Recombinant engineered bacterium co-expressing trans-anethole oxygenase and formate dehydrogenase and application thereof in production of piperonal

Abstract

The present application discloses a method for producing piperonal by using a recombinant engineered bacterium co-expressing trans-anethole oxygenase and formate dehydrogenase, and an engineered bacterium thereof, including constructing a formate dehydrogenase gene fdh and trans-anethole oxygenase gene tao or trans-anethole oxygenase mutant gene co-expression recombinant vector; inductively expressing recombinant genetically engineered bacterium; and producing piperonal by using the recombinant genetically engineered bacterium. 15.91 g/L of piperonal with a transformation rate of 79.55% and a time-space transformation rate of 2.27 g/L/h can be finally obtained during catalysis, and the yield is significantly improved compared with the existing piperonal, thereby being more conducive to the smooth realization of industrial production.

Claims

1. A recombinant engineered bacterium for producing piperonal, which comprises: a gene encoding a trans-anethole oxygenase mutant having the nucleotide sequence of SEQ ID NO: 3, and a gene encoding a formate dehydrogenase that when expressed oxidizes formate; wherein the gene encoding the formate dehydrogenase and the gene encoding the trans-anethole oxygenase mutant are connected in series in a bacterial expression vector.

2. The recombinant engineered bacterium of claim 1, wherein the formate dehydrogenase gene is located upstream of the gene encoding the trans-anethole oxygenase mutant in the vector.

3. The recombinant engineered bacterium of claim 1, wherein the formate dehydrogenase gene is from Candida boidinii.

4. The recombinant engineered bacterium of claim 1, wherein the recombinant engineered bacterium is E. coli BL21(DE3) transformed with the vector.

5. The recombinant engineered bacterium of claim 2, wherein the formate dehydrogenase gene has the nucleotide sequence of SEQ ID NO: 1.

6. A method for producing piperonal comprising (a) culturing the recombinant engineered bacterium of claim 1, and (b) incubating safrole as a substrate and sodium formate as a cosubstrate with the recombinant engineered bacterium.

7. The method of claim 6, wherein the recombinant engineered bacterium is E. coli BL21(DE3) transformed with the vector.

8. The method of claim 6, wherein the formate dehydrogenase gene is from Candida boidinii.

9. The method of claim 6, wherein said method further comprises: (i) transforming an E. coli BL21(DE3) strain with a bacterial expression vector that comprises a gene encoding a trans-anethole oxygenase mutant having the nucleotide sequence of SEQ ID NO: 3, and a gene encoding a formate dehydrogenase that when expressed oxidizes formate, wherein the gene encoding the formate dehydrogenase and the gene encoding the trans-anethole oxygenase mutant are connected in series in the bacterial expression vector; (ii) incubating the transformed E. coli BL21(DE3) strain in a Luria broth medium comprising 30 to 100 mg/L ampicillin; (iii) culturing the transformed E. coli BL21(DE3) strain to obtain a seed solution; (iv) inoculating a second Luria broth medium comprising 30 to 100 mg/L ampicillin with the seed solution, wherein the inoculated second Luria broth medium comprises between 2% to 5% of seed solution; (v) incubating the second Luria broth medium of (iv) at a temperature of 37 C. until OD.sub.600 is 0.5 to 1.8; (vi) adding isopropyl--D-1-thiogalactopyranoside (IPTG) to the second Luria broth medium of (v) to a final concentration of 0.1 to 1 mmol/L to induce expression from the bacterial expression vector; (vii) culturing the second Luria broth medium of (vi) at a temperature of 16 C. to 28 C. for 6 to 10 hours; and (viii) centrifuging the second Luria broth medium of (viii) to collect the recombinant engineered bacterium.

10. The method of claim 9, wherein safrole and sodium formate are in a buffer solution with a pH of 3 to 9, and wherein safrole and sodium formate are incubated with the recombinant engineered bacterium at a temperature of 20 C. to 40 C.

11. The method of claim 10, wherein safrole is added at a concentration of from 1 to 30 g/L, sodium formate is added at a concentration of from 1 to 60 g/L, and the recombinant engineered bacterium is present at a concentration of 10 to 100 g/L.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is a vector map of a trans-anethole oxygenase and formate dehydrogenase co-expression vectorpETDuet-1-fdh-tao in the present application.

(2) FIG. 2 is production of piperonal by E. coli BL21(DE3)pETDuet-1-fdh-tao.sub.3g2 using safrole as a substrate.

DETAILED DESCRIPTION

Example 1

(3) Cloning of formate dehydrogenase gene fdh and trans-anethole oxygenase gene tao (and mutant gene thereof) and construction of a co-expression recombinant system:

(4) A nucleotide sequence of the gene fdh encoding the formate dehydrogenase was shown in SEQ ID NO.1, which was derived from Candida boidinii (GenBank: AJ011046.2) and codon-optimized, and the codon-optimized nucleotide sequence was shown in SEQ ID NO.1. The codon-optimized gene fdh encoding the formate dehydrogenase was connected to a vector pET-28a to obtain a recombinant vector pET-28a-fdh; a nucleotide sequence of the gene tao encoding the trans-anethole oxygenase was shown in SEQ ID NO.2, which was derived from Pseudomonas putida, and a nucleotide sequence of the trans-anethole oxygenase mutant gene tao.sub.3g2 is shown in SEQ ID NO.3. The trans-anethole oxygenase activity of the mutant encoded by the genetao.sub.3g2 was improved by about 1.4 times compared with the activity of the enzyme encoded by the gene tao.

(5) (1) Construction of Co-Expression Recombinant Vectors pETDuet-1-Fdh-Too and pETDuet-1-Fdh-Tao.sub.3g2:

(6) PCR amplification (using PrimeSTAR Max DNA Polymerase, TaKaRa) was carried out by using pET-28a-fdh as a template through upstream and downstream primers (upstream: 5-ccacagccaggatccgaattcatgaaaattgtgctggtgctgta-3, see SEQ ID NO.4; downstream: 5-cgacttaagcattatgcggccgcttatttcttatcatgtttgccataggc-3, see SEQ ID NO.5) to obtain a product containing the formate dehydrogenase gene. A plasmid pETDuet-1 (MSC1) was double-digested with EcoRI and Notl, and a linear plasmid was recovered, and then a recombinant plasmid pETDuet-1-fdh was obtained by onestep cloning. Subsequently, by designing upstream and downstream primers (upstream: 5-agatatacatatggcagatctatggaggacatcatgcaaggc-3, see SEQ ID NO.6; downstream: 5-ggtttctttaccagactcgagtcagttagtcctcaagtcggaattg-3, SEQ ID NO.7) to carry out the PCR amplification to obtain the trans-anethole oxygenase gene tao (and a mutant gene tao.sub.3g2 thereof). A plasmid pETDuet-1-fdh (MSC2) was first double-digested with BglII and XhoI, and a linear plasmid was recovered, and then a recombinant plasmid pETDuet-1-fdh-tao was obtained by one step cloning. By using the same method, a co-expression recombinant vector pETDuet-1-fdh-tao.sub.3g2 of the formate dehydrogenase and the trans-anethole oxygenase mutant was obtained by construction (as shown in FIG. 1). In the above process, the PCR reaction conditions were: 94 C. for 5 min; 98 C. for 10 s, 56 C. for 5 s, 72 C. for 15 s (35 cycles); and 72 C. for 10 min. After the end of PCR, a target gene fragment was recovered by using a PCR product purification kit (PCR product purification kit, GENERay Company). The specific method of onestep cloning (using One Step Cloning Kit, Vazyme Company) includes steps of mixing the target gene fragment (0.04 to 0.08 ng/bp) with the vector (0.005 to 0.01 ng/bp) in the same PCR tube, adding 2 to 4 L of a recombinase buffer (5CEllBuffer) and 1 to 2 L of recombinase (Exnase II), adding ddH.sub.2O to supplement to 10 to 20 L, and reacting at a temperature of 37 C. for 30 min to obtain a recombinant plasmid. PETDuet-1-tao-fdhis obtained by exchanging locations of multiple cloning sites of the above formate dehydrogenase and trans-anethole oxygenase.

(7) (2) Construction of Double Plasmids Expression Vector:

(8) In consideration of the fact that the trans-anethole oxygenase plays a major role in the catalytic synthesis reaction of piperonal, the formate dehydrogenase was cloned into a plasmid pCDFDuet-1 with a lower copy number to finally obtain pCDFDuet-1-fdh. The gene tao was connected to pETDuet-1 to obtain pETDuet-1-tao. PCDFDuet-1-fdh constitutes a double plasmid expression vector with pETDuet-1-tao and pETDuet-1-fdh-tao, respectively.

(9) (3) Construction of Co-Expression Recombinant Vector pETDuet-1-Tao-Fdh:

(10) On the basis of pETDuet-1-fdh-tao, the order of fdh and tao was adjusted to obtain pETDuet-1-tao-fdh.

(11) On the basis of pETDuet-1-fdh-tao.sub.3g2, the order of fdh and tao was adjusted to obtain pETDuet-1-tao.sub.3g2-fdh.

(12) (4) Construction of Other Expression Vectors

(13) Referring to Tables 3 to 5, tao (or tao.sub.3g2) and fdh were cloned into pCDFDuet-1, pACYCDuet-1, pRSFDuet-1, and pCOLADuet-1 plasmids by using a method similar to the above (1) to obtain different co-expression systems.

(14) The vectors constructed in the above (1) to (4) were separately transferred into E. coli BL21(DE3), and screened to obtain positive transformants, namely, co-expression strains.

Example 2

(15) Inducible expression of recombinant genetically engineered bacteria of different co-expression systems:

(16) Different co-expression strains obtained in the above Example 1 were inoculated respectively in an LB medium containing 30 to 100 mg/L ampicillin, and cultured at a temperature of 37 C. overnight to obtain a seed solution; then the seed solution was inoculated in the LB medium containing 30 to 100 mg/L ampicillin in an amount of 2 to 5% until OD.sub.600 nm was 0.5 to 1.8, and was added with IPTG with a final concentration of 0.1 to 1 mmol/L and inductively cultured at a temperature of 16 to 28 C. for 6 to 10 h. Finally, the culture was centrifuged at a temperature of 4 C. and a speed of 5,000-8,000 rpm for 8 to 15 min, and a supernatant was discarded to obtain the cells, and the cells were washed twice with normal saline, and centrifuged to collect cells, namely, a biocatalyst.

Example 3

(17) Comparison of trans-anethole oxygenase activity of different co-expression strains:

(18) The determination method of trans-anethole oxygenase (TAO) activity includes the following steps: adding an appropriate amount of biocatalyst as well as safrole and sodium formate in a reaction system, reacting at a temperature of 30 C. for 20 min, taking a certain volume of reaction solution, adding an equal volume of methanol and uniformly mixing, terminating the reaction, centrifuging at a speed of 10,000 rpm for 5 min, filtering a supernatant through a 0.22 m membrane, and detecting the content of piperonal by HPLC. The trans-anethole oxygenase activity (U) was defined as the amount of enzyme required to produce 1 mol/L piperonal per minute when the thallus OD.sub.600 nm is equal to 1.

(19) The determination method of formate dehydrogenase activity (FDH) includes the following steps: carrying out the determination method at a temperature of 30 C. for consecutive 10 min, measuring OD.sub.340 nm at an interval of 1 min, placing 200 L of a reaction buffer containing 10 mmol/L sodium phosphate buffer (pH=7.5), 167 mmol/L sodium formate, and 1.67 mmol/L NAD.sup.+ in ELIASA and preheating for 10 min, and adding 100 L of a crude enzyme solution to start the reaction. The formate dehydrogenase activity (U) was defined as the amount of enzyme required to produce 1 mol of NADH per minute.

(20) The determination method of piperonal content was an HPLC method. HPLC detection conditions were as follows: an Amethst C18-H reverse column (4.6 mm250 mm, 5 m) was adopted, 60% acetonitrile, and 0.1% formic acid were used as a mobile phase, the column oven temperature was 35 C., the injection volume was 10 L, and the piperonal content was detected at 270 nm.

(21) The strains for comparing the enzyme activity were as follows: E. coli BL21(DE3)pETDuet-1-tao, E. coli BL21(DE3)pETDuet-1-fdh-tao, E. coli BL21(DE3)pETDuet-1-tao-fdh, E. coli BL21(DE3)pETDuet-1-tao.sub.3g2-fdh, E. coli BL21(DE3)pETDuet-1-tao/pCDFDuet-1-fdh, E. coli BL21(DE3)pETDuet-1-fdh-tao/pCDFDuet-1-fdh, etc. (Tables 1 to 5).

(22) By comparing the TAO enzyme activity with the FDH enzyme activity, it was found that the trans-anethole oxygenase activity and the formate dehydrogenase activity were shown at different levels in co-expression systems constructed based on different plasmids (pETDuet-1, pCDFDuet-1, pACYCDuet-1, pRSFDuet-1, and pCOLADuet-1). Compared with other plasmids, pETDuet-1 was selected as a plasmid to co-express FDH and TAO, and better results were obtained. PETDuet-1 was selected as a plasmid to co-express FDH and TAO mutants to obtain a recombinant genetically engineered strain E. coli BL21(DE3)pETDuet-1-fdh-tao.sub.3g2. The enzyme activity of the trans-anethole oxygenase mutant was 205U.

(23) TABLE-US-00001 TABLE 1 Comparison of trans-anetholee monooxygenase activity and formate dehydrogenase activity of different co-expression strains constructed by mainly using pETDuet-1 plasmid Co-expression strains TAO(U) FDH(U) E. coli BL21(DE3)pETDuet-1-tao 38.48 E. coli BL21(DE3)pET-28a-fdh 67.39 E. coli BL21(DE3)pET-28a-fdh-tao 20.21 11.73 E. coli BL21(DE3)pET-28a-tao-fdh 32.42 2.83 E. coli BL21(DE3)pETDuet-1-tao-fdh 62.34 5.74 E. coli BL21(DE3)pETDuet-1-fdh-tao 182.82 26.03 E. coli BL21(DE3)pETDuet-1-tao&pCDFDuet-1- 66.79 14.98 fdh E. coli BL21(DE3)pETDuet-1-fdh- 168.51 27.66 tao&pCDFDuet-1-fdh E. coli BL21(DE3)pETDuet-1-fdh-tao.sub.3g2 205.26 27.20 Note: means that no activity was detected.

(24) TABLE-US-00002 TABLE 2 Comparison of trans-anetholee monooxygenase activity and formate dehydrogenase activity of different co-expression strains constructed by mainly using pCDFDuet-1 plasmid Co-expression strains TAO(U) FDH(U) E. coli BL21 (DE3) pCDFDuet-1-tao 20.74 E. coli BL21 (DE3) pCDFDuet-1-tao-fdh 33.96 4.22 E. coli BL21 (DE3) pCDFDuet-1-fdh-tao 91.05 18.43 E. coli BL21 (DE3) pCDFDuet-1-tao&pETDuet-1- 53.28 19.57 fdh E. coli BL21 (DE3) pCDFDuet-1-fdh- 101.71 24.58 tao&pETDuet-1-fdh E. coli BL21 (DE3) pCDFDuet-1-fdh- 115.73. 22.59 tao.sub.3g2&pETDuet-1-fdh Note: means that no vitality was detected.

(25) TABLE-US-00003 TABLE 3 Comparison of trans-anethole oxygenase activity and formate dehydrogenase activity of different co-expression strains constructed by mainly using pACYCDuet-1 plasmid Co-expression strains TAO(U) FDH(U) E. coli BL21 (DE3) pACYDuet-1-tao 9.07 E. coli BL21 (DE3) pACYDuet-1-tao-fdh 12.54 2.68 E. coli BL21 (DE3) pACYDuet-1-fdh-tao 30.89 9.36 E. coli BL21 (DE3) pACYDuet-1-tao&pETDuet- 10.36 20.64 1-fdh E. coli BL21 (DE3) pACYDuet-1-fdh- 5.38 23.82 tao&pETDuet-1-fdh E. coli BL21 (DE3) pACYDuet-1-fdh-tao.sub.3g2 42.37 7.01 Note: means that no activity was detected.

(26) TABLE-US-00004 TABLE 4 Comparison of trans-anethole oxygenase activity and formate dehydrogenase activity of different co-expression strains constructed by mainly using pRSFDuet-1 plasmid Co-expression strains TAO(U) FDH(U) E. coli BL21(DE3)pRSFDuet-1-tao 19.77 E. coli BL21(DE3)pRSFDuet-1-tao-fdh 33.69 2.71 E. coli BL21(DE3)pRSFDuet-1-fdh-tao 127.03 20.82 E. coli BL21(DE3)pRSFDuet-1-tao&pETDuet-1- 60.42 26.79 fdh E. coli BL21(DE3)pRSFDuet-1-fdh-tao&pETDuet- 93.45 28.00 1-fdh E. coli BL21(DE3)pRSFDuet-1-fdh-tao.sub.3g2 162.44 16.93 Note: means that no activity was detected.

(27) TABLE-US-00005 TABLE 5 Comparison of trans-anethole oxygenase activity and formate dehydrogenase activity of different co-expression strains constructed by mainly using pCOLADuet-1 plasmid Co-expression strains TAO(U) FDH(U) E. coli BL21(DE3)pCOLADuet-1-tao 17.58 E. coli BL21(DE3)pCOLADuet-1-tao-fdh 29.35 2.03 E. coli BL21(DE3)pCOLADuet-1-fdh-tao 100.63 18.26 E. coli BL21(DE3)pCOLADuet-1- 58.27 23.47 tao&pETDuet-1-fdh E. coli BL21(DE3)pCOLADuet-1-fdh- 101.93 31.84 tao&pETDuet-1-fdh E. coli BL21(DE3)pCOLADuet-1-fdh- 112.30 29.04 tao.sub.3g2&pETDuet-1-fdh

Example 4

(28) Production of piperonal by E. coli BL21(DE3)pETDuet-1-fdh-tao.sub.3g2 using safrole as a substrate:

(29) The strain E. coli BL21(DE3)pETDuet-1-fdh-tao.sub.3g2 with the highest trans-anethole oxygenase activity obtained in Example 3 was inductively expressed according to the method in Example 2 to prepare a biocatalyst. In a PBS buffer with pH of 7.4, 7.5% of biocatalyst, 20 g/L safrole and 40 g/L sodium formate were added, the transformation was carried out at a temperature of 30 C. and a speed of 110 rpm for 8 h, and the product generation situation was observed by sampling every 1 h. As shown in FIG. 2, when the transformation was carried out by using the 20 g/L substrate, the piperonal with a yield of 15.91 g/L, a transformation rate of 79.55%, and a space-time transformation rate of 2.27 g/L/h was finally obtained.

(30) In summary, the recombinant genetically engineered bacterium is obtained by constructing the recombinant vector co-expressing the formate dehydrogenase gene and the trans-anethole oxygenase gene (and the mutant gene thereof) according to the present application. The mechanism is as follows: during the catalytic reaction, the concentration of FADH in the catalytic reaction is improved by providing NADH to reduce the prosthetic group FAD of TAO. The experimental results show that the method provided by the present application greatly improves the overall catalytic efficiency, 15.91 g/L piperonal with the transformation rate of 79.55% and the time-space transformation rate of 2.27 g/L/h is finally obtained during catalysis, and the yield is significantly improved compared with the existing piperonal, thereby being more conducive to the smooth realization of industrial production.