Genetically engineered bacteria for producing cinnamic acid and its derivatives, construction method and application thereof

Abstract

The application discloses a genetically engineered bacterium for producing cinnamic acid and derivatives thereof, a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium co-expresses vanillyl alcohol oxidase and oxidoreductase. The genetically engineered bacterium can be used as a biological catalyst to convert a substrate into cinnamic acid and derivatives thereof. The method for producing cinnamic acid and derivatives thereof has the advantages of high production efficiency, short reaction period, low cost and good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a genetically engineered bacterium for producing cinnamic acid and its derivatives, its construction method, and its applications. Background Technology

[0002] Cinnamic acid, also known as β-phenylacrylic acid or 3-phenyl-2-acrylic acid, is an organic compound with the chemical formula C9H8O2. It is an organic acid isolated from cinnamon bark or benzoin. It is produced in plants by the deamination and degradation of phenylalanine. Cinnamic acid is mainly used in fragrances, food additives, pharmaceuticals, cosmetics, pesticides, and organic synthesis.

[0003] 3,4-Dihydroxycinnamic acid, also known as caffeic acid, is widely found in various plants. It can be used as a natural antioxidant and is widely applied in cosmetics, food and pharmaceutical industries. At the same time, it is also an important precursor compound for the synthesis of caffeic acid derivatives, and can be used to synthesize chlorogenic acid and caffeic acid phenethyl ester and other products with high added value.

[0004] The main methods for preparing cinnamic acid and its derivatives (such as caffeic acid) include extraction, chemical synthesis, and biotransformation. Extraction methods primarily use natural plants as raw materials, extracting caffeic acid through steps such as extraction and soaking. However, the high cost, low yield, and low purity of this method limit its industrial application. Chemical synthesis methods suffer from drawbacks such as numerous byproducts, difficult purification, and uncontrollable reaction conditions, making them unsuitable for large-scale industrial production. Existing biosynthetic routes for cinnamic acid and its derivatives have disadvantages such as low yield, low production efficiency, high substrate cost, and long reaction cycles. For example, Chinese patent CN106701843 uses catechol, pyruvate, and ammonia as substrates, generating caffeic acid through the action of tyrosine phenyl lyase and tyrosine amino lyase, but the maximum caffeic acid yield obtained is only 35.5 mg / L. Therefore, developing a method for preparing cinnamic acid and its derivatives with high yield, high production efficiency, and low cost is of greater practical value.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a genetically engineered bacterium for the production of cinnamic acid and its derivatives, its construction method, and its applications. Using this genetically engineered bacterium as a biocatalyst, substrates can be converted into cinnamic acid and its derivatives. This method features high production efficiency, short reaction cycle, high yield of the target product, and low cost, and has promising prospects for industrial application.

[0007] This invention provides a new preparation route to improve the shortcomings of cinnamic acid and its derivatives in the prior art, such as low yield, low production efficiency, high substrate cost, and long reaction cycle.

[0008] Regarding cinnamic acid, this invention uses styrene as a raw material. Vanillyl alcohol oxidase converts styrene to cinnamyl alcohol, coniferyl alcohol dehydrogenase converts cinnamaldehyde to cinnamaldehyde, and coniferyl aldehyde dehydrogenase generates cinnamic acid. A coenzyme is also required during the reaction. The reaction principle is as follows: Figure 1 As shown. Of course, any enzyme capable of converting phenylpropene to cinnamyl alcohol, or cinnamyl alcohol to cinnamaldehyde, or cinnamaldehyde to cinnamic acid, is applicable to this invention; for example, alcohol dehydrogenase (EC1.1.1.1) or cinnamyl alcohol dehydrogenase (EC1.1.1.195) can replace CalA, acetaldehyde dehydrogenase (EC1.2.1.3) or acetaldehyde oxidase (EC1.2.3.1) can replace CalB, etc.; this invention does not list these enzymes one by one. This invention does not limit itself to any specific enzyme.

[0009] Regarding 3,4-dihydroxycinnamic acid, this invention uses eugenol as a raw material. Eugenol is converted to 4-allyl catechol by O-demethylase, 4-allyl catechol is converted to 3,4-dihydroxycinnamicol by vanillyl alcohol oxidase, and 3,4-dihydroxycinnamicol is converted to 3,4-dihydroxycinnamaldehyde by coniferyl alcohol dehydrogenase. 3,4-dihydroxycinnamic acid is then generated under the action of coniferyl alcohol dehydrogenase. A coenzyme is also required during the reaction. The reaction principle is as follows: Figure 2 As shown. Of course, any enzyme capable of converting eugenol to 4-allylcatechol, or 4-allylcatechol to 3,4-dihydroxycinnamate, or 3,4-dihydroxycinnamate to 3,4-dihydroxycinnamaldehyde, or 3,4-dihydroxycinnamaldehyde to 3,4-dihydroxycinnamic acid, is applicable to this invention; for example, alcohol dehydrogenase (EC1.1.1.1) or cinnamate dehydrogenase (EC1.1.1.195) can replace CalA, acetaldehyde dehydrogenase (EC1.2.1.3) or acetaldehyde oxidase (EC1.2.3.1) can replace CalB, etc.; this invention does not list these enzymes one by one. This invention does not limit itself to any specific enzyme.

[0010] Based on the above preparation route, the present invention provides a genetically engineered bacterium that co-expresses vanillyl oxidase (VaoA), coniferyl dehydrogenase (CalA) and coniferyl aldehyde dehydrogenase (CalB), or co-expresses O-demethylases, vanillyl oxidase (VaoA), coniferyl dehydrogenase (CalA) and coniferyl aldehyde dehydrogenase (CalB).

[0011] Based on the above reaction principle, the inventors introduced genes encoding vanillyl oxidase and oxidoreductase into host bacteria to obtain a genetically engineered bacterium. Using this genetically engineered bacterium as a biocatalyst, the substrate phenylpropene can be converted into cinnamic acid. The inventors also introduced genes encoding O-demethylase, vanillyl oxidase and oxidoreductase into host bacteria to obtain another genetically engineered bacterium. Using this genetically engineered bacterium as a biocatalyst, the substrate eugenol can be converted into 3,4-dihydroxycinnamic acid.

[0012] In some embodiments, the O-demethylase includes SpOdem and SmOdem; the amino acid sequence of SpOdem is shown in SEQ ID NO.1; the amino acid sequence of SmOdem is shown in SEQ ID NO.2.

[0013] In this invention, SpOdem is derived from *Sphingomonas paucimobilis*, GenBank accession number BAD61059.1; SmOdem is derived from *Stenotrophomonas maltophilia*, GenBank accession number AAV53699.1. After obtaining the amino acid sequences of the O-demethylases SpOdem and SmOdem, the inventors optimized the codons based on *E. coli* preferences and synthesized two optimized nucleotide sequences using a total synthesis method.

[0014] In some embodiments, the nucleotide sequence of SpOdem is shown in SEQ ID NO.3; the nucleotide sequence of SmOdem is shown in SEQ ID NO.4.

[0015] In some embodiments, vanillyl oxidase comprises FvVaoA and SsVaoA; the amino acid sequence of FvVaoA is shown in SEQ ID NO.5; and the amino acid sequence of SsVaoA is shown in SEQ ID NO.6.

[0016] In this invention, FvVaoA is derived from *Fusarium verticillioides*, GenBank accession number AFJ11909.1; SsVaoA is derived from *Sphaerobolus stellatus*, GenBank accession number KIJ23712.1. After obtaining the amino acid sequences of vanillyl oxidases FvVaoA and SsVaoA, the inventors optimized the codons based on *E. coli* preferences and synthesized the two optimized nucleotide sequences using a total synthesis method.

[0017] In some embodiments, the nucleotide sequence of FvVaoA is shown in SEQ ID NO.7; the nucleotide sequence of SsVaoA is shown in SEQ ID NO.8.

[0018] In some embodiments, the coniferyl alcohol dehydrogenase includes PpCalA and SsCalA; the amino acid sequence of PpCalA is shown in SEQ ID NO.9; and the amino acid sequence of SsCalA is shown in SEQ ID NO.10.

[0019] In this invention, PpCalA is derived from *Sphingomonas paucimobilis*, GenBank accession number AAN68038.1; SsCalA is derived from *Stenotrophomonas maltophilia*, GenBank accession number BAN09098.1. After obtaining the amino acid sequences of the coniferyl alcohol dehydrogenases PpCalA and SsCalA, the inventors optimized the codons based on *E. coli* preferences and synthesized the two optimized nucleotide sequences using a total synthesis method.

[0020] In some embodiments, the nucleotide sequence of PpCalA is shown in SEQ ID NO.11; the nucleotide sequence of SsCalA is shown in SEQ ID NO.12.

[0021] In some embodiments, coniferaldehyde dehydrogenase includes AbCalB and XtCalB; the amino acid sequence of AbCalB is shown in SEQ ID NO.13; and the amino acid sequence of XtCalB is shown in SEQ ID NO.14.

[0022] In this invention, AbCalB is derived from *Sphingomonas paucimobilis*, GenBank accession number BAD61059.1; XtCalB is derived from *Stenotrophomonas maltophilia*, GenBank accession number AAV53699.1. After obtaining the amino acid sequences of the coniferaldehyde dehydrogenases AbCalB and XtCalB, the inventors optimized the codons based on *E. coli* preferences and synthesized the two optimized nucleotide sequences using a total synthesis method.

[0023] In some embodiments, the nucleotide sequence of AbCalB is shown in SEQ ID NO.15; the nucleotide sequence of XtCalB is shown in SEQ ID NO.16.

[0024] In some embodiments, the host of the genetically engineered bacteria is Escherichia coli.

[0025] In some embodiments, Escherichia coli is selected from any one of Escherichia coli BL21(DE3), Escherichiacoli DH5α, and Escherichiacoli XL-Blue.

[0026] In some embodiments, the genetically engineered bacteria contain a dual promoter expression vector, which includes pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

[0027] The present invention also provides a method for constructing the above-mentioned genetically engineered bacteria. The method includes two methods: one is to link the genes of vanillyl oxidase and oxidoreductase to an expression vector, and then introduce the obtained recombinant expression vector into Escherichia coli to obtain genetically engineered bacteria; the other is to link the genes of O-demethylase, vanillyl oxidase and oxidoreductase to an expression vector, and then introduce the obtained recombinant expression vector into Escherichia coli to obtain genetically engineered bacteria.

[0028] In this invention, the construction of the genetically engineered bacteria that produce cinnamic acid or 3,4-dihydroxycinnamic acid involves selecting one enzyme from each of the above enzymes and co-expressing a combination of three or four enzymes. As for the method of introducing plasmids carrying the coding genes of the above enzymes, it can be that any two coding genes exist on the same plasmid, or that the three or four genes exist on different plasmids, or other introduction methods. This invention does not limit these methods.

[0029] Preferably, the pCDFDuet-1 plasmid and the pACYCDuet-1 plasmid are used to co-express the encoding genes of multiple enzymes.

[0030] In some embodiments, EcoRI and HindIII restriction sites are added to both ends of the nucleotide sequence of O-demethylase, NdeI and XhoI restriction sites are added to both ends of the nucleotide sequence of vanillyl oxidase, EcoRI and HindIII restriction sites are added to both ends of the nucleotide sequence of alcohol oxidoreductase, and NdeI and XhoI restriction sites are added to both ends of the nucleotide sequence of aldehyde oxidoreductase.

[0031] This invention also provides a whole-cell catalyst containing the aforementioned genetically engineered bacteria. The whole-cell catalyst can be used to convert a substrate into a target product.

[0032] This invention also provides the application of the above-mentioned genetically engineered bacteria in the production of cinnamic acid and its derivatives, comprising adding the above-mentioned genetically engineered bacteria to a solution containing a substrate for whole-cell transformation to obtain cinnamic acid and its derivatives. The substrate is at least one of styrene and its derivatives, such as styrene, 4-allylphenol, eugenol, 4-allyl-2-hydroxyphenol, etc. Finally, cinnamic acid and its derivatives are obtained, including cinnamic acid and 3,4-dihydroxycinnamic acid.

[0033] Reacting 3,4-dihydroxycinnamic acid with other compounds yields 3,4-dihydroxycinnamic acid derivatives. Alternatively, replacing eugenol (i.e., 2-methoxy-4-(2-propenyl)phenol) with its derivatives (e.g., at least one hydrogen atom at positions 3, 5, or 6 of the benzene ring of eugenol is replaced by an alkyl, hydroxyl, or alkoxy group; or the hydroxyl group at position 1 of the benzene ring of eugenol is replaced by an alkyl, carboxyl, or alkoxy group) yields 3,4-dihydroxycinnamic acid derivatives.

[0034] In some embodiments, the above-mentioned genetically engineered bacteria are added to a solution containing eugenol for whole-cell transformation to obtain the target product.

[0035] In some embodiments, the whole-cell transformation production system includes: substrate 1-15 g / L, glucose 5-30 g / L, and genetically engineered bacterial cell mass 1-20 g / L.

[0036] In some embodiments, the temperature of the whole-cell transformation production system is 15-40°C.

[0037] In some embodiments, the reaction time of the whole-cell transformation production system is 10-24 hours.

[0038] The present invention has the following beneficial effects:

[0039] This invention provides a novel method for producing cinnamic acid and its derivatives, utilizing a recombinant *Escherichia coli* strain. The enzyme selected in this invention possesses advantages such as high activity and strong optical specificity. Furthermore, the production of cinnamic acid and its derivatives using the recombinant strain of this invention yields high output, high conversion rate, high production efficiency, is environmentally friendly, operates under mild reaction conditions, requires no multi-step separation and purification, and is low-cost, demonstrating promising prospects for industrial application. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is the preparation route of cinnamic acid in this invention;

[0042] Figure 2 This is the preparation route for 3,4-dihydroxycinnamic acid in this invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] 1. Selection of bacteria and plasmids

[0046] The bacteria and plasmids used in the embodiments of this invention: pCDFDuet-1 plasmid, pACYCDuet-1 plasmid, Escherichia coli BL21(DE3), Escherichia coli DH5α, and Escherichia coli XL-Blue were all purchased from Novagen.

[0047] 2. Enzyme selection

[0048] (1) Selection of O-demethylase

[0049] The amino acid sequences of the O-demethylases SpOdem and SmOdem were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0050] (2) Selection of vanillyl alcohol oxidase

[0051] The amino acid sequences of vanillyl oxidases FvVaoA and SsVaoA were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. Enzyme cleavage sites NdeI and XhoI were added to both ends of the nucleotide sequences.

[0052] (3) Selection of coniferol dehydrogenase

[0053] The amino acid sequences of coniferyl alcohol dehydrogenases PpCalA and SsCalA were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. EcoRI and HindIII restriction sites were added to both ends of the nucleotide sequences.

[0054] (4) Selection of coniferaldehyde dehydrogenase

[0055] The amino acid sequences of coniferaldehyde dehydrogenases AbCalB and XtCalB were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and a nucleotide sequence was synthesized using standard genetic engineering methods, as shown in SEQ ID NO.15 and SEQ ID NO.16. The amino acid sequence encoding the enzyme is shown in SEQ ID NO.13 and SEQ ID NO.14. Enzyme cleavage sites NdeI and XhoI were added to both ends of the nucleotide sequence.

[0056] 3. Construction of a four-enzyme co-expression system and cell culture

[0057] One enzyme from each of the selected O-demethylase, vanillyl oxidase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase was chosen for co-expression. The encoding genes of all four enzymes were co-expressed using the pCDFDuet-1 and pACYCDuet-1 double plasmids; pCDFDuet-1 contained O-demethylase and vanillyl oxidase, while pACYCDuet-1 contained coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase. After obtaining the co-expression recombinant plasmids, both plasmids were simultaneously transformed into *E. coli* BL21(DE3) competent cells. Positive transformants were obtained by screening with streptomycin and chloramphenicol plates, thus obtaining recombinant *E. coli*. The obtained recombinant bacteria were inoculated into fresh liquid culture medium, induced, centrifuged, and wet cells were obtained.

[0058] 4. Preparation of 3,4-dihydroxycinnamic acid from eugenol via whole-cell conversion

[0059] Transformation system: Eugenol concentration 1-15 g / L, glucose concentration 5-30 g / L, pH adjusted between 6.0 and 9.0, fresh cell mass 1-20 g / L, then transformed at 15-40℃ and 200 rpm for 10-24 h. After transformation, the yield of 3,4-dihydroxycinnamic acid was determined by liquid chromatography.

[0060] 5. Sample detection and analysis

[0061] The conversion solution was analyzed using a Shimadzu 2030C high-performance liquid chromatograph (HPLC). The chromatographic conditions were as follows: mobile phase: methanol:water (V / V = 1:1), Inertsustain C18 column (4.6 × 250 mm, 5 μm), flow rate: 1 mL / min, column temperature: 30 °C, injection volume: 20 μL, and detection wavelength: 323 nm.

[0062] Example 1

[0063] This embodiment provides a method for constructing genetically engineered bacteria, the specific steps of which are as follows:

[0064] (1) The fully synthesized Odem recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. The fully synthesized VaoA recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. The Odem and VaoA from different sources were ligated into the pCDFDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 1.

[0065] (2) The fully synthesized KDC recombinant plasmid and pACYCDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. The fully synthesized BLDH recombinant plasmid and pACYCDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. CalA and CalB from different sources were ligated into the pACYCDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 2.

[0066] (3) Different recombinant plasmids 1 and 2 were combined in pairs and transformed into E.coli BL21(DE3) competent cells to obtain genetically engineered bacteria.

[0067] Example 2

[0068] This embodiment describes the induction culture of the genetically engineered bacteria constructed in Example 1. The specific steps are as follows:

[0069] (1) The genetically engineered bacteria were inoculated into LB medium containing 50 mg / L streptomycin and 50 mg / L chloramphenicol and cultured at 37°C and 200 rpm for 12 h to obtain seed culture.

[0070] (2) Inoculate the seed culture into fresh LB medium at an inoculation rate of 2%, and culture at 37°C and 200 rpm until the bacterial concentration OD600nm reaches 0.7. Add 0.5 mM IPTG, induce at 28°C for 15 h, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the wet bacterial cells twice with 0.9% physiological saline, centrifuge, and set aside.

[0071] Example 3

[0072] This example compares the transformation capabilities of various genetically engineered bacteria.

[0073] The induced cultured genetically engineered bacteria were resuspended in a 50 mL system with a final cell concentration of 20 g / L, eugenol 15 g / L, glucose 30 g / L, and pH 8.0. The reaction was carried out at 30 °C with a shaking speed of 200 rpm for 24 h. After transformation, the yield of 3,4-dihydroxycinnamic acid was determined by HPLC.

[0074] Table 1 Comparison of 3,4-dihydroxycinnamic acid yields of various genetically engineered bacteria

[0075]

[0076]

[0077] Example 4

[0078] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 1 g / L, eugenol 1 g / L, glucose 5 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 1.07 g / L.

[0079] Example 5

[0080] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, eugenol 3 g / L, glucose 10 g / L, pH 8.0, temperature 35℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 3.21 g / L.

[0081] Example 6

[0082] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, eugenol 5 g / L, glucose 10 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 5.42 g / L.

[0083] Example 7

[0084] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 9 g / L, eugenol 7 g / L, glucose 15 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 7.51 g / L.

[0085] Example 8

[0086] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 10 g / L, eugenol of 8 g / L, glucose of 15 g / L, pH 8.0, temperature of 30 °C, shaking speed of 200 rpm, and transformation time of 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 8.53 g / L.

[0087] Example 9

[0088] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 13 g / L, eugenol 10 g / L, glucose 20 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time of 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 10.9 g / L.

[0089] Example 10

[0090] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 15 g / L, eugenol of 12 g / L, glucose of 25 g / L, pH 8.0, temperature of 30 °C, shaking speed of 200 rpm, and transformation time of 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 13.0 g / L.

[0091] Example 11

[0092] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 17 g / L, eugenol 8 g / L, glucose 20 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time of 12 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 8.61 g / L.

[0093] Example 12

[0094] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 20 g / L, eugenol 9 g / L, glucose 20 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 9.72 g / L.

[0095] Example 13

[0096] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 18 g / L, eugenol 12 g / L, glucose 30 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time of 18 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 12.9 g / L.

[0097] Example 14

[0098] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 15 g / L, eugenol 8 g / L, glucose 20 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time of 15 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 8.71 g / L.

[0099] Example 15

[0100] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, eugenol 2 g / L, glucose 10 g / L, pH 6.0, temperature 35℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 2.16 g / L.

[0101] Example 16

[0102] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 8 g / L, eugenol 3 g / L, glucose 10 g / L, pH 7.0, temperature 35℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 3.25 g / L.

[0103] Example 17

[0104] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 16 g / L, eugenol 7 g / L, glucose 20 g / L, pH 7.5, temperature 35℃, shaking speed 200 rpm, and transformation time of 12 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 12.6 g / L.

[0105] Example 18

[0106] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 14 g / L, eugenol 6 g / L, glucose 20 g / L, pH 8.5, temperature 35℃, shaker speed 200 rpm, and transformation time of 12 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 6.48 g / L.

[0107] Example 19

[0108] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 11 g / L, eugenol 4 g / L, glucose 20 g / L, pH 9.0, temperature 35℃, shaking speed 200 rpm, and transformation time of 12 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 4.34 g / L.

[0109] Example 20

[0110] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 7 g / L, eugenol 6 g / L, glucose 20 g / L, pH 7.5, temperature 15℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 6.45 g / L.

[0111] Example 21

[0112] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 12 g / L, eugenol 10 g / L, glucose 25 g / L, pH 7.5, temperature 25℃, shaking speed 200 rpm, and transformation time of 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 10.8 g / L.

[0113] Example 22

[0114] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 17 g / L, eugenol 13 g / L, glucose 30 g / L, pH 7.5, temperature 40℃, shaking speed 200 rpm, and transformation time of 24 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 14.1 g / L.

[0115] Example 23

[0116] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 30 g / L, eugenol 20 g / L, glucose 50 g / L, pH 7.5, temperature 35℃, shaking speed 200 rpm, and transformation time of 36 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 5.01 g / L.

[0117] Example 24

[0118] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 20 g / L, eugenol 15 g / L, glucose 30 g / L, pH 5.5, temperature 35℃, shaking speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 4.12 g / L.

[0119] Example 25

[0120] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 19 g / L, eugenol 13 g / L, glucose 30 g / L, pH 9.5, temperature 35℃, shaker speed 200 rpm, and transformation time of 36 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 3.26 g / L.

[0121] Example 26

[0122] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 18 g / L, eugenol 12 g / L, glucose 30 g / L, pH 7.5, temperature 10℃, shaking speed 200 rpm, and transformation time of 36 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 2.18 g / L.

[0123] Example 27

[0124] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 16 g / L, eugenol 10 g / L, glucose 30 g / L, pH 8.0, temperature 45℃, shaking speed 200 rpm, and transformation time of 36 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 2.23 g / L.

[0125] Example 28

[0126] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-SpOdem-SsVaoA+pACYCDuet-SsCalA-XtCalB, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 0.5 g / L, eugenol 0.5 g / L, glucose 3 g / L, pH 8.0, temperature 30 °C, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of 3,4-dihydroxycinnamic acid was 0.11 g / L.

[0127] Example 29

[0128] The method for preparing cinnamic acid in this embodiment is similar to the method for preparing 3,4-dihydroxycinnamic acid.

[0129] (1) First, construct a genetically engineered bacterium, wherein the recombinant plasmid 1 lacks the gene for O-demethoxyenzyme Odem, and other implementation methods are consistent with Example 1.

[0130] (2) The induction culture method of the genetically engineered bacteria is the same as that in Example 2.

[0131] (3) The induced cultured genetically engineered bacteria were resuspended in a 50 mL system with a final cell concentration of 20 g / L, 15 g / L phenylpropene, 30 g / L glucose, and pH 8.0. The reaction was carried out at 30 °C with a shaking speed of 200 rpm for 24 h. The yield of cinnamic acid was determined by HPLC after the transformation was completed.

[0132] Table 2 Comparison of cinnamic acid production by various genetically engineered bacteria

[0133]

[0134] As shown in Table 2, the genetically engineered strain E. coli BL21(DE3) / pCDFDuet-SsVaoA+pACYCDuet-PpCalA–AbCalB showed the highest activity in the production of cinnamic acid. After the transformation, the yield of cinnamic acid was determined by HPLC to be 18.1 g / L.

[0135] Comparative Example 1

[0136] In this comparative example, the recombinant plasmid 1 lacked the vanillyl alcohol oxidase gene, and the other steps were the same as in Example 4. After transformation, 3,4-dihydroxycinnamic acid was determined by HPLC, and no 3,4-dihydroxycinnamic acid was detected.

[0137] Comparative Example 2

[0138] In this comparative example, the recombinant plasmid 2 lacked the coniferyl alcohol dehydrogenase gene. The other steps were the same as in Example 4. After transformation, 3,4-dihydroxycinnamic acid was determined by HPLC and no 3,4-dihydroxycinnamic acid was detected.

[0139] Comparative Example 3

[0140] In this comparative example, the recombinant plasmid 2 lacked the coniferaldehyde dehydrogenase gene. The other steps were the same as in Example 4. After transformation, 3,4-dihydroxycinnamic acid was determined by HPLC, and no 3,4-dihydroxycinnamic acid was detected.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria include genes encoding vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase; or The genetically engineered bacteria include genes encoding O-demethylase, vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. The O-demethylase has the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2; The vanillyl alcohol oxidase has the amino acid sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; The coniferol dehydrogenase has the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10; The coniferaldehyde dehydrogenase has the amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14; The host of the genetically engineered bacteria is Escherichia coli.

2. The genetically engineered bacterium according to claim 1, characterized in that, The O-demethylase has the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The genetically engineered bacterium according to claim 1, characterized in that, The vanillyl oxidase has the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.

8.

4. The genetically engineered bacterium according to claim 1, characterized in that, The coniferol dehydrogenase has the nucleotide sequence shown in SEQ ID NO. 11 or SEQ ID NO.

12.

5. The genetically engineered bacterium according to claim 1, characterized in that, The coniferaldehyde dehydrogenase has the nucleotide sequence shown in SEQ ID NO. 15 or SEQ ID NO.

16.

6. The genetically engineered bacterium according to claim 1, characterized in that, The *Escherichia coli* strain is selected from any one of *Escherichiacoli* BL21(DE3), *Escherichia coli* DH5α, and *Escherichia coli* XL-Blue.

7. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria contain expression vectors, including pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

8. The method for constructing genetically engineered bacteria according to any one of claims 1-7, characterized in that, The construction method is selected from either (1) or (2) below: (1) The genes of vanillyl oxidase, coniferyl ol dehydrogenase and coniferyl aldehyde dehydrogenase are ligated into an expression vector, and the obtained recombinant expression vector is introduced into the Escherichia coli to obtain the genetically engineered bacteria; (2) The genes of O-demethylase, vanillyl oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase are ligated into an expression vector, and the obtained recombinant expression vector is introduced into the Escherichia coli to obtain the genetically engineered bacteria.

9. A whole-cell catalyst, characterized in that, It contains the genetically engineered bacteria as described in any one of claims 1-7.

10. The application of the genetically engineered bacteria according to any one of claims 1-7 in the production of cinnamic acid and its derivatives, characterized in that, The derivative is 3,4-dihydroxycinnamic acid; A method for producing cinnamic acid using the genetically engineered bacteria according to any one of claims 1-7 comprises: adding the genetically engineered bacteria to a solution containing styrene for whole-cell transformation to obtain the target product; wherein the genetically engineered bacteria contains genes encoding vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase; A method for producing 3,4-dihydroxycinnamic acid using the genetically engineered bacteria according to any one of claims 1-7 comprises: adding the genetically engineered bacteria to a solution containing eugenol for whole-cell transformation to obtain the target product; wherein the genetically engineered bacteria contains genes encoding O-demethylase, vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase.

11. The application according to claim 10, characterized in that, The whole-cell transformation production system for producing cinnamic acid includes: 1-15 g / L styrene, 5-30 g / L glucose, and 1-20 g / L genetically engineered bacterial cells; The whole-cell transformation production system for producing 3,4-dihydroxycinnamic acid includes: eugenol 1-15 g / L, glucose 5-30 g / L, and genetically engineered bacterial cells 1-20 g / L.

12. The application according to claim 11, characterized in that, The temperature of the whole-cell transformation production system is 15-40℃.

13. The application according to claim 11, characterized in that, The reaction time of the whole-cell transformation production system is 10-24 h.

Images