CN111748532B - Application of novel p-coumaroyl-CoA ligase in biosynthesis of phloretin - Google Patents

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 a new p-coumaroyl-CoA ligase in the biosynthesis of phloretin

technical field

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

Background technique

Phloretin, whose chemical name is 2,4,6-trihydroxy-3(4-hydroxyphenyl)propiophenone, is an important dihydrochalcone pharmaceutical intermediate. Phloretin, a glycosylation product of phloretin, is an SGLT2 inhibitor, a natural diabetes drug; another glycosylation product, trilobulin, improves diabetes through α-glucosidase; the hydroxylation product of phloretin is 3-hydroxyradical It is the substance with the highest antioxidant activity among the phloretin derivatives, and is the synthetic precursor of 3-hydroxyphloridzin and aspatine.

p-coumaroyl-CoA ligase (p-coumaroyl-CoA ligase, 4CL) can catalyze p-dihydrocoumaric acid substrate to generate p-dihydrocoumaroyl-CoA, and then in chalcone synthase (chalcone synthase (chalcone) Under the action of synthase, CHS), it catalyzes the condensation of one molecule of p-dihydrocoumaroyl-CoA and three molecules of malonyl-CoA (malony-CoA) to form phloretin. 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 under the action of chalcone synthase and 3 molecules of malonyl-CoA to synthesize naringenin. Unfortunately, phloretin has not been synthesized in Escherichia coli at present, because the activity of 4CL for p-dihydrocoumaric acid has not been found to be high, so p-coumaroyl-CoA ligase becomes phloretin Rate-limiting step in biosynthesis.

Both Aromatoleum aromaticum and Rhodococcus jostii RHA1 can use p-dihydrocoumaric acid as the sole carbon source, while 4CL in Aromatoleum (Aa4CL) and 4CL in Rhodococcus (Rj4CL) were speculated Involved in the degradation pathway of p-dihydrocoumaric acid. Rj4CL has been shown to use p-coumaric acid as a substrate in vitro. But there is no report that it can synthesize phloretin.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies in the prior art, and provides a kind of application of p-coumaroyl-CoA ligase in the biosynthesis of phloretin, which solves the problem that 4CL in the phloretin synthesis process in the prior art is for p-diol. The problem of low activity of hydrocoumaric acid.

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

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

In one embodiment of the present invention, the p-coumaroyl-CoA ligase is Rj4CL derived from Rhodococcus, and its amino acid sequence is shown in SEQ ID No.2;

Advantages of the present invention:

The beneficial effects of the present invention are that, firstly, the present invention finds that Aa4CL derived from nitrogen-fixing bacteria and Rj4CL derived from Rhodococcus can undergo esterification reaction with p-dihydrocoumaric acid as a substrate, and then in chalcone synthase Under the action of phloretin, its catalytic ability to p-dihydrocoumaric acid cannot be achieved by existing 4CL. Second, Aa4CL and Rj4CL have good substrate selectivity, higher phloretin synthesis capacity and lower naringenin by-product synthesis capacity. Third, the invention realizes the synthesis of phloretin in Escherichia coli, and lays a foundation for the industrialized production of phloretin products.

In the present invention, Aa4CL and Rj4CL are screened experimentally, and it is found that they can use p-dihydrocoumaric acid as a substrate to carry out CoA esterification reaction, and are catalyzed by CHS to generate phloretin, and the substrate selectivity is good. The invention discloses the application of Aa4CL and Rj4CL in the biosynthesis of phloretin for the first time, and expands the application scope of Aa4CL and Rj4CL.

Description of drawings

Figure 1. Liquid chromatogram of strain BPL1-4 fermentation (adding p-dihydrocoumaric acid); 1-phloretin standard; 2-BPL3; 3-BPL1; 4-BPL2; 5-BPL4.

Detailed ways

The E. coli strains E.coli BL21 (DE3) and E.coli DH5α involved in the following examples were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

Medium formulation in the following examples:

LB liquid medium: 10g/L NaCl, 10g/L peptone and 5g/L yeast powder, the balance is water, sterilized at 0.1Mpa pressure at 121°C for 20min. Add 1.5g/100mL agar powder to prepare solid medium.

M9 liquid medium: anhydrous Na2HPO4 6.78g/L, KH2PO4 3g/L, NH4Cl 1g/L, NaCl 0.5g/L, yeast powder 1g/L, the balance is water, sterilized under 0.1Mpa pressure at 121 ℃ for 20min. After sterilization, add sterilized MgSO4 0.24g/L, CaCl2 11.1mg/L, and glucose 5g/L. The antibiotic concentrations were, respectively, ampicillin 100 μg/L; kanamycin sulfate 50 μg/L. The mother liquor 1M MgSO4, 1M CaCl2 and 20% glucose should be autoclaved at 115°C for 30min.

The detection method involved in the embodiment:

After fermentation, take 5mL of fermentation broth into a 15mL centrifuge tube, add an equal volume of ethyl acetate, shake on a MIX-2500 mini mixer for 30min, centrifuge at 8000rpm for 10min, take the supernatant for rotary evaporation, and repeat the extraction and rotary evaporation once. It was dissolved in 1 mL of absolute ethanol, filtered through a 0.22 μm organic microporous membrane, and then detected by HPLC. Detection conditions: 4.6×250mm C18 chromatographic column; mobile phase composition: 45% methanol-55% water-0.1% formic acid, flow rate 1mL/min; UV detector, detection wavelength 290nm; injection volume 10μL; column temperature 25℃.

The present invention will be further described below in conjunction with specific embodiments.

Example 1 Construction of expression vector and recombinant strain of p-coumaroyl-CoA ligase

According to the amino acid sequence of HaCHS (AF315345) of Hypericum (Hypericum androsaemum), combined with the codon preference of Escherichia coli, and removed the commonly used restriction sites, the Hachs gene derived from the full-length hypericum was designed, and the Hachs gene was connected. Between the NdeI/XhoI restriction sites of the pETDuet-1 plasmid, it was synthetically synthesized to obtain the vector plasmid pPL1.

According to the amino acid sequence of Aa4CL (CAI09146.1) of nitrogen-fixing bacteria, combined with the codon preference of Escherichia coli, and removed the commonly used restriction sites, the full-length Aa4cl gene was designed, and its nucleotide sequence is shown in SEQ ID No.3 As shown, the Aa4cl gene was connected between the EcoRI/HindIII restriction sites of the pRSFDuet-1 plasmid, and it was synthetically synthesized, and the obtained vector plasmid was named pPA1.

According to the amino acid sequence of Rj4CL of Rhodococcus (WP_011597362.1), combined with the codon preference of Escherichia coli, and removed the commonly used restriction sites, the full-length Rj4cl gene was designed, and its nucleotide sequence is shown in SEQ ID No.4 As shown, the Rj4cl gene was connected between the EcoRI/HindIII restriction sites of the pRSFDuet-1 plasmid, and the resulting vector plasmid was named pPA2.

According to the amino acid sequence of Pa4CL (AJT43268.1) of moss (Plagiochasma appendiculatum), combined with the codon preference of Escherichia coli and the removal of commonly used restriction sites, a full-length Azotobacter-derived Pa4cl gene was designed. Its nucleotide sequence As shown in SEQ ID No. 5, the Pa4cl gene was ligated between the EcoRI/HindIII restriction sites of the pRSFDuet-1 plasmid, and the resulting vector plasmid was named pPA3.

According to the amino acid sequence of At4CL (U18675) of Arabidopsis thaliana, combined with the codon preference of Escherichia coli, and removed the commonly used restriction sites, the full-length At4cl gene was designed, and its nucleotide sequence is as SEQID No. As shown in 6, the At4cl gene was connected between the EcoRI/HindIII restriction sites of the pRSFDuet-1 plasmid, and the whole was synthesized artificially, and the obtained vector plasmid was named pPA4.

Plasmids pPL1 and pPA1 were simultaneously electroporated into E. coli BL21 (DE3) competent cells, and the competent cells were cultured overnight on LB solid medium containing ampicillin and kanadiab. Positive clones were screened and named as strain BPL1. Save for backup.

The plasmids pPL1 and pPA2 were simultaneously electroporated into E. coli BL21 (DE3) competent cells, and the competent cells were cultured overnight on LB solid medium containing ampicillin and kanadiab, and positive clones were screened and named as strain BPL2. Save for backup.

Plasmids pPL1 and pPA3 were simultaneously electroporated into E. coli BL21 (DE3) competent cells, and the competent cells were cultured overnight on LB solid medium containing ampicillin and kanadiab, and positive clones were screened and named as strain BPL3. Save for backup.

Plasmids pPL1 and pPA4 were simultaneously electroporated into E. coli BL21 (DE3) competent cells, and the competent cells were cultured overnight on LB solid medium containing ampicillin and kanadiab, and positive clones were screened and named as strain BPL4. Save for backup.

Example 2 Biosynthesis of phloretin by recombinant strains

The recombinant strains BPL1-4 were inoculated in LB liquid medium at a 1% inoculation ratio, with ampicillin and kana antibiotics added, 37 ° C, 220 rpm, shaking culture for 12 hours, and then transferred to M9 fermentation medium containing antibiotics, 0.1 M IPTG, adding 300mg/L p-dihydrocoumaric acid as a substrate, 30 ℃, 220rpm, shaking and fermenting for 48h, the production of phloretin and the consumption of p-dihydrocoumaric acid were detected.

After HPLC detection, strains BPL3 and BPL4 showed a small amount of background consumption of p-dihydrocoumaric acid, and no phloretin was detected. Strain BPL1 and BPL2 consumed 220 mg/L and 291 mg/L of p-dihydrocoumaric acid, respectively, producing 0.50 mg/L and 0.15 mg/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).

Conclusion: Aa4CL and Rj4CL have the catalytic ability of p-dihydrocoumaric acid substrate, which is much better than that of At4CL and Pa4CL, and are suitable for the biosynthesis of phloretin.

Table 1 Recombinant strains fermented and synthesized phloretin

Example 3 Biosynthesis of naringenin by recombinant strains

The recombinant strains BPL1-4 were inoculated in LB liquid medium at a 1% inoculation ratio, with ampicillin and kana antibiotics added, 37 ° C, 220 rpm, shaking culture for 12 hours, and then transferred to M9 fermentation medium containing antibiotics, 0.1 M IPTG, adding 300 mg/L p-coumaric acid as a substrate, 30 ℃, 220 rpm, shake flask fermentation for 48 h to detect the production of naringenin and the consumption of p-coumaric acid.

After HPLC detection, strain BPL3 consumed 26 mg/L of p-coumaric acid and synthesized 16 mg/L of naringenin. Strains BPL1 and BPL2 consumed 17 mg/L and 0.5 mg/L of p-coumaric acid, respectively, and synthesized 8.7 mg/L and 1.8 mg/L of naringenin (see Table 2 for details). Therefore, Aa4CL and Rj4CL can synthesize naringenin using p-coumaric acid as a substrate, but the effect is not as good as that of At4CL.

Table 2 Recombinant strains fermented and synthesized naringenin

The present invention has been exemplarily described above. It should be noted that, without departing from the core of the present invention, any simple deformations, modifications or other equivalent replacements that those skilled in the art can do without creative effort fall into the scope of the present invention. the scope of protection of the invention.

sequence listing

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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)

1. the application of a p-coumaroyl-CoA ligase in biosynthesis of phloretin, is characterized in that, the aminoacid sequence of described p-coumaroyl-CoA ligase is such as SEQ ID No.1 or SEQ ID No.2 shown.

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