Non-natural 1, 4-pentanediol biosynthetic pathway construction and application thereof

Abstract

The invention discloses non-natural 1, 4-pentanediol biosynthetic pathway construction and application thereof, starting from beta-ketoadipic acid, beta-ketoacid decarboxylase KDC catalyzes beta-ketoadipic acid to generate levulinic acid, then levulinic acid generates 4-hydroxyvaleric acid under the action of alcohol dehydrogenase ADH, and the 1, 4-pentanediol biosynthetic pathway construction is realized. Then, 4-hydroxyvalerate generates 4-hydroxyvaleraldehyde under the catalytic action of carboxylic acid reductase CAR, and finally, 4-hydroxyvaleraldehyde generates 1, 4-pentanediol under the action of alcohol dehydrogenase BDH. The invention provides a non-natural synthesis way for biosynthesis of 1, 4-pentanediol.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and biomanufacturing, and specifically relates to the construction of a non-natural 1,4-pentanediol biosynthetic pathway and its application. Background Art

[0002] 1,4-Pentanediol (1,4-PDO) is a C5 diol substituted with hydroxyl groups at the 1st and 4th positions of the carbon chain. When combined with long-chain dibasic acids, it produces low-melting-point polyesters that exhibit unique advantages in the field of shape-memory materials and are widely used in the production of high-performance materials such as polyester elastic fibers, polyurethane resins, and coatings. Due to its low toxicity and good solubility, 1,4-PDO is also widely used as a solvent in pharmaceutical synthesis. Currently, 1,4-PDO is primarily produced via the levulinic acid reduction method, which involves multiple organic reagents and high-pressure hydrogenation. This production method not only consumes large amounts of hydrogen but is also subject to fluctuations in fossil feedstock prices, directly impacting the production costs of 1,4-PDO and its downstream products. Therefore, with my country's increasing focus on sustainable industrial processes, the development of green and sustainable 1,4-PDO biosynthesis technologies is crucial for promoting the green manufacturing of medium- and long-chain diols in my country.

[0003] By modifying and optimizing microbial metabolic pathways, researchers have been able to enable microorganisms to efficiently produce some specific diols. For example, the biosynthetic technology of 1,4-butanediol (1,4-BDO) and 1,3-butanediol (1,3-BDO) has also been commercialized by Genomatica, a US company. Although a few biosynthetic technologies for diols have been successfully produced, the biosynthesis of most other diols still faces many challenges. For example, 1,4-PDO lacks a corresponding natural biosynthetic pathway, making it difficult to achieve its biosynthesis. Recently, Zeng et al. first proposed a biosynthetic pathway for 1,4-PDO using norvaline as a substrate. This pathway uses norvaline as a substrate and produces 1,4-PDO through hydroxylation, deamination, decarboxylation, and reduction. Although this pathway reduces the energy barrier of carboxyl / aldehyde reduction through hydroxylation, it can only synthesize 11.9 mg / L of 1,4-PDO (Nat. Commun. 2022, 13(1):1595). At the same time, the synthesis pathway using norvaline as a substrate also increases the cost of industrial production of 1,4-PDO. Therefore, the development of a de novo biosynthesis pathway for 1,4-PDO using inexpensive raw materials such as glucose is crucial for the biosynthesis of 1,4-PDO. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing a 1,4-pentanediol biosynthetic pathway and its application.

[0005] Another object of the present invention is to provide a method for synthesizing 1,4-pentanediol using waste carbon resources.

[0006] The present invention intends to use low-quality carbon resources as raw materials to develop a technology that can synthesize 1,4-PDO using glucose, lignin, and polyethylene terephthalate hydrolyzate (ethylene terephthalate), thereby realizing the conversion of waste carbon resources into high-value chemicals and providing a new path for the green biosynthesis of 1,4-PDO.

[0007] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0008] A method for biosynthesizing 1,4-pentanediol, first constructing a non-natural 1,4-pentanediol biosynthetic pathway based on β-ketoadipate for the first time, wherein β-ketoacid decarboxylase KDC catalyzes β-ketoadipate to produce levulinic acid, and then levulinic acid produces 4-hydroxyvaleric acid under the action of alcohol dehydrogenase ADH, and then 4-hydroxyvaleric acid produces 4-hydroxyvaleraldehyde under the catalysis of carboxylic acid reductase CAR, and finally 4-hydroxyvaleraldehyde produces 1,4-pentanediol under the action of alcohol dehydrogenase BDH.

[0009] The β-ketoadipate decarboxylase KDC is shown as SEQ ID No. 2; the dehydrogenase ADH is shown as SEQ ID No. 3; the carboxylic acid reductase CAR is shown as SEQ ID No. 4; and the alcohol dehydrogenase BDH is shown as SEQ ID No. 5.

[0010] The method for biosynthesizing 1,4-pentanediol comprises biosynthesizing 1,4-pentanediol through a catalytic system prepared by a recombinant Escherichia coli strain that overexpresses the four enzymes mentioned above; or catalyzing the synthesis of 1,4-pentanediol through an in vitro multi-enzyme system comprising various enzymes required for the reaction.

[0011] The β-ketoadipic acid can be synthesized into protocatechuic acid based on the shikimic acid pathway, and then the β-ketoadipic acid is generated by ring opening.

[0012] The multiple enzymes for synthesizing 1,4-pentanediol include:

[0013] 1) β-ketoacid decarboxylase that catalyzes the conversion of β-ketoadipate to levulinic acid;

[0014] 2) Alcohol dehydrogenase that catalyzes the conversion of levulinic acid to 4-hydroxyvaleric acid;

[0015] 3) Carboxylic acid reductase that catalyzes the conversion of 4-hydroxyvaleric acid to 4-hydroxyvaleraldehyde;

[0016] 4) Alcohol dehydrogenase that catalyzes the conversion of 4-hydroxyvaleraldehyde to 1,4-pentanediol.

[0017] The β-ketoacid decarboxylase in step 1) is an enzyme that can catalyze the decarboxylation of the β-ketoacid terminal carboxyl group of β-ketoadipate without catalyzing the decarboxylation of the other end, specifically β-ketoacid decarboxylase KDC (SEQ ID No. 2)

[0018] Step 2) The alcohol dehydrogenase is a reductase that can catalyze the reduction of the keto group in levulinic acid to generate a hydroxyl group, specifically levulinic acid alcohol dehydrogenase ADH (SEQ ID No. 3)

[0019] Step 3) The carboxylic acid reductase is a reductase that can catalyze the reduction of the carboxyl group in 4-hydroxypentanoic acid to generate an aldehyde group, specifically 4-hydroxypentanoic acid carboxylic acid reductase CAR (SEQ ID No. 4)

[0020] Step 4) The alcohol dehydrogenase is a reductase that can catalyze the reduction of the aldehyde group in 4-hydroxyvaleraldehyde to generate a hydroxyl group, specifically 4-hydroxyvaleraldehyde alcohol dehydrogenase BDH (SEQ ID No. 5)

[0021] The recombinant E. coli is constructed by inserting the enzyme encoding gene into the expression plasmid pTrc99a and then introducing it into Escherichia coli MG1655. The culture medium is cultured at 37°C until the OD 600 When the pH value was 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.1 mM, and then 1 g / L β-ketoadipic acid was added after induction culture at 16°C for 10 h. After further culture for 6 h, the 1,4-pentanediol production was detected.

[0022] The Escherichia coli fermentation medium comprises: 20-23 g / L glucose, 2-6 g / L yeast powder, 0.5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, and 0.2 g / L magnesium sulfate.

[0023] The invention discloses an engineered Rhodococcus etherivorans strain for synthesizing 1,4-pentanediol. The engineered Rhodococcus etherivorans strain is obtained by knocking out the coenzyme A transferase encoding gene pcaIJ in Rhodococcus etherivorans and simultaneously screening enzyme elements to obtain genes encoding 3-dehydroshikimate dehydratase AroZ, β-ketoadipate decarboxylase KDC, levulinic acid dehydrogenase ADH, carboxylic acid reductase CAR and dehydrogenase BDH with catalytic function, and realizing their catalytic function in Rhodococcus etherivorans.

[0024] The sequence of the 3-dehydroshikimate dehydratase AroZ is shown in SEQ ID No. 1; the β-ketoadipate decarboxylase KDC is shown in SEQ ID No. 2; the dehydrogenase ADH is shown in SEQ ID No. 3; the carboxylic acid reductase CAR is shown in SEQ ID No. 4; and the dehydrogenase BDH is shown in SEQ ID No. 5.

[0025] The Rhodococcus aetherivorans is Rhodococcus aetherivorans N1, and its preservation number is CCTCC NO: M 20221270.

[0026] The knockout plasmid pK18mobsacB was used to construct the coenzyme A transferase encoding gene pcaIJ, and the knockout strain NK was obtained.

[0027] The gene is introduced into the Rhodococcus etherovorus NK by constructing the Rhodococcus shuttle plasmid pNV-aroZ-kdc-adh-car-bdh through the expression plasmid pNV18.1.

[0028] The method for constructing the engineered Rhodococcus etherivorans comprises first constructing a knockout plasmid pK-FR using pK18mobsacB. The plasmid pK-FR contains 1000 bp upstream and downstream sequences of the A-transferase encoding gene pcaIJ. The expression plasmid pNV18.1 is then used to construct a Rhodococcus shuttle plasmid pNV-aroZ-kdc-adh-car-bdh containing the genes encoding 3-dehydroshikimate dehydratase AroZ, β-ketoadipate decarboxylase KDC, levulinic acid dehydrogenase ADH, carboxylic acid reductase CAR, and dehydrogenase BDH, and a urea-inducible promoter nucleic acid sequence pami.

[0029] The plasmid pK-FR was introduced into Rhodococcus etherivus N1 by electroporation, and the strain NK with the A transferase encoding gene pcaIJ knocked out was obtained based on sucrose plate counter-screening; then the plasmid pNV-aroZ-kdc-adh-car-bdh was introduced into Rhodococcus etherivus NK by electroporation to obtain the engineered Rhodococcus etherivus NK-aroZ-

[0030] kdc-adh-car-bdh.

[0031] The glucose, lignin and PET hydrolyzate bis(2-hydroxyethyl) terephthalate (BHET) described in the present invention are from Sigma-Aldrich Company, with CAS No. 50-99-7, CAS No. 8068-05-1 and CAS No. 959-26-2.

[0032] The application of the engineered Rhodococcus etheriferus in the production of 1,4-pentanediol.

[0033] 1,4-Pentanediol was produced by fermenting engineered Rhodococcus etheriferus under aerobic conditions using glucose, glucose and lignin, or a mixture of glucose and BHET as carbon sources.

[0034] The fermentation culture conditions are:

[0035] 1) Plate culture: Streak the engineered Rhodococcus etherivorans onto LB solid medium containing 50 mg / L kanamycin at 25-35°C for 45-50 hours;

[0036] 2) Seed culture: Inoculate the colony from the solid culture medium into LB seed medium containing 50 mg / L kanamycin and culture at 25-35°C, 180 rpm, for 28-32 hours;

[0037] 3) Fermentation culture: The seed culture solution is inoculated into the fermentation medium at an inoculation amount of 3-5% v / v, the fermentation temperature is 25-35° C., and the fermentation culture time is 120 h.

[0038] The Rhodococcus etherivorans bacteria are activated by streaking on a solid culture medium. The activated strain is transferred to a seed culture medium and cultured on a shaker to obtain a seed solution. The seed solution is then inoculated into a fermentation medium for fermentation. Preferred seed activation conditions include incubating the streaked plate in an incubator at 30°C for 50 hours, then transferring the bacteria to a liquid culture medium and incubating at 30°C with a shaker at 180 rpm for 30 hours. Preferred fermentation conditions include inoculating the fermentation medium with a 5% inoculum of the seed solution.

[0039] The carbon sources of the fermentation medium are 10 g / L glucose, 10 g / L glucose and 10 g / L lignin, or 10 g / L glucose and 10 g / L BHET, the nitrogen sources are 5 g / L corn steep liquor powder and 5 g / L urea, the fermentation pH is 6, the temperature is 30° C., and the fermentation culture time is 120 h.

[0040] After protocatechuic acid is synthesized based on the shikimate pathway, the ring is opened to generate β-ketoadipate, which is then converted into levulinic acid under the action of decarboxylase. Levulinic acid is then converted into 4-hydroxyvaleric acid under the action of alcohol dehydrogenase. 4-hydroxyvaleric acid is then converted into 4-hydroxyvaleraldehyde under the catalysis of carboxylic acid reductase. Finally, 4-hydroxyvaleraldehyde is converted into 1,4-pentanediol under the action of alcohol dehydrogenase.

[0041] Beneficial effects:

[0042] The present invention constructs a new non-natural synthesis pathway for 1,4-pentanediol, which can utilize β-ketoadipate in the metabolism of microbial cells as a substrate to synthesize 1,4-pentanediol.

[0043] The present invention is based on a strain of Rhodococcus etheriferus with excellent performance. By knocking out the endogenous coenzyme A encoding gene pcaIJ and introducing the exogenous genes 3-dehydroshikimate dehydratase AroZ, β-ketoadipate decarboxylase KDC, levulinic acid dehydrogenase ADH, carboxylic acid reductase CAR and dehydrogenase BDH encoding genes, the engineered Rhodococcus etheriferus constructed can use carbon sources such as glucose to synthesize 1,4-pentanediol from scratch. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The plasmid map of pNV-aroZ-kdc-adh-car-bdh is shown.

[0045] Figure 2 This is the liquid phase detection spectrum of the fermentation product of Rhodococcus etherovorans NK-aroZ-kdc-adh-car-bdh.

[0046] Figure 3 This is the mass spectrometry detection spectrum of the fermentation product of Rhodococcus etherovorans NK-aroZ-kdc-adh-car-bdh.

[0047] Figure 4 This is a diagram showing the yield of NK-aroZ-kdc-adh-car-bdh synthesized by Rhodococcus etherovorans using different carbon sources. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to specific embodiments.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0051] The strain used in the present invention is Rhodococcus aetherivorans N1, with a deposit number of CCTCC NO: M 20221270; and the model strain is Escherichia coli MG1655.

[0052] The glucose, lignin and PET hydrolyzate bis(2-hydroxyethyl) terephthalate (BHET) used in the culture medium of the following examples were from Sigma-Aldrich Company, CAS No.: 50-99-7, CAS No.: 8068-05-1 and CAS No.: 959-26-2; the β-ketoadipic acid used was purchased from Yuanye Biotechnology Co., Ltd., CAS No.: 689-31-6.

[0053] Example 1

[0054] Based on the proposed metabolic pathway for converting β-ketoadipate to 1,4-PDO, enzyme elements were compared and analyzed on NCBI and their functions verified. The resulting functional enzyme elements were β-ketoadipate decarboxylase KDC (SEQ ID No. 2); levulinic acid dehydrogenase ADH (SEQ ID No. 3); carboxylic acid reductase CAR (SEQ ID No. 4); and dehydrogenase BDH (SEQ ID No. 5). Based on these enzyme elements, an engineered Escherichia coli strain capable of synthesizing 1,4-PDO from β-ketoadipate was constructed.

[0055] (1) Obtaining target genes: Based on the analysis of the assumed 1,4-PDO synthesis pathway, the functions of the enzymes required for each step were determined, and different gene elements with similar functions were selected through functional analysis and sequence alignment analysis. Finally, it was determined that the decarboxylase (KDC) gene from Staphylococcus condimenti, the dehydrogenase (ADH) gene from Levilactobacillus brevis, the carboxylic acid reductase (CAR) gene from Mycobacterium ulcerans, and the dehydrogenase (BDH) gene from Escherichia coli were selected to construct a synthesis pathway for synthesizing 1,4-PDO using β-ketoadipate in recombinant Escherichia coli. The kdc gene is shown in SEQ ID No. 2; the adh gene is shown in SEQ ID No. 3; the car gene is shown in SEQ ID No. 4; and the bdh gene sequence is shown in SEQ ID No. 5.

[0056] (2) GenScript Biotech Co., Ltd. was commissioned to synthesize the gene sequences of kdc, adh, car and bdh, and ligated them to plasmid pTrc99a, and then introduced into Escherichia coli MG1655, ultimately obtaining engineered Escherichia coli M1 containing the four genes of kdc, adh, car and bdh.

[0057] (2) The engineered strain M1 was cultured in E. coli fermentation medium containing 100 mg / L ampicillin at 37°C until the OD 600 When the pH value was 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.1 mM, and then 1 g / L of β-ketoadipic acid was added after induction culture at 16°C for 10 hours. After further culture for 6 hours, it was detected whether 1,4-pentanediol was produced. The liquid phase detection results showed that 0.6 g / L of 1,4-pentanediol was produced under this condition.

[0058] Example 2

[0059] Compared to E. coli, Rhodococcus can tolerate higher concentrations of organic solvents, so the applicant intends to achieve de novo synthesis of 1,4-pentanediol in Rhodococcus. First, the pcaIJ gene of Rhodococcus etherivorans N1 is knocked out through homologous recombination.

[0060] (1) Using the genome of strain N1 as a template, the homology arms A1 and A2 on both sides of pcaIJ were amplified. Each homology arm was 1000 bp in length. Linearized pK18mobsacB was obtained by inverse PCR. Multi-fragment ligation was performed using the ClonExpress MultiS One Step Cloning Kit. The ratio of vector to fragment in the reaction system was 1:1. The ligated reaction solution was introduced into competent E. coli, incubated in an ice-water bath for 30 minutes, then in a 42°C water bath for 90 seconds. After an ice-water bath for 2 minutes, 900 μL of LB liquid medium was added and incubated at 37°C, 180 rpm, for 45-60 minutes. The plasmid pK-FR was selected using a kanamycin selection plate to obtain the pcaIJ knockout plasmid.

[0061] (2) The constructed and verified correct plasmid was introduced into the original Rhodococcus etherivorans N1 by point transformation, with a voltage of 2000 V, a resistance of 400 Ω, a capacitor of 25 μF, and an electric shock time of 5-6 ms. After the electric shock, 400 μL of recovery medium was immediately added and the cells were cultured on a shaking table at 30°C for 3.5 h. The strains were screened on kanamycin screening plates and the strains were verified.

[0062] (3) Strains that were verified to have successfully undergone homologous recombination were placed in LB tubes and cultured at 30°C, 180 rpm for 10 h. The strains were then plated on LB agar containing 30% sucrose and cultured at 30°C for 4 days. The pcaIJ knockout strain NK was screened by alignment with a marker. All primer sequences are shown in Table 1.

[0063] Table 1

[0064]

[0065] Table 2 PCR system configuration table

[0066] system 50 μL Phanta Max Super-Fidelity DNA Polymerase 1 μL buffer 25 μL distilled water 20 μL dNTP 1 μL Quoted above 1 μL

[0067] Table 3 Conditions required for fragment amplification

[0068]

[0069] Example 3

[0070] Based on the proposed β-ketoadipate biosynthesis pathway, enzyme elements were compared and analyzed on NCBI, and their functions were verified. The final functional enzyme element screened was 3-dehydroshikimate dehydratase AroZ (SEQ ID No. 1). Combined with the previously screened genes β-ketoadipate decarboxylase KDC; levulinic acid dehydrogenase ADH; carboxylic acid reductase CAR; and dehydrogenase BDH, an engineered Rhodococcus etherivorans strain NK-aroZ-kdc-adh-car-bdh capable of synthesizing 1,4-PDO was constructed based on these enzyme elements and containing the pami promoter.

[0071] (1) Obtaining the target gene: Based on the hypothetical β-ketoadipate synthesis pathway, the functions of the enzymes required for each step were determined. Through functional analysis and sequence alignment analysis, different gene elements with similar functions were selected. Finally, the gene encoding 3-dehydroshikimate dehydratase (AroZ) from Acinetobacter ursingii was selected. The aroZ gene sequence is shown in SEQ ID No. 1.

[0072] (2) GenScript Biotech Co., Ltd. was commissioned to synthesize the aroZ gene. Combining the gene sequences of kdc, adh, car, and bdh obtained above, PNV-U and PNV-D were used as primers to obtain a plasmid reverse amplification template. AroZ gene fragment containing homology arms was obtained using aroZ-U and aroZ-D as primers; a kdc gene fragment containing homology arms was obtained using kdc-U and kdc-D as primers; an adh gene fragment containing homology arms was obtained using adh-U and adh-D as primers; a car gene fragment containing homology arms was obtained using car-U and car-D as primers; a bdh gene fragment containing homology arms was obtained using bdh-U and bdh-D as primers; and a pami promoter fragment was obtained using pami-U and pami-D as primers. All primer sequences are shown in Table 4.

[0073] Table 4 Primer sequences required for construction

[0074]

[0075] The PCR enzyme used for amplification was Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozymes Biotechnology Co., Ltd. The system is shown in Table 5

[0076] Table 5 PCR system configuration table

[0077]

[0078]

[0079] Amplification procedure:

[0080]

[0081] The corresponding fragment lengths, primers, reaction temperatures and times for different plasmid templates are shown in Table 6.

[0082] Plasmid template fragment length Upstream primer Downstream primer Temperature a Time b

[0083] Table 6 Conditions required for fragment amplification

[0084]

[0085] Multi-fragment ligation was performed using the ClonExpress MultiS One-Step Cloning Kit, with a 1:1 vector:fragment ratio in the reaction system. The ligated reaction mixture was introduced into competent E. coli cells, incubated on ice for 30 minutes, then in a 42°C water bath for 90 seconds. After a 2-minute ice bath, 900 μL of LB was added and incubated at 37°C, 180 rpm, for 45-60 minutes. The cells were then screened using a kanamycin selection plate.

[0086] The constructed and verified correct plasmid was introduced into the original Rhodococcus etherivorans N1 by point transformation, with a voltage of 1500V, a resistance of 400Ω, a capacitor of 25μF, and an electric shock time of 5-6ms. 400μL LB culture medium was added immediately after electric shock, and the cells were cultured on a shaker at 30°C for 3h. The cells were screened on a kanamycin screening plate and the strain was verified and sent for testing to obtain the engineered Rhodococcus etherivorans NK-aroZ-kdc-adh-car-bdh.

[0087] Example 4

[0088] Growth and fermentation of 1,4-PDO by Rhodococcus etherovorans strain NK-aroZ-kdc-adh-car-bdh using different carbon sources:

[0089] (1) Activation of bacterial strains: The engineered Rhodococcus etherivorans strain NK-aroZ-kdc-adh-car-bdh was cultured on LB solid medium containing 50 mg / L kanamycin at 30°C for 50 h.

[0090] (2) Seed culture: A single colony of the strain NK-aroZ-kdc-adh-car-bdh was picked from a solid plate culture medium and inoculated into a 5 mL LB liquid test tube culture medium containing 50 mg / L kanamycin. The culture was cultured at 30°C and 180 rpm for 30 h.

[0091] (3) Fermentation: The seed solution was inoculated at 5% v / v into a fermentation medium containing 50 mg / L kanamycin and different carbon sources. The fermentation was carried out at 30°C and 180 rpm for 120 h. The carbon sources were: 10 g / L glucose, 10 g / L glucose and 10 g / L lignin, 10 g / L glucose and 10 g / L BHET. The nitrogen sources were 5 g / L corn steep liquor powder and 5 g / L urea. The pH was adjusted to 6.

[0092] (4) Quantitative analysis of 1,4-PDO

[0093] The liquid chromatography model used in this patent is Agilent Technologies 1200 Infinity series; the chromatographic column is: HPLC Organic Acid Analysis HPX-87H Ion Exclusion column; differential detector; mobile phase: 0.25 g / L dilute sulfuric acid solution; injection volume: 5 μL; flow rate: 0.5 mL / min; column temperature: 50°C. Figure 2 This is the HPLC detection chart of the recombinant strain 1,4-PDO.

[0094] (5) LC-MS detection of 1,4-PDO

[0095] This patent uses a Thermo Scientific Q Exactive high-resolution mass spectrometer for electrospray ionization mass spectrometry (ESI-MS) analysis. The ionization source adopts HESI (heated electrospray ionization) mode, and full scan (full MS) is performed in negative mode. The mass spectrometer resolution is tuned to 140,000 FWHM (m / z 200). The spray voltage is set to 3.50 kV (positive) / 3.00 kV (negative), and the sheath gas and auxiliary gas flow rates are set to 45 and 15 (arbitrary units), respectively. The transfer capillary temperature is maintained at a constant 250°C, and the S-Lens RF level is set to 50 V. Data acquisition and processing are completed using the Xcalibur software platform. Figure 3 This is the mass spectrometry detection diagram of the recombinant strain 1,4-PDO.

[0096] The strain NK-aroZ-kdc-adh-car-bdh produced a maximum of 4.7 g / L of 1,4-pentanediol under the conditions of a mixed carbon source of 10 g / L glucose and 10 g / L BHET, which is a 376.7% increase compared to the yield when glucose alone was used as the carbon source. In the process of Rhodococcus using glucose to synthesize 1,4-PDO, the substrate glucose is required to first synthesize protocatechuic acid through the shikimic acid pathway, and then be converted into 1,4-PDO. When lignin is added exogenously, Rhodococcus can generate lignin-derived aromatic compounds, such as p-hydroxybenzoic acid and vanillic acid. These compounds can be directly converted into protocatechuic acid and then into 1,4-PDO. This process has the advantage of a shorter pathway compared to the synthesis using glucose as a substrate. When BHET is added exogenously, Rhodococcus can also directly convert BHET into protocatechuic acid and then into 1,4-PDO. This process has the advantage of a shorter pathway compared to using glucose as a substrate, and has the advantage of not requiring depolymerization compared to using lignin as a substrate.

Claims

1. A non-natural 1,4-pentanediol biosynthetic pathway, characterized in that: Starting from β-ketoadipate, β-ketoacid decarboxylase KDC catalyzes β-ketoadipate to produce levulinic acid, and then levulinic acid produces 4-hydroxyvaleric acid under the action of alcohol dehydrogenase ADH, and then 4-hydroxyvaleric acid produces 4-hydroxyvaleraldehyde under the catalysis of carboxylic acid reductase CAR, and finally 4-hydroxyvaleraldehyde produces 1,4-pentanediol under the action of alcohol dehydrogenase BDH.

2. A non-natural 1,4-pentanediol biosynthetic pathway according to claim 1, characterized in that: The β-ketoadipic acid is synthesized based on the shikimic acid pathway and then the protocatechuic acid is ring-opened to generate β-ketoadipic acid.

3. A non-natural 1,4-pentanediol biosynthetic pathway according to claim 1, characterized in that: The β-ketoadipate decarboxylase KDC is shown as SEQ ID No. 2; the dehydrogenase ADH is shown as SEQ ID No. 3; the carboxylic acid reductase CAR is shown as SEQ ID No. 4; and the alcohol dehydrogenase BDH is shown as SEQ ID No.

5.

4. The non-natural 1,4-pentanediol biosynthetic pathway according to claim 1, characterized in that: 1,4-pentanediol is biosynthesized by a catalytic system prepared by a recombinant strain overexpressing various enzymes required for the reaction, or 1,4-pentanediol is catalyzed and synthesized by an in vitro multi-enzyme system containing various enzymes required for the reaction.

5. An engineered Rhodococcus etheriferus strain for synthesizing 1,4-pentanediol, characterized in that: The engineered Rhodococcus etherivorans is a bacterium in which the coenzyme A transferase encoding gene is knocked out. pcaI , and simultaneously introduced 3-dehydroshikimate dehydratase AroZ, β-ketoadipate decarboxylase KDC, levulinic acid dehydrogenase ADH, carboxylic acid reductase CAR and dehydrogenase BDH encoding genes to construct it.

6. The engineered Rhodococcus etherivorans for synthesizing 1,4-pentanediol according to claim 5, characterized in that: The sequence of the 3-dehydroshikimate dehydratase AroZ is shown in SEQ ID No. 1; the sequence of the β-ketoadipate decarboxylase KDC is shown in SEQ ID No. 2; the sequence of the levulinic acid dehydrogenase ADH is shown in SEQ ID No. 3; the sequence of the carboxylic acid reductase CAR is shown in SEQ ID No. 4; and the sequence of the dehydrogenase BDH is shown in SEQ ID No.

5.

7. The engineered Rhodococcus etherivorans for synthesizing 1,4-pentanediol according to claim 5, characterized in that: The ether-vortex Rhodococcus is ether-vortex Rhodococcus ( Rhodococcus aetherivorans ) N1, the deposit number is CCTCC NO: M 20221270.

8. The engineered Rhodococcus etherivorans for synthesizing 1,4-pentanediol according to claim 1, characterized in that The gene was expressed in plasmid pNV18.1 to construct the Rhodococcus shuttle plasmid pNV- aroZ - kdc-adh-car-bdh Introduced into the Rhodococcus etherivorans.

9. Use of the engineered Rhodococcus etherivorans according to any one of claims 5 to 8 in the production of 1,4-pentanediol.

10. The use according to claim 9, characterized in that Glucose, lignin or polyethylene terephthalate hydrolyzate (ethylene terephthalate) is used as substrate and 1,4-pentanediol is produced by fermentation culture of engineered Rhodococcus etheriferus under aerobic conditions.

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