CN111748532A

CN111748532A - Application of novel p-coumaroyl-CoA ligase in biosynthesis of phloretin

Info

Publication number: CN111748532A
Application number: CN202010449008.1A
Authority: CN
Inventors: 赵广荣; 李玲玲; 刘金丛; 刘雪
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2020-10-09
Anticipated expiration: 2040-05-25
Also published as: CN111748532B

Abstract

The invention discloses application of a novel p-coumaroyl-CoA ligase in biosynthesis of phloretin, and belongs to the technical field of enzyme engineering. The invention discovers that Aa4CL derived from azotobacter (Aromatium aromaticum) with an amino acid sequence shown as SEQ ID No.1 or Rj4CL derived from Rhodococcus jostii RHA1 with an amino acid sequence shown as SEQ ID No.2 can firstly synthesize a phloretin product in engineering escherichia coli by taking p-dihydrocoumaric acid as a substrate. The invention widens the application range of the p-coumaroyl-CoA ligase Aa4CL and Rj4CL, and lays a foundation for the industrial production of phloretin.

Description

Application of novel p-coumaroyl-CoA ligase in biosynthesis of phloretin

Technical Field

The invention belongs to the technical field of enzyme engineering, and relates to application of novel p-coumaroyl-CoA ligase in phloretin biosynthesis.

Background

Phloretin (Phloretin) with the chemical name of 2,4, 6-trihydroxy-3 (4-hydroxyphenyl) propiophenone is an important dihydrochalcone medicine intermediate. Phlorizin, a glycosylation product of phloretin, is an SGLT2 inhibitor and a natural diabetes drug; the trilobatin which is another glycosylation product improves the diabetes through alpha-glucosidase; the hydroxylation product of phloretin, 3-hydroxy phloretin, is the substance with the highest antioxidant activity in phloretin derivatives, and is the synthetic precursor of 3-hydroxy phlorizin and aspartame.

p-coumaroyl-CoA ligase (4 CL) can catalyze p-dihydrocoumaric acid substrate to generate p-dihydrocoumaroyl-CoA, and then under the action of chalcone synthase (CHS), 1 molecule of p-dihydrocoumaroyl-CoA and 3 molecules of malonyl-CoA are catalyzed to be condensed to generate phloretin. The p-dihydrocoumaroyl-CoA ligase can also catalyze p-coumaric acid substrate to generate p-coumaroyl-CoA, and then catalyze 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA to synthesize naringenin under the action of chalcone synthetase. Unfortunately, phloretin has not been synthesized in E.coli, and since 4CL has been found to be poorly active towards p-dihydrocoumaric acid, p-coumaroyl-CoA ligase is the rate-limiting step in the phloretin biosynthesis process.

The azotobacter (Aromatium aromaticum) and Rhodococcus jositii RHA1 strains can use p-dihydrocoumaric acid as the only carbon source, and 4CL (Aa4CL) in azotobacter and 4CL (Rj4CL) in Rhodococcus are supposed to participate in the degradation pathway of p-dihydrocoumaric acid. Rj4CL has been demonstrated in vitro to use p-coumaric acid as a substrate. However, no report is available on the synthesis of phloretin.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides the application of p-coumaroyl-CoA ligase in biosynthesis of phloretin, and solves the problem that 4CL is low in activity on p-dihydrocoumaric acid in the phloretin synthesis process in the prior art.

The technical scheme of the invention is as follows: an application of p-coumaroyl-CoA ligase in biosynthesis of phloretin, wherein the amino acid sequence of the p-coumaroyl-CoA ligase is shown as SEQ ID No.1 or SEQ ID No. 2.

In one embodiment of the invention, the p-coumaroyl-CoA ligase is Aa4CL derived from azotobacter, the amino acid sequence of which is shown in SEQ ID No. 1;

in one embodiment of the invention, the p-coumaroyl-CoA ligase is Rj4CL from rhodococcus and has the amino acid sequence shown in SEQ ID No. 2;

the invention has the advantages that:

the invention has the beneficial effects that firstly, the invention discovers that Aa4CL derived from azotobacter and Rj4CL derived from rhodococcus can carry out esterification reaction by taking p-dihydrocoumaric acid as a substrate, and then, phloretin is synthesized under the action of chalcone synthetase, and the catalytic capability of phloretin on p-dihydrocoumaric acid can not be achieved by the existing 4 CL. Secondly, the substrates of Aa4CL and Rj4CL have good selectivity, and the synthesis capacity of phloretin is higher and the synthesis capacity of naringenin byproducts is lower. Thirdly, the invention realizes the synthesis of the phloretin in the escherichia coli and lays a foundation for the industrial production of the phloretin product.

According to the invention, through experimental screening of Aa4CL and Rj4CL, the p-dihydrocoumaric acid is found to be used as a substrate for CoA esterification reaction, and the CoA esterification reaction is catalyzed by CHS to generate phloretin, and the substrate selectivity is good. The invention discloses the application of Aa4CL and Rj4CL in phloretin biosynthesis for the first time, and expands the application range of Aa4CL and Rj4 CL.

Drawings

FIG. 1 shows the liquid chromatogram of the fermentation of strain BPL1-4 (with p-dihydrocoumaric acid added); wherein 1-phloretin standard; 2-BPL 3; 3-BPL 1; 4-BPL 2; 5-BPL 4.

Detailed Description

Coli strains e.coli BL21(DE3) and e.coli DH5 α referred to in the examples below were purchased from tokyo holojin biotechnology limited.

The media formulations in the following examples:

LB liquid medium: 10g/L NaCl, 10g/L peptone and 5g/L yeast powder, the balance being water, sterilizing at 121 ℃ under 0.1MPa for 20 min. Solid culture medium can be prepared by adding 1.5g/100mL agar powder.

M9 liquid medium: anhydrous Na2HPO46.78g/L, KH2PO 43 g/L, NH4Cl 1g/L, NaCl 0.5g/L, yeast powder 1g/L, and water in balance, and sterilizing at 121 deg.C under 0.1MPa for 20 min. After sterilization, sterilized MgSO40.24g/L, CaCl211.1mg/L and glucose 5g/L are added. The antibiotic concentrations are 100 mug/L ampicillin respectively; kanamycin sulfate 50. mu.g/L. The mother liquor 1M MgSO4, 1M CaCl2 and 20% glucose were autoclaved at 115 ℃ for 30 min.

The detection method involved in the examples:

after fermentation, 5mL of fermentation liquid is taken to be placed in a 15mL centrifuge tube, ethyl acetate with the same volume is added, the mixture is shaken for 30min on a MIX-2500 mini mixer, the mixture is centrifuged for 10min at 8000rpm, supernatant is taken to be subjected to rotary evaporation, and extraction and rotary evaporation are repeated once. Dissolving with 1mL of anhydrous ethanol, filtering with 0.22 μm organic microporous membrane, and detecting by HPLC. Detection conditions 4.6X 250mm C18 chromatographic column; the mobile phase composition is 45 percent of methanol, 55 percent of water and 0.1 percent of formic acid, and the flow rate is 1 mL/min; an ultraviolet detector for detecting the wavelength of 290 nm; the sample volume is 10 mu L; the column temperature was 25 ℃.

The present invention will be further described with reference to the following examples.

Example 1 construction of expression vector and recombinant Strain for p-Coumaroyl-CoA ligase

According to the amino acid sequence of HaCHS (AF315345) of Hypericum (Hypericum anhydrosum), the preference of escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length HaCHS gene derived from Hypericum is designed, the HaCHS gene is connected between NdeI/XhoI enzyme cutting sites of pETDuet-1 plasmid, and the vector plasmid pPL1 is obtained through full-artificial synthesis.

According to the amino acid sequence of Aa4CL (CAI09146.1) of azotobacter, the preference of escherichia coli to codon is combined, common enzyme cutting sites are removed, a full-length Aa4cl gene is designed, the nucleotide sequence of the gene is shown as SEQ ID No.3, the Aa4cl gene is connected between EcoRI/HindIII enzyme cutting sites of pRSFDuet-1 plasmid, and the vector plasmid is completely synthesized and named as pPA 1.

According to the amino acid sequence of Rj4CL (WP _011597362.1) of rhodococcus, the preference of escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length Rj4cl gene is designed, the nucleotide sequence of the full-length Rj4cl gene is shown in SEQ ID No.4, the Rj4cl gene is connected between EcoRI/HindIII enzyme cutting sites of pRSFDuet-1 plasmid, and the vector plasmid is obtained through total artificial synthesis and is named as pPA 2.

According to the amino acid sequence of Pa4CL (AJT43268.1) of moss (Plagiophaga apendiculum), the preference of Escherichia coli to codons is combined, common enzyme cutting sites are removed, a Pa4cl gene derived from full-length azotobacter is designed, the nucleotide sequence of the gene is shown as SEQ ID No.5, the Pa4cl gene is connected between EcoRI/HindIII enzyme cutting sites of pRSFDuet-1 plasmid, and the obtained vector plasmid is named as pPA3 after full-artificial synthesis.

According to the amino acid sequence of At4CL (U18675) of Arabidopsis thaliana (Arabidopsis thaliana), the preference of Escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length At4cl gene is designed, the nucleotide sequence of the full-length At4cl gene is shown as SEQ ID No.6, the At4cl gene is connected between EcoRI/HindIII enzyme cutting sites of pRSFDuet-1 plasmid, and the vector plasmid is obtained through total artificial synthesis and is named as pPA 4.

Plasmids pPL1 and pPA1 were transformed into E.coli BL21(DE3) competent by simultaneous electric shock, cultured overnight on LB solid medium containing ampicillin and kanamycin double antibody, and positive clones were selected, designated strain BPL1, and stored for use.

Plasmids pPL1 and pPA2 were transformed into E.coli BL21(DE3) competent by simultaneous electric shock, cultured overnight on LB solid medium containing ampicillin and kanamycin double antibody, and positive clones were selected, designated strain BPL2, and stored for use.

Plasmids pPL1 and pPA3 were transformed into E.coli BL21(DE3) competent by simultaneous electric shock, cultured overnight on LB solid medium containing ampicillin and kanamycin double antibody, and positive clones were selected, designated strain BPL3, and stored for use.

Plasmids pPL1 and pPA4 were transformed into E.coli BL21(DE3) competent by simultaneous electric shock, cultured overnight on LB solid medium containing ampicillin and kanamycin double antibody, and positive clones were selected, designated strain BPL4, and stored for use.

Example 2 biosynthesis of phloretin by recombinant strains

The recombinant strain BPL1-4 is respectively inoculated in an LB liquid culture medium according to the inoculation proportion of 1 percent, ampicillin and kana antibiotic are added, the culture is carried out at 37 ℃ and 220rpm with shaking for 12h, then the recombinant strain BPL1-4 is transferred to an M9 fermentation culture medium containing the antibiotic, 0.1M IPTG is added, 300mg/L p-dihydrocoumaric acid is used as a substrate, 30 ℃ and 220rpm are carried out with shaking for 48h, and then the production amount of phloretin and the consumption amount of p-dihydrocoumaric acid are detected.

By HPLC detection, strains BPL3 and BPL4 showed a small background consumption of p-dihydrocoumaric acid, and phloretin was not detected. Strains BPL1 and BPL2 consumed 220mg/L and 291mg/L p-dihydrocoumaric acid, respectively, yielding 0.50mg/L and 0.15mg/L phloretin. Therefore, Aa4CL and Rj4CL can synthesize phloretin using p-dihydrocoumaric acid as a substrate, while At4CL and Pa4CL cannot catalyze the synthesis of phloretin (see table 1 for details).

And (4) conclusion: aa4CL and Rj4CL have p-dihydrocoumaric acid substrate catalytic capability, are greatly superior to At4CL and Pa4CL, and are suitable for phloretin biosynthesis.

TABLE 1 fermentation Synthesis of phloretin by recombinant strains

Example 3 biosynthesis of naringenin by recombinant strains

Respectively inoculating the recombinant strain BPL1-4 in an LB liquid culture medium according to the inoculation proportion of 1%, adding ampicillin and kanamycin antibiotic, culturing at 37 ℃ and 220rpm under shaking for 12h, then transferring the recombinant strain BPL1-4 to an M9 fermentation culture medium containing the antibiotic, 0.1M IPTG, adding 300mg/L p-coumaric acid as a substrate, fermenting at 30 ℃ and 220rpm under shaking for 48h, and then detecting the generation amount of naringenin and the consumption amount of p-coumaric acid.

Through HPLC detection, the strain BPL3 consumes 26mg/L of p-coumaric acid, and synthesizes 16mg/L of naringenin. The strains BPL1 and BPL2 respectively consume 17mg/L and 0.5mg/L of p-coumaric acid, and synthesize 8.7mg/L and 1.8mg/L of naringenin (see Table 2 for details). Therefore, Aa4CL and Rj4CL can synthesize naringenin with p-coumaric acid as a substrate, but the effect is not as good as At4 CL.

TABLE 2 fermentation synthesis of naringenin by recombinant strains

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Sequence listing

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Asp Pro Ala Ala Leu Glu Val Met Phe Ala Ala Gly Leu Leu Gly Ala

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Asp Leu Ser Ser Leu Arg Ala Ile Cys Val Ala Gly Ala Pro Leu Pro

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Ala Tyr Gly Leu Thr Glu Thr Ala Pro Ser Val Thr Met Leu Ser Ser

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gttctggata ccatcgagaa agaacgtatt accaccttct ttgcggtgcc gaccatgatc 780

caggaactgt ggcatcaccc gcgttttgcg gatgcggatc tgagcagcct gcgtgcgatt 840

tgcgttgcgg gtgcgccgct gccggaagcg ctgatcagcc cgtggcagga ccgtgatgtg 900

gcgattaccc aagcgtacgg tctgaccgag accgcgccga gcgtgaccat gctgagcagc 960

gcggacgttc gtaccaagat tggtagcgcg ggcaaacgta ccttctttac cgacgttgat 1020

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ggcccgaacg ttatgctggg ttacctgaac caaccggaag cgaccgcgcg taccattgtg 1140

gacggttggc tgcacaccgg tgatgcgggc tatttcgacg atgaaggttt cctgtttatc 1200

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gaaaaatggg gtgagaccgg catggcgttt gtggttgcgg cggacggcac caccctggat 1380

gaggaaaccg ttcgtgcgcg tctgcgtgaa aagctggcgg gtttcaaaat cccgaccttt 1440

attcaaattg cggaggcgct gccgcgtacc gcgaccggca agattcgtaa accggatctg 1500

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<210>5

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<212>DNA

<213> Artificial sequence ()

<400>5

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ggtgaccgtg ttgctctgat agacgctgtt accggtgcta aactgaccta ctctgaactg 180

cgttctgcta tcgactctgt tgctgctggt ctggctcagt ctggtgttaa acagggtgac 240

gttgttatga tatgcatgcc gaactctatc cactggccga tactgctgtt cggttctctg 300

cgtatcggtg ctgttgttac caccgctaac ccggttggta acgttcagga aatcggtcgt 360

caggctaaag actctcgtac cgcttacctg ataaccgttc cggaactgtg cggtaaactg 420

gcttctctga acatcccgct ggttctgaac gacatggact ctctgggtaa caaaaaacag 480

ttccacggtg ctgctttcgc tcgtttctct gaactgctga aagctgaccg taaaaaagtt 540

ccgcaggttc gtatcaaaga aaaagacacc gctaccctgc tgtactcttc tggtactacc 600

ggtgcttcta aaggtgttat cctgacccac ctgaacctgg ttgaatctat caaccagcgt 660

atggttgctg acccgcgttc tttccaggtt gctgaagaag ttcagatata cggtgttatc 720

atcccgctgt tccacgttat gggtatgatg gctatctgca tgccgaccct gcgtaaaggt 780

gaccagatgg ttgttttccc gaaattcgac ctggctgaaa tgctgggtgc ggttcagagg 840

ttccgtatca ccggcctggc tctggttccg ccgatactgg ttctgctgtc taaatctccg 900

ctggttgaaa aatacgacct gtcttctctg caactgatag gtttcggtgc tgctccggct 960

ggtgacctgt ctggtgtttc taaacgtttc cgtggtgttt acctgcgtca gggttacggt 1020

atgaccgaaa ccgcttcttc tggtactggt gttccgctgg aagacgttga ctacgctaac 1080

aaacacaact cttgcggtct gctggttccg aacatgcagg ctaaagttgt tgacgttctg 1140

accggtaaac cgctgccgcc gggtaaagaa ggtgaactgt ggctgcgtgg tctgaacgtt 1200

atgaaaggtt acctgaacaa cgaaaaagct accgctgaaa ccctggacaa agacggttgg 1260

ctgcacaccg gtgacctggc taaaatcgac gagcgtggct tcgtatacat cgtggaccgt 1320

ctgaaagaac tgataaaata caaaggtttc caggttgctc cggctgaact ggaatctatc 1380

ctgctgtctc actctgacat catggacgct gctgttgttc cgttcccgga cgaagaagct 1440

ggtgaaatcc cggttgcttt cgttgttcgt cgtccgggta ctaccctgac cggtaactct 1500

atcatggaat ttgttgcttc taaagtttct ccgtacaaaa aaatccgtcg tgttatcttc 1560

gttgacacca tcccgaaact ggaatctggt aaaatcctgc gtaaagaact gaaatctaaa 1620

ctgctgccgt cttctaaact gtaa 1644

<210>6

<211>1686

<212>DNA

<213> Artificial sequence ()

<400>6

atggcgccac aagaacaagc agtttctcag gtgatggaga aacagagcaa caacaacaac 60

agtgacgtca ttttccgatc aaagttactg gatatttaca tcccgaacca cctatctctc 120

cacgactaca tcttccaaaa catctccgaa ttcgccacta agccttgcct aatcaacgga 180

ccaaccggcc acgtgtacac ttactccgac gtccacgtca tctcccgcca aatcgccgcc 240

aattttcaca aactcggcgt taaccaaaac gacgtcgtca tgctcctcct cccaaactgt 300

cccgaattcg tcctctcttt cctcgccgcc tccttccgcg gcgcaaccgc caccgccgca 360

aaccctttct tcactccggc ggagatagct aaacaagcca aagcctccaa caccaaactc 420

ataatcaccg aagctcgtta cgtcgacaaa atcaaaccac ttcaaaacga cgacggagta 480

gtcatcgtct gcatcgacga caacgaatcc gtgccaatcc ctgaaggctg cctccgcttc 540

accgagttga ctcagtcgac aaccgaggca tcagaagtca tcgactcggt ggagatttca 600

ccggacgacg tggtggcact accttactcc tctggcacga cgggattacc aaaaggagtg 660

atgctgactc acaagggact agtcacgagc gttgctcagc aagtcgacgg cgagaacccg 720

aatctttatt tccacagcga tgacgtcata ctctgtgttt tgcccatgtt tcatatctac 780

gctttgaact cgatcatgtt gtgtggtctt agagttggtg cggcgattct gataatgccg 840

aagtttgaga tcaatctgct attggagctg atccagaggt gtaaagtgac ggtggctccg 900

atggttccgc cgattgtgtt ggccattgcg aagtcttcgg agacggagaa gtatgatttg 960

agctcgataa gagtggtgaa atctggtgct gctcctcttg gtaaagaact tgaagatgcc 1020

gttaatgcca agtttcctaa tgccaaactc ggtcagggat acggaatgac ggaagcaggt 1080

ccagtgctag caatgtcgtt aggttttgca aaggaacctt ttccggttaa gtcaggagct 1140

tgtggtactg ttgtaagaaa tgctgagatg aaaatagttg atccagacac cggagattct 1200

ctttcgagga atcaacccgg tgagatttgt attcgtggtc accagatcat gaaaggttac 1260

ctcaacaatc cggcagctac agcagagacc attgataaag acggttggct tcatactgga 1320

gatattggat tgatcgatga cgatgacgag cttttcatcg ttgatcgatt gaaagaactt 1380

atcaagtata aaggttttca ggtagctccg gctgagctag aggctttgct catcggtcat 1440

cctgacatta ctgatgttgc tgttgtcgca atgaaagaag aagcagctgg tgaagttcct 1500

gttgcatttg tggtgaaatc gaaggattcg gagttatcag aagatgatgt gaagcaattc 1560

gtgtcgaaac aggttgtgtt ttacaagaga atcaacaaag tgttcttcac tgaatccatt 1620

cctaaagctc catcagggaa gatattgagg aaagatctga ggacaaaact agcaaatgga 1680

ttgtaa 1686

Claims

1. An application of p-coumaroyl-CoA ligase in biosynthesis of phloretin is characterized in that the amino acid sequence of the p-coumaroyl-CoA ligase is shown as SEQ ID No.1 or SEQ ID No. 2.

2. The use of p-coumaroyl-CoA ligase of claim 1 for the biosynthesis of phloretin, wherein the amino acid sequence of SEQ ID No.1 is Aa4CL from azotobacter.

3. Use of p-coumaroyl-CoA ligase according to claim 1 for the biosynthesis of phloretin, wherein the amino acid sequence of SEQ ID No.2 is Rj4CL from rhodococcus.