 152.15): Aladdin, product catalog number X100092. L-xylose (CAS number: 609-06-3; molecular formula: C5H 10 O5; molecular weight: 150.13): Aladdin Company, product catalog number X115551. Unless otherwise specified, the phosphate buffer in the examples is a phosphate buffer of pH 7.0 and 50 mM.

[0091] The schematic diagram of the conversion of xylitol to L-xylose using SCOX and PMCA is shown in Figure 1 .

[0092] The SCOX gene is shown in SEQ ID NO:2, and encodes the protein shown in SEQ ID NO:1.

[0093] The PMCAT gene is shown in SEQ ID NO:4, and encodes the protein shown in SEQ ID NO:3.

[0094] Example 1. Construction of recombinant bacteria 1. Construction of recombinant plasmid Using vector pBAD / HisB as the starting plasmid, the SCOX gene shown in SEQ ID NO: 2 was replaced XOt I and Pst I enzyme cuts the small fragment between the recognition sequences and replaces it with the PMCAT gene shown in SEQ ID NO: 4 Eco RI and Hind III enzyme cut the small fragment between the recognition sequences to obtain the recombinant plasmid pSOXCAT.

[0095] The recombinant plasmid pSOXCAT was verified by sequencing.

[0096] 2. Construction of engineered bacteria LX001 The recombinant plasmid pSOXCAT was introduced into Escherichia coli BW25113 to obtain the recombinant bacteria, which was named engineered bacteria LX001.

[0097] Example 2: Synthesis of L-xylose from xylitol using engineered bacteria LX001 1. Take a single clone of the engineered bacteria LX001 and inoculate it into a liquid LB medium containing 50 μg / mL ampicillin. Cultivate at 37°C and 220 rpm with shaking until the OD 600nm =0.6 (In practical applications, OD 600nm =0.6~0.8).

[0098] 2. After completing step 1, add L-arabinose to the culture system and make its concentration in the system 0.2 g / 100 mL, and culture at 37°C and 200 rpm for 12 hours.

[0099] 3. After completing step 2, take the entire culture system, centrifuge it at 4°C and 6000 rpm for 15 min, and collect the bacterial precipitate.

[0100] 4. Prepare the initial reaction system, and then shake the reaction at 37°C and 220 rpm for 12 hours.

[0101] The initial reaction system consists of the bacterial precipitate obtained in step 3, xylitol and phosphate buffer. In the initial reaction system, the bacterial content is 30 g (wet weight) / L and the xylitol content is 0.7M.

[0102] 5. After completing step 4, take a sample, centrifuge at 6000g for 10 minutes, and collect the supernatant. Dilute the supernatant to 20 times the volume with distilled water, then filter with a filter membrane with a pore size of 0.22µm and collect the filtrate. The filtrate is tested by HPLC.

[0103] The relevant parameters of HPLC detection are as follows: HPLC chromatograph: Agilent 1260 high performance liquid chromatograph; Chromatographic column: Aminex HPX-87H (300 mm × 7.8 mm); Mobile phase: 5mmol / L sulfuric acid aqueous solution; Mobile phase flow rate: 0.5 mL / min; Column temperature: 50°C; Detector:RID.

[0104] The xylitol standard and L-xylose standard were dissolved in distilled water and then tested by HPLC (the relevant parameters of HPLC test are the same as above). The chromatogram is shown in Figure 2 .

[0105] For HPLC under the same condition parameters, the peaks in the chromatogram with a retention time of ±0.1 min from the standard peak can be identified as peaks of the same substance.

[0106] The L-xylose standard was dissolved in distilled water to obtain standard solutions of various concentrations, and then HPLC detection was performed (the relevant parameters of HPLC detection are the same as above). A standard curve of L-xylose content and peak area was prepared, and the obtained standard curve equation was: Y=142613X+1597.12, R 2 =0.9998925.

[0107] The L-xylose content in the system after step 4 was calculated according to the standard curve equation, and then the conversion rate was calculated. The results are shown in Table 1.

[0108] Table 1

[0109] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Recombinant microorganisms with SCOX gene and PMCAT gene; The SCOX gene encodes the protein shown in SEQ ID NO: 1; The PMCAT gene encodes the protein shown in SEQ ID NO:

3.

2. The recombinant microorganism according to claim 1, characterized in that: The SCOX gene is derived from Streptomyces coelicolor; The PMCAT gene is derived from Proteus mirabilis.

3. The recombinant microorganism according to claim 1, characterized in that: The coding region of the SCOX gene is shown in SEQ ID NO: 2; The coding region of the PMCAT gene is shown in SEQ ID NO:

4.

4. A method for preparing a recombinant microorganism for producing L-xylose, comprising the following steps: introducing SCOX gene and PMCAT gene into a microorganism to obtain a recombinant microorganism having SCOX gene and PMCAT gene, i.e., a recombinant microorganism for producing L-xylose; the SCOX gene is the SCOX gene described in claim 1, 2 or 3; the PMCAT gene is the PMCAT gene described in claim 1, 2 or 3.

5. Use of the recombinant microorganism according to any one of claims 1 to 3 in the preparation of L-xylose.

6. A method for preparing L-xylose, comprising the following steps: using xylitol as a raw material and the recombinant microorganism according to any one of claims 1 to 3 as a whole-cell catalyst to prepare L-xylose.

7. Application of biomaterial I in the preparation of recombinant microorganisms for producing L-xylose; The biological material I includes SCOX gene-related biological materials and PMCAT gene-related biological materials; the SCOX gene-related biological materials are SCOX genes, expression cassettes with SCOX genes or recombinant vectors with SCOX genes; the PMCAT gene-related biological materials are PMCAT genes, expression cassettes with PMCAT genes or recombinant vectors with PMCAT genes; The SCOX gene is the SCOX gene described in claim 1, 2 or 3; the PMCAT gene is the PMCAT gene described in claim 1, 2 or 3.

8. Application of biomaterial II in the preparation of recombinant microorganisms for producing L-xylose; The biological material II includes a double gene related biological material; the double gene is the SCOX gene and the PMCAT gene; the double gene related biological material is the double gene, an expression cassette having the double gene or a recombinant vector having the double gene; The SCOX gene is the SCOX gene described in claim 1, 2 or 3; the PMCAT gene is the PMCAT gene described in claim 1, 2 or 3.

9. Application of biomaterial III in the preparation of L-xylose; The biological material III includes SCOX gene-related biological materials and PMCAT gene-related biological materials; the SCOX gene-related biological materials are SCOX genes, expression cassettes with SCOX genes, recombinant vectors with SCOX genes, or recombinant microorganisms with SCOX genes; the PMCAT gene-related biological materials are PMCAT genes, expression cassettes with PMCAT genes, recombinant vectors with PMCAT genes, or recombinant microorganisms with PMCAT genes; The SCOX gene is the SCOX gene described in claim 1, 2 or 3; the PMCAT gene is the PMCAT gene described in claim 1, 2 or 3.

10. Application of biomaterial IV in the preparation of L-xylose; The biological material IV includes a double gene related biological material; the double gene is the SCOX gene and the PMCAT gene; the double gene related biological material is the double gene, an expression cassette having the double gene, a recombinant vector having the double gene, or a recombinant microorganism having the double gene; The SCOX gene is the SCOX gene described in claim 1, 2 or 3; the PMCAT gene is the PMCAT gene described in claim 1, 2 or 3.

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