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 PDF

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WO2020258896A1
WO2020258896A1 PCT/CN2020/076363 CN2020076363W WO2020258896A1 WO 2020258896 A1 WO2020258896 A1 WO 2020258896A1 CN 2020076363 W CN2020076363 W CN 2020076363W WO 2020258896 A1 WO2020258896 A1 WO 2020258896A1
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rosmarinic acid
acid
polyphosphate kinase
recombinant
recombinant cells
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French (fr)
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蔡宇杰
燕毅
丁彦蕊
白亚军
郑晓辉
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陕西鸿道生物分析科学技术研究院有限公司
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Priority to EP20830994.8A priority Critical patent/EP3992298A4/en
Priority to JP2021577280A priority patent/JP2022540791A/ja
Priority to KR1020227002342A priority patent/KR20220038352A/ko
Priority to MX2022000059A priority patent/MX2022000059A/es
Priority to CA3145416A priority patent/CA3145416A1/en
Priority to AU2020303019A priority patent/AU2020303019A1/en
Priority to BR112021026392A priority patent/BR112021026392A2/pt
Publication of WO2020258896A1 publication Critical patent/WO2020258896A1/zh
Priority to US17/209,449 priority patent/US20210207105A1/en
Priority to IL289360A priority patent/IL289360A/en

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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

提供了一种生产迷迭香酸的菌株及方法。构建了表达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的重组细胞或者重组细胞的组合，并利用重组细胞或者重组细胞的组合催化丹参素和咖啡酸合成迷迭香酸。

Description

一种生产迷迭香酸的菌株及方法 技术领域
本发明涉及一种生产迷迭香酸的菌株及方法，属于生物工程技术领域。
背景技术
迷迭香酸(Rosmarinic acid，RA)，是一种天然的多酚类化合物。RA是由咖啡酸(Caffeic acid)和D-丹参素(D-Danshensu)所组成的酯，广泛分布于唇形科、紫草科、葫芦科、椴树科、伞形科等植物中。相比于其他植物，RA在唇形科和紫草科中含量最高。RA作为迷迭香中最有效的抗氧化成分，已被美国食品药物管理局(FDA)认可为“公众安全食品”。早在1998年Nakamura等就提出邻二酚羟基是清除自由基活性的结构基础，而C3位的共轭双键具有增效作用(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)。近几年的研究表明，迷迭香酸对多种疾病有明显的疗效，迷迭香酸在抗氧化、抑制病原微生物、抗癌抗肿瘤、抗炎活性和免疫抑制活性、抗血栓、抗血小板凝集、抗抑郁、防辐射、防止细胞损伤及记忆损伤等方面都具有良好的生物活性。随着人们对迷迭香酸生物学活性及其在体内的代谢特征的不断研究，RA的越来越多的生物活性被开发出来。因此，进一步地合理开发、利用迷迭香酸使其为人类所用，将成为一种研究热点。
目前，工业化生产迷迭香酸的方法还不成熟，且大多处于实验室阶段。小量制备迷迭香酸的方法主要有以下两种：1)通过对唇形科植物进行纤维素酶酶解法、超声法、回流法处理，再提取获得迷迭香酸；2)利用化学合成的方法制备迷迭香酸。第1)种从植物中提取迷迭香酸的方法，简便、易于实现，产品质量有保障。但是，此方法需要使用大量的有机溶剂，提取过程需要耗费较长的时间并且迷迭香酸的回收率不高。第2)种化学合成迷迭香酸的方法，虽然能成功合成迷迭香酸，但由于原料和试剂成本昂贵、副产物多并且合成步骤较长、反应剧烈不易控制等缺点，这种方法不适合工业化生产。除此之外，由于迷迭香酸是重要的食品、药物、保健品的原材料，因此，通过化学方法得到的产品不受人们欢迎。目前市场上的迷迭香酸主要还是从植物中提取得到的，例如，中国发明专利CN 108658769 A公开了一种基于响应面法夏枯草迷迭香酸的提取工艺；中国发明专利CN 107935855 A公开了一种通过回流法从迷迭香中提取迷迭香酸的方法。然而，由于植物资源有限、植物中迷迭香酸有限，并且提取 过程繁琐、复杂，使得从植物中提取的迷迭香酸的价格昂贵。因此，通过微生物法生产受到了广泛的关注。
2014年，Bloch等人提出以大肠杆菌自身代谢产生的酪氨酸和4-羟基苯丙酮酸为底物，在羟基酸脱氢酶的作用下将内源的4-羟基苯丙酮酸转化成4-羟基苯乳酸，随后通过从大肠杆菌克隆的羟化酶HpaBC进行间羟基化，得到3,4-二羟基苯乳酸；同时，以内源性的酪氨酸为底物生成咖啡酸，首先以酪氨酸在酪氨酸氨裂解酶作用下生成对香豆酸，然后也是通过从大肠杆菌克隆的羟化酶HpaBC进行间羟基化得到咖啡酸，得到咖啡酸后在4-香豆酸-CoA连接酶的作用下生成咖啡酰辅酶A；最终，咖啡酰辅酶A和3,4-二羟基苯乳酸在迷迭香酸合成酶作用下经过72小时后转化得到1.8±0.3μm迷迭香酸(Construction of a chimeric biosynthetic pathway for the de novo biosynthesis of rosmarinic acid in Escherichia coli.Chembiochem,15(16):2393-2401(2014))。但该方法中HpaBC酶活力较低，导致迷迭香酸的产量极低。也有人尝采用过表达4-香豆酸-CoA连接酶和迷迭香酸合成酶的大肠杆菌来转化咖啡酸和丹参素生成迷迭香酸，但由于缺少辅酶及快速再生体系，产量也极低(Synthesis of rosmarinic acid analogues in Escherichia coli,Biotechnol Lett38:619–627(2016))。
发明内容
本发明公开了以酚酸为底物合成迷迭香酸，提高生物法或酶法合成迷迭香酸的产量的方法。同时，本发明公开的方法能合成L-迷迭香酸。
本发明提供了以酚酸为底物合成迷迭香酸的方法，所述酚酸为咖啡酸和丹参素；咖啡酸被4-香豆酸辅酶A连接酶(4-coumarate:CoA ligase)与辅酶A(CoA)连接生成咖啡酰辅酶A(Caffeyl-CoA)，迷迭香酸合成酶(Rosmarinic acid synthase)利用ATP的能量将咖啡酰辅酶A和丹参素合成为迷迭香酸。在此过程中CoA被释放，ATP被水解成AMP。多聚磷酸激酶2-II(polyphosphate kinase 2-II，PPK2-II)将AMP生成为ADP，进一步ADP被多聚磷酸盐激酶2-I(polyphosphate kinase 2-I,PPK2-I)实现ATP的再生。
在一种实施方式中，所述4-香豆酸辅酶A连接酶为来自于Scutellaria baicalensis、Ocimum tenuiflorum、Ocimum basilicum、Arabidopsis thaliana、Penicillium chrysogenum、Streptomyces coelicolor A3(2)、Rhodococcus jostii RHA1。或者，所述4-香豆酸辅酶A连接酶，其氨基酸序列是NCBI上accession NO.为BAD90936.1、ADO16242.1、AGP02119.1、AAD47193.1、CAA04820.1、CAB95894.1、ABG96911.1的序列。或者，4-香豆酸辅酶A连接酶的核苷酸序列是NCBI上accession NO.为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)的序列。
在一种实施方式中，所述多聚磷酸盐激酶2-I为来自于Sinorhizobium_meliloti。或者，所述多聚磷酸盐激酶2-I，其氨基酸序列是NCBI上accession NO.为NP_384613.1的序列。或者，多聚磷酸盐激酶2-I的核苷酸序列是NCBI上accession NO.为NC_003047 REGION:complement(564142..565044)的序列。
在一种实施方式中，所述迷迭香酸合成酶为来自于Plectranthus scutellarioides、Lavandula angustifolia、Melissa officinalis、Salvia miltiorrhiza、Coffea canephora、Nicotiana tabacum、Dianthus caryophyllus。或者，所述迷迭香酸合成酶，其氨基酸序列是NCBI上accession NO.为CAK55166.1、ABI48360.1、CBW35684.1、ADA60182.1、ABO47805.1、CAE46932.1、CAB06430.1的序列。或者，迷迭香酸合成酶的核苷酸序列是NCBI上accession NO.为AM283092.1、DQ886904.1 REGION:51..1433、FR670523.1、FJ906696.1、EF137954.1 REGION:3..1307、AJ582651.1、Z84386.1 REGION:137..1477的序列。
在一种实施方式中，所述多聚磷酸盐激酶2-II为来自于Acinetobacter johnsonii。或者，所述多聚磷酸盐激酶2-II，其氨基酸序列是NCBI上accession NO.为BAC76403.1的序列。或者，多聚磷酸盐激酶2-II的核苷酸序列是NCBI上accession NO.为AB092983 REGION:339..1766的序列。
在一种实施方式中，所述丹参素是D-丹参素或者L-丹参素。当丹参素是L-丹参素时，迷迭香酸合成酶选择来自Coffea canephora或Dianthus caryophyllus的迷迭香酸合成酶。
本发明还提供了一种能够以酚酸为底物合成迷迭香酸的重组细胞或一种能够以酚酸为底物合成迷迭香酸的重组细胞的组合。所述重组细胞表达了4-香豆酸辅酶A连接酶(4-coumarate:CoA ligase)、迷迭香酸合成酶(Rosmarinic acid synthase)、多聚磷酸激酶2-II(polyphosphate kinase 2-II，PPK2-II)、多聚磷酸盐激酶2-I(polyphosphate kinase 2-I,PPK2-I)。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。
所述重组细胞或者重组细胞的组合都可以选择大肠杆菌为宿主，例如Escherichia coli BL21(DE3)。
所述4种酶可以在宿主中借助载体进行表达融合表达或共表达，或者整合到宿主的基因组上进行表达。所述4种酶借助载体进行表达时，可以选择一个或多个载体表达4种酶中的 一种或多种。
对于所述重组细胞，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。例如，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。又例如，以大肠杆菌为宿主，表达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；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。
对于所述重组细胞的组合，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。例如，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。又例如，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。
特别地，本发明还提供了一种能够以咖啡酸、L-丹参素为底物合成L-迷迭香酸的重组细胞，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。每个基因前均包含T7启动子和RBS结合点，每个基因后带有T7终止子。
本发明还提供了一种全细胞催化生产迷迭香酸的方法，包括步骤：(1)制备所述的重组细胞或者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，以咖啡酸和D-丹参素(L-丹参素)为底物合成迷D-迭香酸(L-迭香酸)。步骤(1)所述制备，是培养、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。使用所述全细胞催化剂时，除了提供底物，还需要维持适当的温度和pH，必要时还提供一些辅酶或者营养物质，以帮助全细胞催化剂更好地发挥催化作用。
在一种实施方式中，所述全细胞转化生产的体系中，包括细胞湿重为1-200g/L，丹参素(D或L)1-100g/L，咖啡酸1-100g/L，ATP 0-1g/L，CoA0-1g/L，六聚偏磷酸钠300g/L，pH 5.0-9.0；于15-40℃反应，反应时间1-48小时。
本发明构建了强化表达4种酶的基因工程菌应用于迷迭香酸的生产。本发明采用的底物为咖啡酸和丹参素，咖啡酸和丹参素这两种酚酸易于得到。
本发明通过合理的表达策略，在表达4-香豆酸辅酶A连接酶、迷迭香酸合成酶的同时，还表达了多聚磷酸激酶2-II、多聚磷酸盐激酶2-I，实现了ATP和CoA的双辅酶再生，有效地保证了酶催化反应的持续进行，并提高了迷迭香酸的产量。
自然界中迷迭香酸的丹参素基团部分均匀为D型，因此，常见的迷迭香酸均是D型的。本发明获得了能够以L-丹参素为底物的迷迭香酸合成酶，在此基础上，以L-丹参素和咖啡酸为原料，通过生物法合成得到了L-迷迭香酸。
附图说明
图1实施例4中合成得到的迷迭香酸的液相图谱。
图2实施例6中合成得到的迷迭香酸的液相图谱。
具体实施方式
1、下述实施例所涉及的菌株及质粒
pRSFDuet-1、pETDuet-1、pCDFDuet-1、pACYCDuet-1质粒和Escherichia coli BL21(DE3)，均购自Novagen公司。
2、多基因共表达体系的构建及细胞的培养
目前大肠杆菌多基因共表达的有多种方法(例如：“大肠杆菌多基因共表达策略，中国生物工程杂志，2012，32(4):117-122”这篇文章所记载的方法)，本发明采用刘向磊的博士论文(合成生物学技术改造大肠杆菌生产莽草酸及白藜芦醇，2016，上海医药工业研究院)所记 载的方法来构建重组大肠杆菌。下述实施例中，共表达多基因时，每个基因前均包含T7启动子和RBS结合点，每个基因后带有T7终止子。理论上来讲，因为每个基因前都有T7和RBS，因此，基因的表达强度受基因在质粒上的排列次序的影响不大。将构建好的质粒热转导入大肠杆菌感受态细胞中，并涂布于单抗或混合抗生素固体平板上，筛选得到阳性转化子，即得到重组大肠杆菌。
细胞的培养：根据经典的重组大肠杆菌培养及诱导表达方案，将重组大肠杆菌按体积比为2％的量转接到LB发酵培养基(蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L)中，当细胞OD 600达到0.6-0.8后，加入终浓度为0.4mM的IPTG，在20℃诱导表达培养8h。诱导表达结束后，4℃、8000rpm、20分钟离心收集细胞。
3、相关酶的选择
(1)多聚磷酸盐激酶2-I
选择源于Sinorhizobium_meliloti的编码多聚磷酸盐激酶2-I的基因smpkk，基因smpkk在NCBI上accession NO为NC_003047 REGION:complement(564142..565044)，对应的氨基酸序列是NP_384613.1。
(2)多聚磷酸盐激酶2-II
选择源于Acinetobacter johnsonii的编码多聚磷酸盐激酶2-II的基因ajpkk，基因ajpkk NCBI上accession NO.为AB092983 REGION:339..1766的序列，对应的氨基酸序列是BAC76403.1。
(3)4-香豆酸辅酶A连接酶
参见实施例1。
(4)迷迭香酸合成酶
参见实施例2。
4、样品的检测分析
迷迭香酸含量的测定方法参考文献：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。丹参素、咖啡酸、迷迭香酸的溶解度较低，本发明中的转化过程会加入过量底物，底物会边反应边溶解，产物则在高浓度情况下会边反应边析出，测定时加入纯水使之完全溶解后测定。
迷迭香酸合成酶活力根据文献测定：Rosmarinic acid synthase is a new member of the  superfamily of BAHD acyltransferases,Planta,2006,224:1503–1510。
4-香豆酸辅酶A连接酶活力根据文献测定：4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm.Arch.Biochem.Biophys.1977,184,237-248。
比酶活(U mg -1)定义为每mg酶所具有的酶活力单位。一个酶活力单位(U)定义为1min生成1μmol产物所需的酶量。
实施例1：4-香豆酸辅酶A连接酶的筛选与表达
4-香豆酸辅酶A连接酶广泛各类生物体中，依据Scutellaria baicalensis、Ocimum tenuiflorum、Ocimum basilicum、Arabidopsis thaliana、Penicillium chrysogenum、Streptomyces coelicolor A3(2)、Rhodococcus jostiid在NCBI上的4-香豆酸辅酶A连接酶的基因信息全合成得到4-香豆酸辅酶A基因sb4cl、ot4cl、ob4cl、at4cl、pc4cl、sc4cl、rj4cl。与这些基因相对应的氨基酸序列在NCBI上accession NO为：BAD90936.1、ADO16242.1、AGP02119.1、AAD47193.1、CAA04820.1、CAB95894.1、ABG96911.1。将合成得到的基因连接到pETDuet-1载体上，并在Escherichia coli BL21(DE3)中得到诱导表达。诱导表达方法：将重组大肠杆菌按体积比为2％的量转接到LB发酵培养基(蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L)中，进行发酵培养，当细胞OD 600达到0.6-0.8后，加入终浓度为0.4mM的IPTG，在20℃诱导表达培养8h。诱导表达结束后，在4℃、8000rpm、20分钟条件下，对发酵液进行离心处理，收集细胞。破碎所收集得到的细胞，并利用Histag标签从细胞破碎液中纯化得到纯酶，得到纯酶后测定纯酶的活性。
当以咖啡酸和辅酶A为底物时，4-香豆酸辅酶A连接酶基因sb4cl、ot4cl、ob4cl、at4cl、pc4cl、sc4cl、rj4cl各自表达的酶的比酶活分别为：152、143、161、179、202、174、88U/mg。
实施例2：迷迭香酸合成酶的筛选与表达
迷迭香酸合成酶主要存在于植物中，根据Plectranthus scutellarioides、Lavandula angustifolia、Melissa officinalis、Salvia miltiorrhiza、Coffea canephora、Nicotiana tabacum、Dianthus caryophyllus在NCBI上的迷迭香酸合成酶基因信息全合成了迷迭香酸合成酶基因psras、laras、moras、smras、ccras、ntras、dcras。与这些基因相对应的氨基酸序列在NCBI上accession NO为：CAK55166.1、ABI48360.1、CBW35684.1、ADA60182.1、ABO47805.1、CAE46932.1、CAB06430.1的序列。将合成得到的基因分别连接到pETDuet-1载体上，与实施例1同样的方法表达纯化。
以咖啡酰辅酶A和D-丹参素为底物时，迷迭香酸合成酶基因psras、laras、moras、smras、ccras、ntras、dcras各自表达的酶的比酶活分别为：410、320、414、233、361、521、371U/mg。
以咖啡酰辅酶A和L-丹参素为底物时，迷迭香酸合成酶基因psras、laras、moras、smras、ccras、ntras、dcras各自表达的酶的比酶活分别为：0、0、0、0、120、0、142U/mg。可见，只有ccras、dcras编码的迷迭香酸合成酶具有以L-丹参素为底物合成迷迭香酸的能力。
实施例3:同时表达4种酶的重组大肠杆菌的构建
重组大肠杆菌构建：
如表1所示，从pETDuet-1、pACYCDuet-1、pRSFDuet-1、pCDFduet-1四种质粒中进行选择，将编码四种酶的基因连接到同一个质粒上，或者分别连接到两个质粒上(每个质粒上表达2个基因)，或者分别连接到四个质粒上(每个质粒上表达1个基因)。每个基因前均包含T7启动子和RBS结合点，每个基因后均带有T7终止子。将构建得到的重组质粒转化入大肠杆菌Escherichia coli BL21中，利用混合抗生平板筛选得到阳性转化子，即得到可强化表达4个基因的重组大肠杆菌。
对重组大肠杆菌进行诱导表达，诱导表达完成后收集菌体。构建100mL的反应体系，其中，细胞湿重为200g/L，D-丹参素20g/L，咖啡酸20g/L，CoA 1g/L，ATP 1g/L，六聚偏磷酸钠60g/L，pH 8。将100mL的反应体系置于30℃反应24小时。丹参素和咖啡酸的溶解度均很小，因此，在反应过程中，它们是边消耗边溶解。反应结束后将反应液稀释后利用液相色谱测定反应液中迷迭香酸的浓度，结果如表1所示。
表1
Figure PCTCN2020076363-appb-000001
Figure PCTCN2020076363-appb-000002
实施例4：应用四种酶体外合成迷迭香酸
将smpkk、ajpkk、pc4cl、ntras四个基因分别连接到pEDTDuet-1载体上，得到4个重组载体，将4个重组载体分别转化到Escherichia coli BL21中，得到分别表达4种酶的重组大肠杆菌。采用与实施例1同样的方法表达纯化后得到四种纯酶。然后于100mL反应体系中加入这四种纯酶各2mg，D-丹参素20g/L，咖啡酸20g/L，CoA 1g/L，ATP 1g/L，六聚偏磷酸钠60g/L，pH 8；于30℃反应5小时。最终，液相色谱测定结果表明反应液中迷迭香酸浓度为36g/L，其液相图谱如说明书附图1。
实施例5：应用分别表达smpkk、ajpkk、pc4cl、ntras基因的重组细胞的组合合成迷迭香酸
将smpkk、ajpkk、pc4cl、ntras四个基因分别连接到pEDTDuet-1载体上，得到4个重组载体，将4个重组载体分别转化到Escherichia coli BL21中，得到分别表达4种酶中的一种酶的重组大肠杆菌。采用与实施例1同样的方法诱导重组大肠杆菌表达酶。然后，于100mL反应体系中加入这四种重组细胞各20g/L，D-丹参素20g/L，咖啡酸20g/L，CoA 1g/L，ATP 1g/L，六聚偏磷酸钠60g/L，pH 8；于30℃反应5小时。最终，液相色谱测定结果表明反应液中迷迭香酸浓度为23g/L。
实施例6：以重组大肠杆菌全细胞催化合成L-迷迭香酸
自然界中迷迭香酸的丹参素基团部份均匀为D型，本实施例以L-丹参素和咖啡酸为原料合成L-迷迭香酸(L-迷迭香酸与D-迷迭香酸的不同之处在于丹参素基团部份为L型)。此前，L-迷迭香酸一直未能以生物法合成得到。
选择Coffea canephora、Dianthus caryophyllus来源的编码迷迭香酸合成酶的基因ccras、dcras，与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连接酶的基因一起，构建重组菌Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk+pTDuet-1-pc4cl-dcras。根据实施例1所述的方法，将其诱导表达，然后收集菌体。
于100mL反应体系中，细胞湿重为100g/L，L-丹参素20g/L，咖啡酸20g/L，CoA 1g/L， ATP 1g/L，六聚偏磷酸钠60g/L，pH 8；于30℃反应24小时。转化结束后，利用液相色谱法测得反应液中L-迷迭香酸浓度为33g/L，其液相图谱如说明书附图2。采用江苏汉邦科技有限公司的DAC-HB50制备色谱柱制备纯化样品，制备色谱条件为：流动相50％甲醇、柱温自然、流速3mL/min、进样量5mL。首次制得的样品的色谱纯达到99.9％，将经过反复进样分离得到的产品于50℃下真空旋转蒸干。称取蒸干得到的样品0.5g，溶于去离子水中并定容至50mL，采用日本爱宕AP-300全自动旋光仪测定旋光度，其旋光度为
Figure PCTCN2020076363-appb-000003
因此，我们可以确认本实施例制备得到的迷迭香酸为L-迷迭香酸。
实施例7：以重组大肠杆菌全细胞催化合成迷迭香酸
构建如下2种重组菌Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk(合名为E1)，Escherichia coli BL21(DE3)/pETDuet-1-sc4cl-ccras(命名为E2)。
根据实施例1所述的方法，将E1和E2分别诱导表达，然后收集菌体。于100mL反应体系中，E1细胞湿重为30g/L，E2细胞湿重为50g/L、D-丹参素100g/L，咖啡酸10g/L，六聚偏磷酸钠300g/L，CoA 1g/L，ATP 1g/L，pH 9；于40℃反应48小时。转化结束后液相色谱测定迷迭香酸含量为162g/L。
实施例8：以重组大肠杆菌全细胞催化合成迷迭香酸
构建如下2种重组菌Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk-at4cl(合名为E3)，Escherichia coli BL21(DE3)/pACYCDuet-1-ccras(命名为E4)。
根据实施例1所述的方法，将E3和E4分别诱导表达，然后收集菌体。于100mL反应体系中，E3细胞湿重为100g/L，E4细胞湿重为100g/L，D-丹参素1g/L，咖啡酸1g/L，CoA 0.5g/L，ATP 1g/L，六聚偏磷酸钠3g/L，pH 5；于15℃反应48小时。转化结束后液相色谱测定迷迭香酸含量为1.3g/L。
实施例9：以重组大肠杆菌全细胞催化合成迷迭香酸
构建如下2种重组菌Escherichia coli BL21(DE3)/pRSFDuet-1-ntras-at4cl(合名为E5)，Escherichia coli BL21(DE3)/pACYCDuet-1-smpkk-ajpkk(命名为E6)。
根据实施例1所述的方法，将E5和E6分别诱导表达，然后收集菌体。于100mL反应体系中，E5细胞湿重为100g/L，E6细胞湿重为100g/L，D-丹参素1g/L，咖啡酸1g/L，六聚偏磷酸钠3g/L，CoA 1g/L，ATP 0.5g/L，pH7；于15℃反应，反应时间1小时。转化结束后液相色谱测定迷迭香酸含量为1.5g/L。
实施例10：以重组大肠杆菌全细胞催化合成迷迭香酸
构建重组菌Escherichia coli BL21(DE3)/pRSFDuet-1-ntras-at4cl+pCDFDuet-1-smpkk-ajpkk。 根据实施例1所述的方法，将重组菌诱导表达，然后收集菌体。于100mL反应体系中，细胞湿重为1g/L，D-丹参素1g/L，咖啡酸1g/L，ATP 1g/L，CoA 1g/L，六聚偏磷酸钠20g/L，pH 8；于40℃反应，时间48小时。转化结束后，利用液相色谱测的迷迭香酸的含量为1.6g/L。若该反应体积中的ATP和CoA浓度为0g/L，则在其他转化条件不变的情况下，迷迭香酸含量为0.4g/L。
实施例11：以重组大肠杆菌全细胞催化合成迷迭香酸
构建如下2种重组菌Escherichia coli BL21(DE3)/pRSFDuet-1-smpkk-ajpkk(合名为E1)，Escherichia coli BL21(DE3)/pETDuet-1-sc4cl-ccras(命名为E2)。
根据实施例1所述的方法，将E1和E2分别诱导表达，然后收集菌体。于100mL反应体系中，E1细胞湿重为30g/L，E2细胞湿重为50g/L、D-丹参素100g/L，咖啡酸10g/L，六聚偏磷酸钠300g/L，CoA 1g/L，ATP 0.1g/L，pH 9；于40℃反应，反应时间48小时。转化结束后，利用液相色谱测得迷迭香酸的含量为146g/L。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 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。
  2. 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。
  3. 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)。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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)。
  10. 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。
  11. 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。
  12. 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。
  13. 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。
  14. 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。
  15. 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。
  16. 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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