Patent Application
20200010864
The disclosure herein relates to the field of biocatalysis, and particularly relates to a method for producing piperonal by using a recombinant engineered bacterium co-expressing trans-anethole oxygenase and formate dehydrogenase and an engineered bacterium thereof.
Piperonal, also known as heliotropin, has a sweet cherry and vanilla aroma. The piperonal is widely applied in food flavor, cosmetic, pharmaceutical, agricultural and electroplating industries. Natural heliotropinis mainly found in plants such as melon, Robinia pseudoacacia, allspice, and vanilla beans, but the content is extremely small, and people cannot directly extract the natural heliotropin from plants.
At present, the heliotropin is industrially produced mainly by a semi-synthesis method using safrole as a raw material and a total synthesis method using catechol as a raw material. The method seriously pollutes the environment and consumes much energy. With the increasing attention of people on the material production process and the increasing requirements for food additives, flavor substances synthesized by a bio-fermentation method and enzymatic method are increasingly favored by people.
Microbial synthesis of piperonal is currently not applied to industrial production. The Santos team in Brazil found several strains of microbes of producing piperonal with isosafrole. The most efficient strain is Paecilomyces variot bainier, but only under the condition that H2O2 is added, the highest yield is 64 mg/L, and the transformation rate is only 20%. In Chinese patent CN105779363A, it is found that a strain of Serratia liquefaciens produces 282 mg/L of piperonal.
Therefore, how to improve enzyme activity of trans-anethole oxygenase and accelerate catalytic synthesis rate of piperonal is a technical problem to be solved urgently in the field of microbial synthesis of piperonal.
The present disclosire provides a recombinant engineered bacterium co-expressing trans-anethole oxygenase and formate dehydrogenase used for producing piperonal, wherein a gene fdh encoding the formate dehydrogenase, and a gene tao encoding the trans-anethole oxygenase or a gene tao3g2 encoding a trans-anethole oxygenase mutant are connected in series to a pETDuet-1 vector. Preferably, the gene fdh encoding the formate dehydrogenase is located at a first multiple cloning site MSC1 of the pETDuet-1 vector, and the gene tao encoding the trans-anethole oxygenase or the gene tao3g2 encoding the trans-anethole oxygenase mutant is located at a second multiple cloning site MSC2 of the pETDuet-1 vector.
In an embodiment of the present application, a nucleotide sequence of the trans-anethole oxygenase gene tao is shown in SEQ ID NO.2; a nucleotide sequence of the trans-anethole oxygenase mutant gene tao3g2 is shown in SEQ. ID NO.3; the formate dehydrogenase gene fdh is derived from Candida boidinii, and a nucleotide sequence thereof is shown in SEQ ID NO.1 after codon optimization.
In an embodiment of the present application, the recombinant engineered bacterium uses E. coli BL21(DE3) as a host.
The present application also provides a method for producing piperonal by using the recombinant engineered bacterium co-expressing Trans-Anethole Oxygenase and formate dehydrogenase. The method comprises the following steps:
(1) preparing a biocatalyst,
(2) by using safrole as a substrate and sodium formate as a cosubstrate, catalytically synthesizing piperonal with the biocatalyst.
In an embodiment of the present application, in step (1), the formate dehydrogenase gene fdh and the trans-anethole oxygenase gene tao or the trans-anethole oxygenase mutant gene tao3g2 co-expression recombinant vector transform the E. coli BL21(DE3) strain, then are inoculated in an LB medium containing 30 to 100 mg/L ampicillin, and cultured to obtain a seed solution; and then the seed solution is inoculated in the LB medium containing 30 mg/L to 100 mg/L ampicillin in an amount of 2% to 5% and cultured at a temperature of 37° C. until OD600 nm is 0.5 to 1.8, and IPTG with a final concentration of 0.1 mmol/L to 1 mmol/L is added, inductively cultured at a temperature of 16° C. to 28° C. for 6 h to 10 h, and centrifuged to collect thallus cells.
In an embodiment of the present application, in step (2), a reaction system is constituted by using the safrole as the substrate, the sodium formate as the cosubstrate, and a buffer with pH of 3 to 9 as a reaction medium, the transformation is carried out at a temperature of 20° C. to 40° C. and a speed of 50 rpm˜240 rpm for 0.5 h to 12 h.
In an embodiment of the present application, in step (2), the substrate concentration is 1 g/L to 30 g/L, the cosubstrate concentration is 1 g/L to 60 g/L, and the amount of the biocatalyst is 10 to 100 g/L calculated by a wet weight of a thallus.
The present application has the beneficial effects that are combinant genetically engineered bacterium is obtained by constructing the recombinant vectors co-expressing the formate dehydrogenase gene and the trans-anethole oxygenase gene or the trans-anethole oxygenase mutant gene, 15.91 g/L piperonal with a transformation rate of 79.55% and a 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.
Cloning of formate dehydrogenase gene fdh and trans-anethole oxygenase gene tao (and mutant gene thereof) and construction of a co-expression recombinant system:
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 tao3g2 is shown in SEQ ID NO.3. The trans-anethole oxygenase activity of the mutant encoded by the genetao3g2 was improved by about 1.4 times compared with the activity of the enzyme encoded by the gene tao.
(1) Construction of Co-Expression Recombinant Vectors pETDuet-1-Fdh-Too and pETDuet-1-Fdh-Tao3g2:
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: 5T-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 tao3g2 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-tao3g2 of the formate dehydrogenase and the trans-anethole oxygenase mutant was obtained by construction (as shown in
(2) Construction of Double Plasmids Expression Vector:
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.
(3) Construction of Co-Expression Recombinant Vector pETDuet-1-Tao-Fdh:
On the basis of pETDuet-1-fdh-tao, the order of fdh and tao was adjusted to obtain pETDuet-1-tao-fdh.
On the basis of pETDuet-1-fdh-tao3g2, the order of fdh and tao was adjusted to obtain pETDuet-1-tao3g2-fdh.
(4) Construction of Other Expression Vectors
Referring to Tables 3 to 5, tao (or tao3g2) 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.
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.
Inducible expression of recombinant genetically engineered bacteria of different co-expression systems:
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 OD600 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.
Comparison of trans-anethole oxygenase activity of different co-expression strains:
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 μm 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 μmol/L piperonal per minute when the thallus OD600 nm is equal to 1.
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 OD340 nm at an interval of 1 min, placing 200 μL 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+ in ELIASA and preheating for 10 min, and adding 100 μL 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 μmol of NADH per minute.
The determination method of piperonal content was an HPLC method. HPLC detection conditions were as follows: an Amethst C18-H reverse column (4.6 mm×250 mm, 5 μm) 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 μL, and the piperonal content was detected at 270 nm.
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-tao3g2-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).
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-tao3g2. The enzyme activity of the trans-anethole oxygenase mutant was 205U.
E. coli BL21(DE3)pETDuet-1-tao
E. coli BL21(DE3)pET-28a-fdh
E. coli BL21(DE3)pET-28a-fdh-tao
E. coli BL21(DE3)pET-28a-tao-fdh
E. coli BL21(DE3)pETDuet-1-tao-fdh
E. coli BL21(DE3)pETDuet-1-fdh-tao
E. coli BL21(DE3)pETDuet-1-tao&pCDFDuet-1-
E. coli BL21(DE3)pETDuet-1-fdh-
E. coli BL21(DE3)pETDuet-1-fdh-tao3g2
E. coli BL21 (DE3) pCDFDuet-1-tao
E. coli BL21 (DE3) pCDFDuet-1-tao-fdh
E. coli BL21 (DE3) pCDFDuet-1-fdh-tao
E. coli BL21 (DE3) pCDFDuet-1-tao&pETDuet-1-
E. coli BL21 (DE3) pCDFDuet-1-fdh-
E. coli BL21 (DE3) pCDFDuet-1-fdh-
E. coli BL21 (DE3) pACYDuet-1-tao
E. coli BL21 (DE3) pACYDuet-1-tao-fdh
E. coli BL21 (DE3) pACYDuet-1-fdh-tao
E. coli BL21 (DE3) pACYDuet-1-tao&pETDuet-
E. coli BL21 (DE3) pACYDuet-1-fdh-
E. coli BL21 (DE3) pACYDuet-1-fdh-tao3g2
E. coli BL21(DE3)pRSFDuet-1-tao
E. coli BL21(DE3)pRSFDuet-1-tao-fdh
E. coli BL21(DE3)pRSFDuet-1-fdh-tao
E. coli BL21(DE3)pRSFDuet-1-tao&pETDuet-1-
E. coli BL21(DE3)pRSFDuet-1-fdh-tao&pETDuet-
E. coli BL21(DE3)pRSFDuet-1-fdh-tao3g2
E. coli BL21(DE3)pCOLADuet-1-tao
E. coli BL21(DE3)pCOLADuet-1-tao-fdh
E. coli BL21(DE3)pCOLADuet-1-fdh-tao
E. coli BL21(DE3)pCOLADuet-1-
E. coli BL21(DE3)pCOLADuet-1-fdh-
E. coli BL21(DE3)pCOLADuet-1-fdh-
Production of piperonal by E. coli BL21(DE3)pETDuet-1-fdh-tao3g2 using safrole as a substrate:
The strain E. coli BL21(DE3)pETDuet-1-fdh-tao3g2 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
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019103219780 | Apr 2019 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2018/094507 | Jul 2018 | US |
| Child | 16517792 | US |
