WO2020258896A1 - 一种生产迷迭香酸的菌株及方法 - Google Patents
一种生产迷迭香酸的菌株及方法 Download PDFInfo
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Definitions
- the invention relates to a strain and method for producing rosmarinic acid, and belongs to the technical field of bioengineering.
- Rosmarinic acid is a natural polyphenol compound.
- RA is an ester composed of Caffeic acid and D-Danshensu. It is widely distributed in plants such as Lamiaceae, Boraginaceae, Cucurbitaceae, Lindenaceae, Umbelliferae and other plants. Compared with other plants, RA has the highest content in Lamiaceae and Boraginaceae. As the most effective antioxidant ingredient in rosemary, RA has been recognized by the US Food and Drug Administration (FDA) as a "public safety food". As early as 1998, Nakamura et al.
- FDA US Food and Drug Administration
- the o-diphenol hydroxyl group is the structural basis for free radical scavenging activity, and the conjugated double bond at the C3 position has a synergistic effect (Ohto Y, Murakami A, Nakamura Y, et al. Superoxide scavenging activity of rosmarinic acid from Perilla frutescens Britton var.acuta f.viridis[J].Journal of Agricultural and Food Chemistry,1998,46(11):4545-4550). Research in recent years has shown that rosmarinic acid has obvious effects on many diseases.
- Rosmarinic acid has anti-oxidation, inhibition of pathogenic microorganisms, anti-cancer and anti-tumor, anti-inflammatory and immunosuppressive activities, anti-thrombotic and anti-platelet activities. It has good biological activity in agglutination, anti-depression, radiation protection, cell damage and memory damage. As people continue to study the biological activity of rosmarinic acid and its metabolic characteristics in the body, more and more biological activities of RA have been developed. Therefore, further rational development and utilization of rosmarinic acid for human use will become a research hotspot.
- rosmarinic acid is not yet mature, and most of them are in the laboratory stage.
- the first) method for extracting rosmarinic acid from plants is simple and easy to implement, and the product quality is guaranteed. However, this method requires a large amount of organic solvents, the extraction process takes a long time and the recovery rate of rosmarinic acid is not high.
- the second) method of chemically synthesizing rosmarinic acid although it can successfully synthesize rosmarinic acid, but due to the high cost of raw materials and reagents, many by-products, long synthesis steps, and violent reactions that are difficult to control, this method is not Suitable for industrial production.
- rosmarinic acid is an important raw material for foods, drugs, and health products, products obtained through chemical methods are not welcome. At present, rosmarinic acid on the market is mainly extracted from plants.
- Chinese invention patent CN 108658769 A discloses an extraction process of Prunella vulgaris rosmarinic acid based on response surface method
- Chinese invention patent CN 107935855 A discloses A method of extracting rosmarinic acid from rosemary by reflux method is presented.
- Bloch et al. proposed that tyrosine and 4-hydroxyphenylpyruvate produced by E. coli's own metabolism were used as substrates to convert endogenous 4-hydroxyphenylpyruvate into 4 under the action of hydroxy acid dehydrogenase.
- the invention discloses a method for synthesizing rosmarinic acid by using phenolic acid as a substrate, and improving the yield of synthetic rosmarinic acid by biological or enzymatic method. At the same time, the method disclosed in the present invention can synthesize L-rosmarinic acid.
- the present invention provides a method for synthesizing rosmarinic acid using phenolic acid as a substrate.
- the phenolic acid is caffeic acid and danshensu; caffeic acid is combined with 4-coumarate: CoA ligase Coenzyme A (CoA) is connected to generate Caffeyl-CoA (Caffeyl-CoA), and Rosmarinic acid synthase uses the energy of ATP to synthesize caffeyl-CoA and Danshensu into rosmarinic acid. During this process, CoA is released and ATP is hydrolyzed into AMP.
- Polyphosphate kinase 2-II (polyphosphate kinase 2-II, PPK2-II) generates AMP into ADP, and further ADP is regenerated by polyphosphate kinase 2-I (polyphosphate kinase 2-I, PPK2-I) to achieve ATP regeneration .
- the 4-coumarate Coenzyme A ligase is derived from Scutellaria baicalensis, Ocimum tenuiflorum, Ocimum basilicum, Arabidopsis thaliana, Penicillium chrysogenum, Streptomyces coelicolor A3(2), Rhodococcus jostii RHA1.
- the 4-coumaric acid-CoA ligase has an amino acid sequence of accession NO on NCBI. It is BAD90936.1, ADO16242.1, AGP02119.1, AAD47193.1, CAA04820.1, CAB95894.1, ABG96911. 1 sequence.
- nucleotide sequence of 4-coumarate-CoA ligase is accession NO. on NCBI. It is AB166767.1 REGION: 42..1691, HM990148.1 REGION: 1..1704, KC576841.1 REGION: 1. .1704, AF106086.1 REGION: 67..1737, AJ001540.1 REGION: 89..1825, AL645882 REGION: complement (4799896..4801464), CP000431.1 REGION: complement (5466961..5468496) sequence.
- the polyphosphate kinase 2-I is from Sinorhizobium_meliloti.
- the amino acid sequence of the polyphosphate kinase 2-I is the sequence of accession NO. NP_384613.1 on the NCBI.
- the nucleotide sequence of polyphosphate kinase 2-I is the sequence of accession NO. NC_003047 REGION: complement (564142..565044) on NCBI.
- the rosmarinic acid synthase is derived from Plectranthus scutellarioides, Lavandula angustifolia, Melissa officinalis, Salvia miltiorrhiza, Coffea canephora, Nicotiana tabacum, Dianthus caryophyllus.
- the amino acid sequence of the rosmarinic acid synthetase is the accession NO on NCBI. It is the sequence of CAK55166.1, ABI48360.1, CBW35684.1, ADA60182.1, ABO47805.1, CAE46932.1, CAB06430.1 .
- the nucleotide sequence of rosmarinic acid synthase is the accession NO. on NCBI.
- the polyphosphate kinase 2-II is from Acinetobacter johnsonii.
- the amino acid sequence of the polyphosphate kinase 2-II is the sequence of accession NO. of BAC76403.1 on NCBI.
- the nucleotide sequence of polyphosphate kinase 2-II is the sequence of accession NO. AB092983 REGION: 339..1766 on NCBI.
- the Danshensu is D-danshensu or L-danshensu.
- rosmarinic acid synthase is selected from Coffea canephora or Dianthus caryophyllus.
- the present invention also provides a recombinant cell capable of synthesizing rosmarinic acid using phenolic acid as a substrate or a combination of recombinant cells capable of synthesizing rosmarinic acid using phenolic acid as a substrate.
- the recombinant cell expresses 4-coumarate: CoA ligase (4-coumarate: CoA ligase), rosmarinic acid synthase (Rosmarinic acid synthase), and polyphosphate kinase 2-II (polyphosphate kinase 2-II, PPK2-II), polyphosphate kinase 2-I (polyphosphate kinase 2-I, PPK2-I).
- the combination of the recombinant cells is composed of recombinants expressing one or more of 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphokinase 2-II, and polyphosphate kinase 2-I.
- Cell composition each recombinant cell does not repeatedly express one of 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphokinase 2-II, and polyphosphate kinase 2-I with other recombinant cellskind.
- E. coli can be selected as the host for the recombinant cell or the combination of recombinant cells, such as Escherichia coli BL21 (DE3).
- the four enzymes can be expressed in the host by means of a vector for expression, fusion, expression or co-expression, or integrated into the genome of the host for expression.
- a vector for expression fusion, expression or co-expression, or integrated into the genome of the host for expression.
- one or more vectors can be selected to express one or more of the four enzymes.
- 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphokinase 2-I, and polyphosphate kinase 2-II are co-expressed or integrated into the host by means of a vector. Expression in the host genome; when 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphokinase 2-I, and polyphosphate kinase 2-II are expressed with the aid of vectors, multiple vectors are selected, Each vector expresses one or more of the 4 enzymes, or alternatively, one vector is selected to express these 4 enzymes simultaneously.
- the genes encoding 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II are allocated to pETDuet-1, pACYCDuet-1, and pRSFDuet. -1, pCDFduet-1 on one or more of the four plasmids, each of which carries coding for 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphokinase 2-I, One or more of the genes of polyphosphate kinase 2-II.
- Escherichia coli express genes encoding rosmarinic acid synthase derived from Cofea canephora or Dianthus caryophyllus, as well as encoding polyphosphate kinase 2-I, polyphosphate kinase 2-II, 4-flavor
- the gene of soybean acid coenzyme A ligase; pRSFDuet-1 is used as a vector to express genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II
- pTDuet-1 is used as a vector to express genes encoding 4-coumaric acid Genes for coenzyme A ligase and rosmarinic acid synthase; genes encoding 4-coumarate coenzyme A ligase, rosmarinic acid synthase, polyphosphokinase 2-I, and polyphosphate kinase 2-II
- the front contains the T7 promoter and RBS binding point
- 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphokinase 2-I, and polyphosphate kinase 2-II are co-expressed in the host by means of a vector or Integrate into the host genome for expression.
- the genes encoding 4-coumarate-CoA ligase, rosmarinic acid synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II are allocated to pETDuet-1, pACYCDuet-1, and pRSFDuet.
- pRSFDuet-1 is used as a vector to express genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II
- pTDuet-1 is used as a vector to express genes encoding 4-coumaric acid Genes for coenzyme A ligase and rosmarinic acid synthase; genes encoding 4-coumarate coenzyme A ligase, rosmarinic acid synthase, polyphosphokinase 2-I, and polyphosphate kinase 2-II
- the front contains the T7 promoter and RBS binding point, and the rear of the gene has a T7 terminator.
- the present invention also provides a recombinant cell capable of synthesizing L-rosmarinic acid using caffeic acid and L-danshensu as substrates, the recombinant cell expressing Coffea canephora or Dianthus caryophyllus-derived encoding rosemary Acid synthase genes, and genes encoding polyphosphate kinase 2-I, polyphosphate kinase 2-II, 4-coumarate-CoA ligase.
- the recombinant cell uses Escherichia coli as a host, uses pRSFDuet-1 as a vector to express genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II, and uses pTDuet-1 as a vector to express genes encoding 4-couma.
- the present invention also provides a method for whole-cell catalytic production of rosmarinic acid, which includes the steps: (1) preparing the recombinant cell or the combination of the recombinant cells, (2) preparing the recombinant cell in step (1)
- a combination of cells or recombinant cells is used as a catalyst to synthesize rosmarinic acid (L-marinic acid) with caffeic acid and D-danshensu (L-danshensu) as substrates.
- the preparation in step (1) is to cultivate and propagate the recombinant cells or a combination of recombinant cells, and allow the recombinant cells or the combination of recombinant cells to express the four enzymes, and then collect the recombinant cells.
- the whole-cell catalyst in addition to providing a substrate, it is also necessary to maintain an appropriate temperature and pH, and if necessary, provide some coenzymes or nutrients to help the whole-cell catalyst perform a better catalytic effect.
- the whole cell transformation production system includes cell wet weight of 1-200g/L, danshensu (D or L) 1-100g/L, caffeic acid 1-100g/L, ATP 0-1g/L, CoA0-1g/L, sodium hexametaphosphate 300g/L, pH 5.0-9.0; reaction at 15-40°C, reaction time 1-48 hours.
- the present invention constructs a genetically engineered bacteria expressing four kinds of enzymes to be applied to the production of rosmarinic acid.
- the substrates used in the invention are caffeic acid and danshensu, and the two phenolic acids of caffeic acid and danshensu are easily available.
- the present invention uses a reasonable expression strategy to express 4-coumarate-CoA ligase and rosmarinic acid synthase, as well as polyphosphokinase 2-II and polyphosphate kinase 2-I.
- the double coenzyme regeneration of ATP and CoA effectively ensures the continuous progress of the enzyme-catalyzed reaction and increases the production of rosmarinic acid.
- the Danshensu group of rosmarinic acid is uniformly D-type, so common rosmarinic acid is D-type.
- the present invention obtains rosmarinic acid synthetase capable of using L-danshensu as a substrate. On this basis, L-danshensu and caffeic acid are used as raw materials to obtain L-rosmarinic acid through biological synthesis.
- Figure 1 The liquid phase spectrum of rosmarinic acid synthesized in Example 4.
- Figure 2 The liquid phase spectrum of rosmarinic acid synthesized in Example 6.
- pRSFDuet-1 pETDuet-1
- pCDFDuet-1 pACYCDuet-1 plasmids and Escherichia coli BL21 (DE3) were all purchased from Novagen.
- E. coli multi-gene co-expression strategy Chinese Journal of Bioengineering, 2012, 32(4):117-122
- This article describes the method
- this The invention uses the method described in Liu Xianglei's doctoral thesis (Synthetic Biology Technology to Transform E. coli to Produce Shikimic Acid and Resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry) to construct recombinant E.
- each gene contains a T7 promoter and an RBS binding site, and each gene is followed by a T7 terminator.
- each gene has T7 and RBS before it, the expression intensity of the gene is not greatly affected by the sequence of the gene on the plasmid.
- the constructed plasmid is thermally transduced into E. coli competent cells, and spread on a monoclonal antibody or mixed antibiotic solid plate, and positive transformants are screened to obtain recombinant E. coli.
- the recombinant E. coli was transferred to the LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/ In L), when the cell OD 600 reaches 0.6-0.8, IPTG with a final concentration of 0.4 mM is added, and expression is induced at 20° C. and cultured for 8 hours. After the induction of expression, the cells were collected by centrifugation at 4°C, 8000 rpm, and 20 minutes.
- the gene smpkk encoding polyphosphate kinase 2-I from Sinorhizobium_meliloti was selected.
- the access NO of gene smpkk on NCBI is NC_003047 REGION: complement (564142..565044), and the corresponding amino acid sequence is NP_384613.1.
- the gene ajpkk encoding polyphosphate kinase 2-II from Acinetobacter johnsonii was selected.
- the accession NO. of the gene ajpkk NCBI was the sequence of AB092983 REGION: 339..1766, and the corresponding amino acid sequence was BAC76403.1.
- the determination method of rosmarinic acid content Reference 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 radiation. Journal of food of 2(2010) 66–70.
- the solubility of Danshensu, caffeic acid, and rosmarinic acid is relatively low.
- an excessive amount of substrate will be added. The substrate will be dissolved while reacting, and the product will be precipitated while reacting under high concentration conditions. Measure after adding pure water to completely dissolve it.
- Rosmarinic acid synthase activity is measured according to the literature: Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases, Planta, 2006, 224:1503-1510.
- 4-coumarate-CoA ligase was determined according to the literature: 4-Coumarate: CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm.Arch.Biochem.Biophys.1977,184,237-248.
- Specific enzyme activity (U mg -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 generate 1 ⁇ mol product in 1 minute.
- 4-coumaric acid coenzyme A ligase is widely used in various organisms, according to the 4-coumaric acid on NCBI of Scutellaria baicalensis, Ocimum tenuiflorum, Ocimum basilicum, Arabidopsis thaliana, Penicillium chrysogenum, Streptomyces coelicolor A3(2), Rhodococcus jostiid Coenzyme
- a ligase gene information is fully synthesized to obtain 4-coumarate-CoA genes sb4cl, ot4cl, ob4cl, at4cl, pc4cl, sc4cl, rj4cl.
- accession NOs of the amino acid sequences corresponding to these genes on NCBI are: BAD90936.1, ADO16242.1, AGP02119.1, AAD47193.1, CAA04820.1, CAB95894.1, ABG96911.1.
- the synthesized gene was ligated to the pETDuet-1 vector and expressed in Escherichia coli BL21 (DE3). Induced expression method: transfer the recombinant E.
- coli to LB fermentation medium peptone 10g/L, yeast powder 5g/L, NaCl 10g/L
- IPTG IPTG with a final concentration of 0.4 mM was added, and the expression was induced and cultured at 20°C for 8 hours.
- the fermentation broth was centrifuged at 4°C, 8000 rpm, and 20 minutes to collect the cells. Crush the collected cells, and use Histag tag to purify the pure enzyme from the cell crushing liquid. After obtaining the pure enzyme, measure the activity of the pure enzyme.
- the specific enzyme activities of the enzymes expressed by the 4-coumarate-CoA ligase genes sb4cl, ot4cl, ob4cl, at4cl, pc4cl, sc4cl, and rj4cl are respectively: 152, 143, 161, 179, 202, 174, 88 U/mg.
- Rosmarinic acid synthase mainly exists in plants. According to Plectranthus scutellarioides, Lavandula angustifolia, Melissa officinalis, Salvia miltiorrhiza, Coffea canephora, Nicotiana tabacum, Dianthus caryophyllus, the rosmarinic acid synthase gene information on NCBI is fully synthesized. Fraction acid synthase genes psras, laras, moras, smras, ccras, ntras, dcras.
- amino acid sequences corresponding to these genes on the NCBI accession NO are: CAK55166.1, ABI48360.1, CBW35684.1, ADA60182.1, ABO47805.1, CAE46932.1, CAB06430.1 sequence.
- the synthesized genes were respectively ligated to pETDuet-1 vector, and expressed and purified in the same way as in Example 1.
- the specific enzyme activities of the enzymes expressed by the rosmarinic acid synthase genes psras, laras, moras, smras, ccras, ntras, and dcras are respectively: 410, 320, 414, 233, 361, 521, 371 U/mg.
- the specific enzyme activities of the enzymes expressed by the rosmarinic acid synthase genes psras, laras, moras, smras, ccras, ntras, and dcras are respectively: 0, 0, 0, 0, 120, 0, 142 U/mg. It can be seen that only 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 E. coli expressing 4 enzymes simultaneously
- plasmids As shown in Table 1, select from four plasmids: pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and ligate the genes encoding the four enzymes to the same plasmid, or ligate to two plasmids separately (2 genes expressed on each plasmid), or ligated to four plasmids (1 gene expressed on each plasmid). Each gene contains a T7 promoter and RBS binding point, and each gene has a T7 terminator.
- the constructed recombinant plasmid was transformed into Escherichia coli BL21, and positive transformants were obtained by screening with a mixed antibiotic plate to obtain a recombinant Escherichia coli capable of expressing 4 genes.
- the recombinant E. coli was induced to express, and the bacteria were collected after the induced expression was completed. Construct a 100mL reaction system, in which the wet cell weight is 200g/L, D-danshensu 20g/L, caffeic acid 20g/L, CoA 1g/L, ATP 1g/L, sodium hexametaphosphate 60g/L, pH 8.
- the 100mL reaction system was placed at 30°C for 24 hours.
- the solubility of Danshensu and caffeic acid is very small, so in the reaction process, they dissolve while being consumed.
- 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.
- Example 4 Using four enzymes to synthesize rosmarinic acid in vitro
- the four genes of smpkk, ajpkk, pc4cl, and ntras were respectively ligated to the pEDTDuet-1 vector to obtain 4 recombinant vectors, and the 4 recombinant vectors were transformed into Escherichia coli BL21 to obtain recombinant E. coli expressing the 4 enzymes respectively.
- Four pure enzymes were obtained after expression and purification using the same method as in Example 1.
- Example 5 Synthesis of rosmarinic acid using a combination of recombinant cells expressing smpkk, ajpkk, pc4cl, and ntras genes
- the four genes of smpkk, ajpkk, pc4cl, and ntras were respectively ligated to pEDTDuet-1 vector to obtain 4 recombinant vectors, and the 4 recombinant vectors were transformed into Escherichia coli BL21 to express one of the 4 enzymes.
- recombinant E. coli The same method as in Example 1 was used to induce recombinant E. coli to express the enzyme.
- the Danshensu group of rosmarinic acid is uniformly D-type.
- L-danshensu and caffeic acid are used as raw materials to synthesize L-rosmarinic acid (L-rosmarinic acid and D-rosmarinic acid).
- the difference between scented acid is that the Danshensu group is L-shaped).
- L-rosmarinic acid has not been synthesized by biological methods.
- the wet cell weight is 100g/L, L-danshensu 20g/L, caffeic acid 20g/L, CoA 1g/L, ATP 1g/L, sodium hexametaphosphate 60g/L, pH 8; React at 30°C for 24 hours.
- concentration of L-rosmarinic acid in the reaction solution was measured by liquid chromatography to be 33 g/L, and its liquid spectrum was as shown in Figure 2 of the specification.
- the DAC-HB50 preparative chromatographic column of Jiangsu Hanbang Technology Co., Ltd. was used to prepare the purified samples.
- the preparative chromatographic conditions were: mobile phase 50% methanol, natural column temperature, flow rate 3mL/min, and injection volume 5mL.
- the chromatographic purity of the sample prepared for the first time reached 99.9%, and the product obtained after repeated injection and separation was evaporated to dryness under vacuum at 50°C.
- Example 7 Using recombinant E. coli whole cells to catalyze the synthesis of rosmarinic acid
- E1 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk
- E2 Escherichia coli BL21(DE3)/pETDuet-1-sc4cl-ccras
- E1 and E2 were induced to express respectively, and then the cells were collected.
- the wet weight of E1 cells is 30g/L
- the wet weight of E2 cells is 50g/L
- D-danshensu 100g/L caffeic acid 10g/L
- sodium hexametaphosphate 300g/L CoA 1g/L L
- ATP 1g/L pH 9
- react at 40°C for 48 hours the content of rosmarinic acid determined by liquid chromatography was 162g/L.
- Example 8 Using recombinant E. coli whole cells to catalyze the synthesis of rosmarinic acid
- E3 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk-at4cl
- E4 Escherichia coli BL21(DE3)/pACYCDuet-1-ccras
- E3 and E4 were induced to express respectively, and then the cells were collected.
- the wet weight of E3 cells is 100g/L
- the wet weight of E4 cells is 100g/L
- D-danshensu 1g/L is 100g/L
- caffeic acid 1g/L is 100g/L
- CoA 0.5g/L
- ATP ATP 1g/L
- the content of rosmarinic acid determined by liquid chromatography was 1.3 g/L.
- Example 9 Using recombinant E. coli whole cells to catalyze the synthesis of rosmarinic acid
- E6 Escherichia coli BL21(DE3)/pRSFDuet-1-ntras-at4cl
- E6 Escherichia coli BL21(DE3)/pACYCDuet-1-smpkk-ajpkk
- E5 and E6 were induced to express respectively, and then the cells were collected.
- the wet weight of E5 cells is 100g/L
- the wet weight of E6 cells is 100g/L
- D-danshensu 1g/L is 100g/L
- caffeic acid 1g/L sodium hexametaphosphate 3g/L
- CoA 1g/L L ATP 0.5g/L
- pH7 react at 15°C for 1 hour.
- the content of rosmarinic acid determined by liquid chromatography was 1.5g/L.
- Example 10 Using recombinant E. coli whole cells to catalyze the synthesis of rosmarinic acid
- the recombinant bacteria are induced to express, and then the bacteria are collected.
- the wet cell weight is 1g/L, D-danshensu 1g/L, caffeic acid 1g/L, ATP 1g/L, CoA 1g/L, sodium hexametaphosphate 20g/L, pH 8; React at 40°C for 48 hours.
- the content of rosmarinic acid measured by liquid chromatography was 1.6 g/L. 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 conversion conditions remain unchanged.
- Example 11 Using recombinant E. coli whole cells to catalyze the synthesis of rosmarinic acid
- E1 Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk
- E2 Escherichia coli BL21(DE3)/pETDuet-1-sc4cl-ccras
- E1 and E2 were induced to express respectively, and then the cells were collected.
- the wet weight of E1 cells is 30g/L
- the wet weight of E2 cells is 50g/L
- D-danshensu 100g/L caffeic acid 10g/L
- sodium hexametaphosphate 300g/L CoA 1g/L L
- ATP 0.1g/L pH 9
- react at 40°C for 48 hours the content of rosmarinic acid measured by liquid chromatography was 146 g/L.
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Abstract
Description
Claims (16)
- 一种合成迷迭香酸的方法,其特征在于,以为酚酸底物,所述酚酸为丹参素和咖啡酸;咖啡酸被4-香豆酸辅酶A连接酶与辅酶A连接生成咖啡酰辅酶A;迷迭香酸合成酶利用ATP的能量将咖啡酰辅酶A和丹参素合成为迷迭香酸,ATP释放能量后成为AMP,多聚磷酸激酶2-II将AMP生成为ADP,多聚磷酸盐激酶2-I进一步将ADP生成为ATP。
- 一种重组细胞,其特征在于,所述重组细胞能够以酚酸为底物合成迷迭香酸,所述重组细胞表达了4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II。
- 根据权利要求2所述的一种能够以酚酸为底物合成迷迭香酸的重组细胞,其特征在于,所述重组细胞选择大肠杆菌为宿主,包括Escherichia coli BL21(DE3)。
- 根据权利要求2或3所述的一种能够以酚酸为底物合成迷迭香酸的重组细胞,其特征在于,4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II在宿主中借助载体进行共表达或整合到宿主基因组中进行表达;4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II借助载体进行表达时,选择多个载体,每个载体表达4种酶中的一种或多种,或者,选择一个载体,由一个载体同时表达这4种酶。
- 根据权利要求4所述的一种能够以酚酸为底物合成迷迭香酸的重组细胞,其特征在于,将编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II的基因分配到pETDuet-1、pACYCDuet-1、pRSFDuet-1、pCDFduet-1这4种质粒中的一种或多种上,每种质粒上携带编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II的基因中1种基因或多种。
- 根据权利要求2或3所述的一种能够以酚酸为底物合成迷迭香酸的重组细胞,其特征在于,所述迷迭香酸包括D-迷迭香酸、L-迷迭香酸。
- 根据权利要求6所述的一种能够以酚酸为底物合成迷迭香酸的重组细胞,其特征在于,所述重组细胞表达了Coffea canephora或Dianthus caryophyllus来源的编码迷迭香酸合成酶的基因,以及编码多聚磷酸盐激酶2-I、多聚磷酸盐激酶2-Ⅱ、4-香豆酸辅酶A连接酶的基因;所述重组细胞以大肠杆菌为宿主,以pRSFDuet-1为载体表达编码多聚磷酸盐激酶2-I、多聚磷酸盐激酶2-Ⅱ的基因,以pTDuet-1为载体表达编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶的基因;编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II的基因前均包含T7启动子和RBS结合点,基因后带有T7终止子。
- 重组细胞的组合,其特征在于,所述重组细胞的组合能够以酚酸为底物合成迷迭香酸,所述重组细胞的组合由分别表达4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-II、多聚磷酸盐激酶2-I中的一种或以上的重组细胞组成,每个重组细胞不与其他重组细胞重复 表达4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-II、多聚磷酸盐激酶2-I中的一种。
- 根据权利要求8所述的重组细胞的组合,其特征在于,所述重组细胞选择大肠杆菌为宿主,包括Escherichia coli BL21(DE3)。
- 根据权利要求8或9所述的重组细胞的组合,其特征在于,4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II在宿主中借助载体进行共表达或整合到宿主基因组中进行表达。
- 根据权利要求10所述的重组细胞的组合,其特征在于,将编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II的基因分配到pETDuet-1、pACYCDuet-1、pRSFDuet-1、pCDFduet-1这4种质粒中的一种或多种上,每种质粒上携带编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II的基因中1种基因或多种。
- 根据权利要求8或9所述的重组细胞的组合,其特征在于,所述迷迭香酸包括D-迷迭香酸、L-迷迭香酸。
- 根据权利要求12所述的重组细胞的组合,其特征在于,所述重组细胞的宿主选择大肠杆菌,以pRSFDuet-1为载体表达编码多聚磷酸盐激酶2-I、多聚磷酸盐激酶2-Ⅱ的基因,以pTDuet-1为载体表达编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶的基因;编码4-香豆酸辅酶A连接酶、迷迭香酸合成酶、多聚磷酸激酶2-I、多聚磷酸盐激酶2-II的基因前均包含T7启动子和RBS结合点,基因后带有T7终止子。
- 一种生产迷迭香酸的方法,其特征在于,包括步骤:(1)制备权利要求2~7任一所述的重组细胞或者权利要求8~13任一所述的重组细胞的组合,(2)以步骤(1)制备的重组细胞或重组细胞的组合作为催化剂,以丹参素和咖啡酸为底物合成迷迭香酸。
- 根据权利要求14所述的一种全细胞催化生产迷迭香酸的方法,其特征在于,步骤(1)所述制备是指培养、繁殖重组细胞或者重组细胞的组合,并使得重组细胞或者重组细胞的组合表达所述4种酶,然后收集重组细胞。
- 权利要求2~7任一所述的重组细胞或者权利要求8~13任一所述的重组细胞的组合在生产迷迭香酸或者含有迷迭香酸的产品或者以迷迭香酸为前体的物质中的应用。
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EP20830994.8A EP3992298A4 (en) | 2019-06-25 | 2020-02-24 | Strain and method for producing rosmarinic acid |
JP2021577280A JP2022540791A (ja) | 2019-06-25 | 2020-02-24 | ロスマリン酸を産生するための株および方法 |
KR1020227002342A KR20220038352A (ko) | 2019-06-25 | 2020-02-24 | 로즈마린산의 생산을 위한 균주 및 방법 |
MX2022000059A MX2022000059A (es) | 2019-06-25 | 2020-02-24 | Cepa y metodo para producir acido rosmarinico. |
CA3145416A CA3145416A1 (en) | 2019-06-25 | 2020-02-24 | Strain and method for producing rosmarinic acid |
AU2020303019A AU2020303019A1 (en) | 2019-06-25 | 2020-02-24 | Strain and method for producing rosmarinic acid |
BR112021026392A BR112021026392A2 (pt) | 2019-06-25 | 2020-02-24 | Cepa e método para produção de ácido rosmarínico |
US17/209,449 US20210207105A1 (en) | 2019-06-25 | 2021-03-23 | Strain and Method for Producing Rosmarinic Acid |
IL289360A IL289360A (en) | 2019-06-25 | 2021-12-23 | strain and method for the preparation of rosmarinic acid |
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