Strain and method for producing rosmarinic acid

Abstract

The disclosure discloses a strain and method for producing rosmarinic acid, and belongs to the technical field of bioengineering. The disclosure constructs a recombinant cell or a combination of recombinant cells expressing 4-coumarate: CoA ligase, rosmarinic acid synthase, polyphosphate kinase 2-I (PPK2-I) and polyphosphate kinase 2-II (PPK2-II), and utilizes the recombinant cell or the combination of recombinant cells to catalyze Danshensu and caffeic acid for synthesizing rosmarinic acid. The disclosure has good industrial application prospects.

Claims

1. A recombinant microbial cell that synthesizes rosmarinic acid from phenolic acids as substrates, comprising: a recombinant Escherichia coli cell comprising genes that encode and are capable of expressing 4-coumarate: coenzyme A (CoA) ligase, rosmarinic acid synthase, polyphosphate kinase 2-I (PPK2-I), and polyphosphate kinase 2-II (PPK2-II); wherein the gene encoding the 4-coumarate: CoA ligase is selected from the group consisting of: sb4cl, ot4cl, ob4cl, pc4cl, sc4cl, and rj4cl; wherein the gene encoding the rosmarinic acid synthase is selected from the group consisting of: psras, laras, moras, smras, ccras, ntras, and dcras; wherein the gene encoding the PPK2-I is smpkk; and wherein the gene encoding the PPK2-II is ajpkk.

2. The recombinant microbial cell according to claim 1, wherein the E. coli cell is E. coli BL21 (DE3).

3. The recombinant microbial cell according to claim 2, wherein the genes encoding the 4-coumarate: CoA ligase, the rosmarinic acid synthase, the PPK2-I, and the PPK2-II are co-expressed in the recombinant E. coli cell from one or more vectors or are integrated into a genome of the recombinant E. coli cell.

4. The recombinant microbial cell according to claim 3, wherein the genes encoding the 4-coumarate: CoA ligase, the rosmarinic acid synthase, the PPK2-I, and the PPK2-II are encoded by one or more of four plasmids selected from pETDuet-1, pACYCDuet-1, pRSFDuet-1, and pCDFduet-1, and wherein each of the one or more of four plasmids encodes one or more of the genes encoding the 4-coumarate: CoA ligase, the rosmarinic acid synthase, the PPK2-I, and the PPK2-II.

5. The recombinant microbial cell according to claim 1, wherein the recombinant E. coli cell comprises pRSFDuet-1 and pTDuet-1 vectors comprising the genes that encode the PPK2-I, the PPK2-II, and the 4-coumarate: CoA ligase; wherein the pRSFDuet-1 vector expresses genes encoding the PPK2-I and the PPK2-II, and the pTDuet-1 vector expresses the gene encoding the 4-coumarate: CoA ligase and the gene encoding the rosmarinic acid synthase; and wherein the gene encoding the 4-coumarate: CoA ligase, the gene encoding the rosmarinic acid synthase, the gene encoding the PPK2-I, and the gene encoding the PPK2-II in the recombinant E. coli cell each comprise T7 promoters, RBS binding sites, and T7 terminators.

6. A method of producing rosmarinic acid from phenolic acid substrates in a recombinant microbial cell, which comprises: providing the recombinant microbial cell according to claim 1, culturing the recombinant microbial cell; and adding Danshensu and caffeic acid as phenolic substrates to the recombinant microbial cell to produce rosmarinic acid.

7. The method according to claim 6, wherein the culturing step further comprises: culturing and propagating the recombinant microbial cell, inducing the recombinant microbial cell to express the 4-coumarate: CoA ligase, the rosmarinic acid synthase, the PPK2-I, and the PPK2-II, and collecting the recombinant cells.

8. The method according to claim 6, wherein the recombinant cells are incubated at 15 C. to 40 C. for 1 hour to 48 hours in a reaction system comprising: 1 g/L to 100 g/L of the Danshensu (D or L), 1 g/L to 100 g/L of the caffeic acid, 0 g/L to 1 g/L of adenosine triphosphate (ATP), 0 g/L to 1 g/L of coenzyme A (CoA), and 300 g/L of sodium hexametaphosphate, at a pH value of 5.0 to 9.0.

9. The method according to claim 6, wherein the recombinant cells are incubated at 15 C. to 30 C. for 5 hours to 48 hours in a reaction system comprising D-Danshensu, caffeic acid, coenzyme A (CoA), adenosine triphosphate (ATP), and sodium hexametaphosphate.

10. The method according to claim 6, wherein the recombinant cells are incubated at 40 C. for 48 hours in a reaction system comprising D-Danshensu, caffeic acid, coenzyme A (CoA), adenosine triphosphate (ATP), and sodium hexametaphosphate.

11. Two or more recombinant cells that synthesize rosmarinic acid from phenolic acids as substrates, comprising: two or more recombinant cells that are Escherichia coli cells that comprise genes that encode and are capable of expressing one or more of 4-coumarate: CoA ligase, rosmarinic acid synthase, polyphosphate kinase 2-II (PPK2-II), and polyphosphate kinase 2-I (PPK2-I); wherein each recombinant E. coli cell does not repeatedly express one of the 4-coumarate: CoA ligase, the rosmarinic acid synthase, the PPK2-II, and the PPK2-I expressed by the other recombinant E. coli cells; wherein the gene encoding the 4-coumarate: CoA ligase is selected from the group consisting of: sb4cl, ot4cl, ob4cl, pc4cl, sc4cl, and rj4cl; wherein the gene encoding the rosmarinic acid synthase is selected from the group consisting of: psras, laras, moras, smras, ccras, ntras, and dcras; wherein the gene encoding the PPK2-I is smpkk; and wherein the gene encoding the PPK2-II is ajpkk.

12. The two or more recombinant cells according to claim 11, wherein the E. coli is E. coli BL21 (DE3).

13. The two or more recombinant cells according to claim 12, wherein the genes encoding the 4-coumarate: CoA ligase, the rosmarinic acid synthase, the PPK2-I, and the PPK2-II are encoded by one or more of four plasmids selected from pETDuet-1, pACYCDuet-1, pRSFDuet-1, and pCDFduet-1, and wherein each of the one or more of four plasmids encodes one or more of; the gene encoding the 4-coumarate: CoA ligase, the gene encoding the rosmarinic acid synthase, the gene encoding the PPK2-I, and the gene encoding the PPK2-II.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is the liquid chromatogram of rosmarinic acid synthesized in Example 4.

(2) FIG. 2 is the liquid chromatogram of rosmarinic acid synthesized in Example 6.

DETAILED DESCRIPTION

(3) 1. Strains and Plasmids Involved in the Following Examples

(4) pRSFDuet-1, pETDuet-1, pCDFDuet-1 and pACYCDuet-1 plasmids, and Escherichia coli BL21 (DE3) were all purchased from Novagen.

(5) 2. Construction of Multi-Gene Co-Expression System and Cell Culture

(6) At present, there are many methods for co-expression of multiple genes in Escherichia coli (for example, the method described in the article Multi-gene co-expression strategy in Escherichia coli, China Biotechnology, 2012, 32(4): 117-122). The disclosure uses the method described in Liu Xianglei's doctoral dissertation (Production of Shikimic Acid and Resveratrol by Transformation of Escherichia coli by Synthetic Biology Technology, 2016, Shanghai Institute of Pharmaceutical Industry) to construct recombinant Escherichia coli. In the following examples, when multiple genes are co-expressed, each gene includes a T7 promoter and an RBS binding site before the gene, and a T7 terminator behind the gene. Theoretically, because each gene has T7 and RBS in front, the expression intensity of the gene is not affected by the order of the gene on a plasmid. The constructed plasmid is thermally transduced into Escherichia coli competent cells, and spread on a monoclonal antibody or mixed antibiotic solid plate, and positive transformants are screened to obtain recombinant Escherichia coli.

(7) Culture of cells: According to a classical recombinant Escherichia coli culture and induction expression scheme, recombinant Escherichia coli is transferred to an LB fermentation medium (10 g/L peptone, 5 g/L yeast powder, and 10 g/L NaCl) at a volume percentage of 2%. After the cell OD.sub.600 reaches 0.6-0.8, IPTG with a final concentration of 0.4 mM is added, and expression culture is induced at 20 C. for 8 h. After expression induction is completed, the cells are collected by centrifugation at 4 C. and 8000 rpm for 20 minutes.

(8) 3. Selection of Related Enzymes

(9) (1) Polyphosphate Kinase 2-1 (PPK2-1)

(10) The gene smpkk encoding PPK2-I derived from Sinorhizobium_meliloti was selected. The accession NO. of the gene smpkk on NCBI is NC_003047 REGION: complement (564142 . . . 565044), and the corresponding amino acid sequence is NP_384613.1.

(11) (2) Polyphosphate Kinase 2-II (PPK2-II)

(12) The gene ajpkk encoding PPK2-II derived from Acinetobacter johnsonii was selected. The accession NO. of the sequence of the gene ajpkk on NCBI is AB092983 REGION: 339 . . . 1766, and the corresponding amino acid sequence is BAC76403.1.

(13) (3) 4-Coumarate: CoA Ligase

(14) Refer to example 1.

(15) (4) Rosmarinic Acid Synthase

(16) Refer to example 2.

(17) 4. Detection and Analysis of Samples

(18) Reference for the measuring method of rosmarinic acid content: Enhanced accumulation of caffeic acid, rosmarinic acid and luteolin-glucoside in red Perilla cultivated under red diode laser and blue LED illumination followed by UV-A irradiation. Journal of functional foods 2 (2010) 66-70. The solubility of Danshensu, caffeic acid, and rosmarinic acid is relatively low. In the transformation process of the disclosure, an excessive amount of substrate is added, the substrate will dissolve while reacting, and the product will precipitate while reacting under high concentration conditions. When measuring, pure water is added to make the product completely dissolved before measuring.

(19) Reference for measuring the activity of rosmarinic acid synthase: Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases, Planta, 2006, 224:1503-1510.

(20) Reference for measuring the activity of 4-coumarate: CoA ligase: 4-Coumarate: CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm. Arch. Biochem. Biophys. 1977, 184, 237-248.

(21) Specific enzyme activity (U mg.sup.1) is defined as the unit of enzyme activity per mg enzyme. An enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 mol of product in 1 min.

Example 1: Screening and Expression of 4-Coumarate: CoA Ligase

(22) 4-coumarate: CoA ligase is widely present in various organisms. According to the gene information of the 4-coumarate: CoA ligase in Scutellaria baicalensis, Ocimum tenuiflorum, Ocimum basilicum, Arabidopsis thaliana, Penicillium chrysogenum, Streptomyces coelicolor A3 (2), and Rhodococcus jostiid on NCBI, 4-coumarate: CoA genes sb4cl, ot4cl, ob4cl, at4cl, pc4cl, sc4cl, and rj4cl were obtained by complete synthesis. The accession NOs. of the amino acid sequences corresponding to the genes on NCBI are: BAD90936.1, ADO16242.1, AGP02119.1, AAD47193.1, CAA04820.1, CAB95894.1, and ABG96911.1. The synthesized genes were ligated to the pETDuet-1 vector and induced for expression in Escherichia coli BL21 (DE3). The expression induction method is: recombinant Escherichia coli was transferred to an LB fermentation medium (containing 10 g/L peptone, 5 g/L yeast powder and 10 g/L NaCl) at a volume percentage of 2% for performing fermentation culture; and after the cell OD.sub.600 reached 0.6-0.8, IPTG with a final concentration of 0.4 mM was added, and expression culture was induced at 20 C. for 8 h. After expression induction is completed, the fermentation broth was centrifuged at 4 C. and 8000 rpm for 20 minutes, and cells were collected. The collected cells were crushed, and a Histag tag was used to purify the cell crushing liquid to obtain the pure enzyme. After the pure enzyme was obtained, the activity of the pure enzyme was measured.

(23) When caffeic acid and coenzyme A were used as substrates, the specific enzyme activity of the enzymes expressed by the 4-coumarate: CoA ligase genes sb4cl, ot4cl, ob4cl, at4cl, pc4cl, sc4cl, and rj4cl was respectively: 152, 143, 161, 179, 202, 174, and 88 U/mg.

Example 2: Screening and Expression of Rosmarinic Acid Synthase

(24) Rosmarinic acid synthase mainly exists in plants. According to the gene information of the rosmarinic acid synthase in Plectranthus scutellarioides, Lavandula angustifolia, Melissa officinalis, Salvia miltiorrhiza, Coffea canephora, Nicotiana tabacum, and Dianthus caryophyllus on NCBI, rosmarinic acid synthase genes psras, laras, moras, smras, ccras, ntras, and dcras were obtained by complete synthesis. The accession NOs. of the amino acid sequences corresponding to the genes on NCBI are: CAK55166.1, ABI48360.1, CBW35684.1, ADA60182.1, ABO47805.1, CAE46932.1, and CAB06430.1. The synthesized genes were respectively ligated to pETDuet-1 vector, and expressed and purified in the same way as in Example 1.

(25) When caffeyl-CoA and D-Danshensu were used as substrates, the specific enzyme activity of the enzymes expressed by rosmarinic acid synthase genes psras, laras, moras, smras, ccras, ntras, and dcras was respectively: 410, 320, 414, 233, 361, 521, and 371 U/mg.

(26) When caffeyl-CoA and L-Danshensu were used as substrates, the specific enzyme activity of the enzymes expressed by rosmarinic acid synthase genes psras, laras, moras, smras, ccras, ntras, and dcras was respectively: 0, 0, 0, 0, 120, 0, and 142 U/mg. It can be seen that only the rosmarinic acid synthase encoded by ccras and dcras has the ability to synthesize rosmarinic acid with L-Danshensu as a substrate.

Example 3: Construction of Recombinant Escherichia coli Expressing Four Enzymes Simultaneously

(27) Construction of Recombinant Escherichia coli:

(28) As shown in Table 1, selection was made from four plasmids pETDuet-1, pACYCDuet-1, pRSFDuet-1, and pCDFduet-1, and the genes encoding the four enzymes were ligated to the same plasmid, or ligated to two plasmids separately (2 genes expressed on each plasmid), or ligated to the four plasmids (1 gene expressed on each plasmid). Each gene included a T7 promoter and an RBS binding site before the gene, and a T7 terminator behind the gene. The constructed recombinant plasmids were transformed into Escherichia coli BL21, and positive transformants were obtained by screening with a mixed antibiotic plate to obtain recombinant Escherichia coli capable of strengthening expression of 4 genes.

(29) The recombinant Escherichia coli was induced for expression, and bacterial cells were collected after the expression induction was completed. A 100 mL reaction system, containing 200 g/L (wet weight) cells, 20 g/L D-Danshensu, 20 g/L caffeic acid, 1 g/L CoA, 1 g/L ATP, and 60 g/L sodium hexametaphosphate, and having a pH of 8, was constructed. The 100 ml reaction system was placed at 30 C. for reaction for 24 hours. The solubility of the Danshensu and caffeic acid is very small, so in the reaction process, the Danshensu and caffeic acid were dissolved while being consumed. After the reaction, the reaction solution was diluted and the concentration of rosmarinic acid in the reaction solution was measured by liquid chromatography. The results are shown in Table 1.

(30) TABLE-US-00001 TABLE 1 Rosmarinic Recombinant strains acid g/L Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-pc4cl -ntras 34 Escherichia coli BL21(DE3)/pCDFDuet-1-smpkk-ajpkk+pACYCDuet-1-pc4cl- 32 ntras Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk+pETDuet-1-ajpkk+pCDFDuet- 22 1-pc4cl+pACYCDuet-1-ntras Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk-pc4cl-ntras 26 Escherichia coli BL21(DE3)/pETDuet-1-smpkk-ajpkk-pc4cl-ntras 28 Escherichia coli BL21(DE3)/pACYCDuet-1-smpkk-ajpkk-pc4cl-ntras 14 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk-pc4cl+pETDuet-1-ntras 19 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-pc4cl+pETDuet-1-ntras-ajpkk 33 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ntras+pETDuet-1-pc4cl-ajpkk 31 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-sb4cl-psras 9 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-ot4cl-laras 30 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-ob4cl -moras 31 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-at4cl-smras 33 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-pc4cl-ccras 29 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-rj4cl-dcras 28 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pETDuet-1-sc4cl-ccras 16

Example 4: Synthesis of Rosmarinic Acid In Vitro Using Four Enzymes

(31) The 4 genes smpkk, ajpkk, pc4cl, and ntras were respectively ligated to pEDTDuet-1 vectors to obtain 4 recombinant vectors. The 4 recombinant vectors were transformed into Escherichia coli BL21 respectively to obtain recombinant Escherichia coli expressing 4 enzymes. 4 pure enzymes were obtained after expression and purification using the same method as in Example 1. Then 2 mg of each of these four pure enzymes was added to the 100 mL reaction system containing 20 g/L D-Danshensu, 20 g/L caffeic acid, 1 g/L CoA, 1 g/L ATP, and 60 g/L sodium hexametaphosphate, and having a pH of 8; and reaction was performed at 30 C. for 5 hours. Finally, the liquid chromatographic measurement result indicated that the concentration of rosmarinic acid in the reaction solution was 36 g/L, and the liquid chromatogram was as shown in FIG. 1 of the specification.

Example 5: Synthesis of Rosmarinic Acid Using the Combination of Recombinant Cells Expressing smpkk, ajpkk, pc4cl, and ntras Genes Respectively

(32) The 4 genes smpkk, ajpkk, pc4cl, and ntras were respectively ligated to pEDTDuet-1 vectors to obtain 4 recombinant vectors. The 4 recombinant vectors were transformed into Escherichia coli BL21 respectively to obtain recombinant Escherichia coli respectively expressing one of 4 enzymes. Recombinant Escherichia coli expression enzymes were induced using the same method as in Example 1. Then four kinds of 20 g/L recombinant cells were added in a 100 mL reaction system containing 20 g/L D-Danshensu, 20 g/L caffeic acid, 1 g/L CoA, 1 g/L ATP, and 60 g/L sodium hexametaphosphate, and having a pH of 8, and reaction was performed at 30 C. for 5 hours. Finally, the liquid chromatographic measurement result indicated that the concentration of rosmarinic acid in the reaction solution was 23 g/L.

Example 6: Synthesis of L-Rosmarinic Acid by Recombinant Escherichia coli Whole-Cell Catalysis

(33) The Danshensu group of rosmarinic acid in nature is D-type. In the present example, L-Danshensu and caffeic acid were used as raw materials to synthesize L-rosmarinic acid (the difference between L-rosmarinic acid and D-rosmarinic acid is that the Danshensu group of L-rosmarinic acid is L-type). Previously, L-rosmarinic acid has not been synthesized by biological methods.

(34) The genes ccras and dcras encoding the rosmarinic acid synthase derived from Coffea canephora and Dianthus caryophyllus were selected, together with the genes encoding PPK2-I, PPK2-II, and 4-coumarate: CoA ligase, to construct a recombinant strain Escherichia coli BL21 (DE3)/pRSFDuet-1-smpkk-ajpkk+pTDuet-1-pc4cl-dcras. According to the method in Example 1, the recombinant strain was induced for expression, and then bacterial cells were collected.

(35) In a 100 mL reaction system containing 100 g/L (wet weight) cells, 20 g/L L-Danshensu, 20 g/L caffeic acid, 1 g/L CoA, 1 g/L ATP, and 60 g/L sodium hexametaphosphate, and having a pH of 8, reaction was performed at 30 C. for 24 hours. After the transformation, the concentration of L-rosmarinic acid in the reaction solution was measured as 33 g/L by liquid chromatography, and the liquid chromatogram was as shown in FIG. 2 of the specification. The DAC-HB50 preparative chromatographic column of Jiangsu Hanbon Science & Technology Co., Ltd. was used to prepare purified samples. The preparative chromatographic conditions were: the mobile phase was 50% methanol, the column temperature was natural, the flow rate was 3 mL/min, and the injection volume was 5 mL. The chromatographic purity of the sample prepared for the first time reached 99.9%, and the product obtained after repeated injection and separation was spin-evaporated to dryness under vacuum at 50 C. 0.5 g of the sample evaporated to dryness was weighed and dissolved in deionized water and diluted to 50 mL, and the optical rotation was measured with the Japanese Atago AP-300 automatic polarimeter. The optical rotation is [].sub.D.sup.20=15.5. Therefore, we can confirm that the rosmarinic acid prepared in the present example is L-rosmarinic acid.

Example 7: Synthesis of Rosmarinic Acid by Recombinant Escherichia coli Whole-Cell Catalysis

(36) The following 2 kinds of recombinant strains were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-smpkk-ajpkk (named E1), and Escherichia coli BL21 (DE3)/pETDuet-1-sc4cl-ccras (named E2).

(37) According to the method in Example 1, E1 and E2 were induced for expression respectively, and then bacterial cells were collected. In a 100 mL reaction system containing 30 g/L (wet weight) E1 cells, 50 g/L (wet weight) E2 cells, 100 g/L D-Danshensu, 10 g/L caffeic acid, 300 g/L sodium hexametaphosphate, 1 g/L CoA, and 1 g/L ATP, and having a pH of 9, reaction was performed at 40 C. for 48 hours. After the transformation, the content of rosmarinic acid was measured as 162 g/L by liquid chromatography.

Example 8: Synthesis of Rosmarinic Acid by Recombinant Escherichia coli Whole-Cell Catalysis

(38) The following 2 kinds of recombinant strains were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-smpkk-ajpkk-at4cl (named E3), Escherichia coli BL21 (DE3)/pACYCDuet-1-ccras (named E4).

(39) According to the method in Example 1, E3 and E4 were induced for expression respectively, and then bacterial cells were collected. In a 100 mL reaction system containing 100 g/L (wet weight) E3 cells, 100 g/L (wet weight) E4 cells, 1 g/L D-Danshensu, 1 g/L caffeic acid, 0.5 g/L CoA, 1 g/L ATP, and 3 g/L sodium hexametaphosphate, and having a pH of 5, reaction was performed at 15 C. for 48 hours. After the transformation, the content of rosmarinic acid was measured as 1.3 g/L by liquid chromatography.

Example 9: Synthesis of Rosmarinic Acid by Recombinant Escherichia coli Whole-Cell Catalysis

(40) The following 2 kinds of recombinant strains were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-ntras-at4cl (named E5), and Escherichia coli BL21 (DE3)/pACYCDuet-1-smpkk-ajpkk (named E6).

(41) According to the method in Example 1, E5 and E6 were induced for expression respectively, and then bacterial cells were collected. In a 100 mL reaction system containing 100 g/L (wet weight) E5 cells, 100 g/L (wet weight) E6 cells, 1 g/L D-Danshensu, 1 g/L caffeic acid, 3 g/L sodium hexametaphosphate 1 g/L CoA, and 0.5 g/L ATP, and having a pH of 7, reaction was performed at 15 C. for 1 hour. After the transformation, the content of rosmarinic acid was measured as 1.5 g/L by liquid chromatography.

Example 10: Synthesis of Rosmarinic Acid by Recombinant Escherichia coli Whole-Cell Catalysis

(42) The following recombinant strain was constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-ntras-at4cl+pCDFDuet-1-smpkk-ajpkk. According to the method in Example 1, the recombinant strain was induced for expression, and then bacterial cells were collected. In a 100 mL reaction system containing 1 g/L (wet weight) cells, 1 g/L D-Danshensu, 1 g/L caffeic acid, 1 g/L ATP, 1 g/L CoA, and 20 g/L sodium hexametaphosphate, and having a pH of 8, reaction was performed at 40 C. for 48 hours. After the transformation, the content of rosmarinic acid was measured as 1.6 g/L by liquid chromatography. If the concentration of ATP and CoA in the reaction volume is 0 g/L, the content of rosmarinic acid is 0.4 g/L under the condition that other transformation conditions remain unchanged.

Example 11: Synthesis of Rosmarinic Acid by Recombinant Escherichia coli Whole-Cell Catalysis

(43) The following 2 kinds of recombinant strains were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-smpkk-ajpkk (named E1), and Escherichia coli BL21 (DE3)/pETDuet-1-sc4cl-ccras (named E2).

(44) According to the method in Example 1, E1 and E2 were induced for expression respectively, and then bacterial cells were collected. In a 100 mL reaction system containing 30 g/L (wet weight) E1 cells, 50 g/L (wet weight) E2 cells, 100 g/L D-Danshensu, 10 g/L caffeic acid, 300 g/L sodium hexametaphosphate, 1 g/L CoA, and 0.1 g/L ATP, and having a pH of 9, reaction was performed at 40 C. for 48 hours. After the transformation, the content of rosmarinic acid was measured as 146 g/L by liquid chromatography.

(45) Although the disclosure has been disclosed as above in preferred examples, it is not intended to limit the disclosure. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be defined by the claims.