CN107365368B - Asperphenamate synthesis related protein, coding gene and application thereof - Google Patents

Asperphenamate synthesis related protein, coding gene and application thereof Download PDF

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CN107365368B
CN107365368B CN201610312406.2A CN201610312406A CN107365368B CN 107365368 B CN107365368 B CN 107365368B CN 201610312406 A CN201610312406 A CN 201610312406A CN 107365368 B CN107365368 B CN 107365368B
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asperphenamate
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尹文兵
范爱丽
李伟
王龙
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Institute of Microbiology of CAS
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Abstract

The invention provides an asperphenamate synthesis-related protein, which comprises the following components: (1) the amino acid sequence is shown in a sequence table SEQ ID NO: 1 and SEQ ID NO: 2; (2) a protein which is derived from the protein in the sequence (1) and is related to asperphenamate synthesis after the amino acid residue sequence in the sequence is substituted and/or deleted and/or added by one or more amino acid residues; the protein can catalyze phenylalanine and benzoic acid to form N-Benzoylphenylalaninol, and can also accept linear dipeptide as a substrate and catalyze esterification reaction to release a final product. Also provides a coding gene of the protein and a method for constructing gene engineering bacteria for producing asperphinamate, and a method for obtaining a novel amino acid ester compound by feeding precursor substrates such as single amino acid or linear dipeptide and the like by utilizing the constructed gene engineering bacteria. The protein related to the biosynthesis of asperphenamate and the coding gene thereof provided by the invention open up a new way for biosynthesis of asperphenamate, improve the synthesis rate and are helpful for understanding the biosynthesis mechanism of natural products of amino acid ester compounds.

Description

Asperphenamate synthesis related protein, coding gene and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to asperphenamate synthesis related protein, and a coding gene and application thereof.
Background
The amino acid ester compounds and the derivatives thereof have special physicochemical properties and good biological activity, so that the amino acid ester compounds and the derivatives thereof have wide application prospects, and particularly can be used as medicines or medicine intermediates for treating different diseases in the field of medicines. Such as: valacyclovir (valaciclovir) is an amino acid ester drug formed by combining acyclovir (aciclovir) and L-serine, and can overcome the defect of poor oral absorption of acyclovir and better play an antiviral role; benzyl L-alanine is an important intermediate for producing an Angiotensin Converting Enzyme (ACE) inhibitor quinapril hydrochloride (quinapril); the N-maleic-L-valine ester curcumin serving as a curcumin prodrug has the advantages of good water solubility, high bioavailability and the like, and can effectively inhibit the proliferation of human bladder cancer and gastric cancer cells.
Aspephenomate is a fungal secondary metabolite of amino acid esters (fig. 1), which can be produced by a variety of aspergillus niger fungi and shows good antitumor activity, such as: has good inhibitory effect on human lung cancer cell A549, human breast cancer cell MCF-7, estrogen receptor tumor cell T47D and MDA-MB231, human leukemia cell HL60, human cervical cancer cell Hela and human gastric cancer cell SGC-7901, especially on estrogen receptor tumor cell T47D and MDA-MB231, half of themEffective concentration (IC)50) 8.9 and 11.2. mu. mol/L were achieved. The water solubility and the biological activity of the derivative IM23b of asperphyrate are further improved, particularly the derivative has the strongest inhibition effect on human breast cancer cells MCF-7, and the research result shows that asperphyrate has good medicinal development potential and application value. The fully artificial chemical synthesis of Asperphenamate has been reported, but the efficiency of artificial synthesis is low, and the chemical synthesis indicates that phenylalanine, benzoyl substituted phenylalanine and phenylalaninol are precursors for the biosynthesis of Asperphenamate, but the biosynthesis mechanism, particularly the action mechanism of related esterases, is not clear.
In view of the above, there is an urgent need in the art to study genes and biosynthetic mechanisms related to the synthesis of asperphyramate, in order to improve the discovery, modification and production of asperphyramate and related amino acid ester compounds thereof.
Disclosure of Invention
The invention provides an asperphenamate synthesis related protein, a coding gene and application thereof, and opens up a new path for biologically synthesizing asperphenamate.
In one aspect of the present invention, there is provided a protein for synthesizing asperphenamate, wherein the protein comprises the following (1) or (2):
(1) the amino acid sequence is shown in a sequence table SEQ ID NO: 1 and SEQ ID NO: 2;
(2) and (2) the protein which is derived from the protein (1) and is related to asperphenamate synthesis by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequence in the sequence.
The protein comprises an amino acid sequence shown in a sequence table SEQ ID NO: 5 and SEQ ID NO: 6, and the amino acid sequence of the protein is shown as the sequence table SEQ ID NO: 9 and SEQ ID NO: 10.
In another aspect of the present invention, there is provided a gene encoding a protein that synthesizes asperphenamate, wherein the gene comprises any one of the following genes (1) to (3):
(1) the nucleotide sequence is shown in a sequence table SEQ ID NO: 3 and SEQ ID NO: 4;
(2) a gene which hybridizes with the DNA molecule shown in (1) under strict conditions and codes the protein;
(3) a gene which has 90% or more homology with the gene of (1) or (2) and encodes the protein.
The coding gene comprises a nucleotide sequence shown in a sequence table SEQ ID NO: 7 and SEQ ID NO: 8, and also comprises a gene with an amino acid sequence shown as a sequence table SEQ ID NO: 11 and SEQ ID NO: 12.
In a further aspect of the invention, there is provided a gene cluster for the synthesis of asperphyramate comprising the coding gene as described above.
The present invention also provides a recombinant vector comprising the coding gene according to claim 3 or 4.
The invention also provides an engineering bacterium for producing asperphamate, wherein the engineering bacterium comprises the coding gene.
The invention also provides a method for preparing the gene engineering bacteria for producing asperphamate, which comprises the following steps:
(1) the cloning sequences are shown in sequence table SEQ ID NO: 3 and SEQ ID NO: 4;
(2) introducing the gene in the step (1) into a vector to obtain a recombinant vector;
(3) and (3) introducing the recombinant vector in the step (2) into a receptor strain, and obtaining the asperphenamate genetic engineering bacteria after screening and verification and precursor feeding.
The method as described above, wherein the sequence in (1) is as shown in sequence table SEQ ID NO: 3 and SEQ ID NO: the gene shown in 4 can be replaced by the gene shown in (2) or (3) in claim 3, or replaced by a gene with a nucleotide sequence shown in a sequence table SEQ ID NO: 7 and SEQ ID NO: 8, and the amino acid sequence can be replaced by the gene shown in the sequence table SEQ ID NO: 11 and SEQ id no: 12.
The method for preparing linear dipeptide compound by feeding amino acid precursor by the genetically engineered bacteria, wherein N-Benzoylphenylalaninol can be generated by feeding Benzoic Acid (BA) and phenylalanine (Phe).
The method for preparing the novel amino acid ester compound by feeding the amino acid precursor to the genetically engineered bacteria, wherein the genetically engineered bacteria can catalyze benzoic acid and phenylalanine substrates to synthesize N-benzolylalinol after feeding Benzoic Acid (BA) and benzyl isoleucine (PIS); after the benzoic acid and the benzyl isoleucine are fed, the gene engineering bacteria can catalyze the benzoic acid and the benzyl isoleucine to synthesize the amino acid ester compound.
The method for preparing the amino acid ester compound by feeding the linear dipeptide precursor by the genetic engineering bacteria comprises the following steps of feeding the linear dipeptide, and then allowing the genetic engineering bacteria to accept the linear dipeptide as a substrate and catalyze an esterification reaction to release a final product to synthesize the amino acid ester compound asperphenamate; the linear dipeptide is preferably N-Benzoylphenylalanine or N-Benzoylphenylalaninol; also preferred are N-acid-S-N-acetylcysteine (SNAC-acid) and N-Benzoylphenylalaninol.
The genetic engineering bacteria are applied to synthesis of an asperphenamate compound or preparation of an amino acid ester compound.
The protein related to aspherenate biosynthesis and the coding gene thereof provided by the invention open up a new path for aspherenate biosynthesis, and compared with the chemical synthesis method in the prior art, the synthesis rate can be improved. In addition, the engineering bacteria containing the asperphyramate biosynthesis related gene prepared by the invention can also produce amino acid ester compounds under the condition of feeding precursor substrates such as amino acid or linear dipeptide and the like, is beneficial to understanding the biosynthesis mechanism of natural products of the amino acid ester compounds, can also be used for modifying, searching or discovering compounds or genes and proteins for medicines, and has a great industrial application prospect.
Drawings
FIG. 1 shows the chemical structure of asperphamate.
FIG. 2 is HPLC analysis of fermentation components of wild type Penicillium brevicompactum and various mutant strains in example 1 of the present invention. A wild strain of Penicillium brevicompactum and a mutant strain in which a gene of interest has been knocked out are cultured in YES solid medium for 48 hours and then subjected to HPLC analysis. Wild type strains produced by asperphamate (FIG. 2-i); apmA is non-ribosomal peptide synthase (NRPS), and after the apmA gene is knocked out, penicillium brevicompactum does not produce aspenaphenamate any more (figure 2-ii); apmB is non-ribosomal polypeptide synthetase (NRPS), and after apmB gene is deleted, the Penicillium brevicompactum can not produce asperaphenamate any more, but can produce the precursor N-Benzoylphenylalaninol of asperaphenamate (FIG. 2-iii); apmC is an aldolase, apmE is an isomerase or dehydrogenase, and the asperphenamate is still produced by Penicillium brevicompactum after deletion of apmC and apmE genes respectively (FIGS. 2-iv and 2-v). Ordinate: AU, abscissa: and (5) min.
FIG. 3 is a gene alignment chart of genes related to the synthesis of aspergillium brevis (P. brevicompactum) aspeperphenate and non-ribosomal polypeptide synthetase (NRPS) related gene clusters of Aspergillus terreus (Aspergillus terreus) and Aspergillus aculeatus (Aspergillus aculeatus).
FIG. 4 shows HPLC analysis of the precursor feeding of the genetically engineered Aspergillus nidulans strain in example 4 of the present invention. And culturing the aspergillus nidulans control bacteria and the overexpression genetic engineering bacteria obtained by transforming the target genes in a GMM culture medium, adding related precursors, and performing HPLC analysis after 6 days of culture.
The genetically engineered bacterium (AN-apmA) fed benzoic acid with a final concentration of 60 μ g/mL and phenylalanine with a final concentration of 60 μ g/mL can generate N-Benzoylphenylalaninol (FIG. 4-Ai and FIG. 4-Aii); no target compound was produced after the control bacteria were fed benzoic acid at a final concentration of 60. mu.g/mL and phenylalanine at a final concentration of 60. mu.g/mL (FIG. 4-Aiii), and no target compound was produced after the control bacteria were fed N-Benzoylphenylalaninol at a final concentration of 30. mu.g/mL (FIG. 4-Aiv). The asperiphenam can be produced by feeding genetically engineered bacteria (AN-apmB) with benzoic acid at a final concentration of 60. mu.g/mL, phenylalanine at a final concentration of 60. mu.g/mL and N-Benzoylphenylalaninol at a final concentration of 30. mu.g/mL (FIG. 4-Bi), and the control bacteria are free of the target compound (FIG. 4-Bii); genetically engineered bacteria (AN-apmB) are fed with N-benzol phenyl alaninol at a final concentration of 30 mu g/mL and N-benzol phenyl alaninol at a final concentration of 30 mu g/mL13C-labeled N-Benzoylphenylalanine can be produced13C-labeled asperphenamate (FIG. 4-Ci), control bacteria were sterile and free of target compound (FIG. 4-Cii). Ordinate: AU, abscissa: and (5) min.
FIG. 5 shows HPLC analysis of the expression of the genetically engineered Aspergillus nidulans strain (AN-apmA + apmB) in example 4 of the present invention. And culturing the aspergillus nidulans control bacteria and the overexpression genetic engineering bacteria obtained by transforming the target genes in a GMM culture medium, adding related precursors, and performing HPLC analysis after 6 days of culture. The genetically engineered bacteria (AN-apmA + apmB) can generate precursor N-benzolyalaninol and final product asperiphenamide after being fed with benzoic acid with a final concentration of 60 mug/mL and phenylalanine with a final concentration of 60 mug/mL (figure 5-i), and no target compound is generated when not being fed (figure 5-ii); the genetically engineered bacterium (AN-apmA) fed benzoic acid at a final concentration of 60. mu.g/mL as a control and phenylalanine at a final concentration of 60. mu.g/mL produced only the precursor N-benzoylalaninol (FIG. 5-iii), the genetically engineered bacterium (AN-apmB) fed benzoic acid at a final concentration of 60. mu.g/mL and phenylalanine at a final concentration of 60. mu.g/mL did not produce the target compound (FIG. 5-iv), and asperiphenate was produced after N-benzoylalaninol at a final concentration of 30. mu.g/mL (FIG. 5-v). Ordinate: AU, abscissa: and (5) min.
FIG. 6 is a schematic representation of the deduced biosynthetic pathway of asperphyramate.
FIG. 7 shows the analysis of the products of the genetically engineered Aspergillus nidulans strain of the present invention in example 4 after feeding N-acid-S-N-acetyl cysteine (SNAC-acid) and N-Benzoylphenylalaninol. The genetically engineered strain A-apmA of Aspergillus nidulans is cultured in GMM culture medium, and N-acid-S-N-acetyl cysteine (SNAC-acid) and N-benzoyl phenyl alaninol are added, and HPLC analysis is performed after culturing for 6 days at 25 ℃. LC-MS analysis ion flow graph (FIG. 7-i), HPLC analysis (FIG. 7-ii, 210nm), 256.1361 molecular weight extraction analysis (FIG. 7-iii). Ordinate: ion current intensity, abscissa: and (5) min.
FIG. 8 is the analysis of the products of Aspergillus nidulans genetically engineered strain (AN-apmA + apmB) of the present invention after feeding Benzoic Acid (BA) and the precursor analog Phenylisoline (PIS). The genetically engineered strain of Aspergillus nidulans (AN-apmA + apmB) was cultured in GMM medium, and phenylisoline was added, and HPLC analysis was performed after 6 days of culture at 25 ℃. LC-MS analysis ion flow diagram (FIG. 8-i), HPLC analysis diagram (FIG. 8-ii, 210nm), 507.2305 molecular weight extraction analysis (FIG. 8-iii), 539.2182 molecular weight extraction analysis (FIG. 8-iv), 523.2250 molecular weight extraction analysis (FIG. 8-v), 256.1361 molecular weight extraction analysis (FIG. 8-vi). Ordinate: ion current intensity, abscissa: and (5) min.
Detailed Description
The following detailed description of the present invention, taken in conjunction with the accompanying drawings and examples, is provided to enable the invention and its various aspects and advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and the Penicillium brevis strain (Penicillium brevicompactum) is commercially available (e.g., China general microbiological culture Collection center (CGMCC) or the like).
Example 1 Gene Cluster Gene knockout and functional verification of asperphyramate biosynthesis in Penicillium brevicompactum
The penicillium brevicompactum can produce asperphenamate in YES culture medium, and candidate gene clusters related to biosynthesis of the penicillium brevicompactum are obtained through genome scanning and bioinformatics analysis. In this example, genes in candidate NRPS gene clusters of penicillium brevicompactum strains were knocked out by resistance marker screening and based on the principle of homologous recombination, apmA, apmB, apmC, and apmE genes were knocked out, and the knocked-out mutants were cultured in YES medium at 25 ℃ for 48 hours, and the conditions of production of asperphamate and its precursor by wild-type and different gene-deleted mutants were detected by HPLC analysis. The results are shown in FIG. 2.
YES medium composition (1L): 20g of yeast extract, 150g of sucrose and 20g of agar. Sequentially adding the above components, adding distilled water to final volume of 1L, sterilizing at 121 deg.C for 20-30 min, and storing at room temperature.
The cultured cells and the culture medium were extracted under the following conditions: mycelia and the culture medium obtained after 48 hours of culture were subjected to compound extraction. 1 plate of the culture was divided into small pieces and placed in a triangular flask, and 20-40mL of MEA (ethyl acetate: methanol: glacial acetic acid: 89:10:1) was added for ultrasonic extraction (ultrasonic frequency 80Hz, time 1 hour); collecting extractive solution, and performing rotary evaporation (temperature 30 deg.C, rotation speed 100rpm, vacuum degree less than 3 mmHg); evaporating to dryness, dissolving with 0.30-0.60mL methanol, diluting with appropriate concentration, injecting 20-40ul, washing column with 40-100% methanol, and performing HPLC analysis.
HPLC analysis was performed under the following conditions: the device comprises the following steps: waters e2695 system detector: waters 2998(200-600 nm); a chromatographic column: 38020-41COSMOSIL 5C18-MS-II Packed Column, 4.6mm I.D.x250 mm; elution speed: 1mL/min, eluent: water and methanol increased the methanol content from 40% methanol to 100% methanol within 20 min.
Example 2 bioinformatic analysis of asperphyrate biosynthetic Gene Cluster genes
Utilizing BLAST program to make homology search and comparison of DNA and amino acid sequence in NCBI GenBank and Local DNA database and protein database, analyzing related gene in asperphenamate biosynthesis gene cluster, and naming obtained related gene cluster for synthesizing asperphenamate as apm, and naming NRPS gene directly related to its synthesis as apmA and apmB, and 3 genes between NRPS are respectively apmC, apmD and apmE. Gene clusters with higher sequence conservation to the genes in the gene cluster were also found, including corresponding gene clusters of aspergillus terreus (a. terreus) and aspergillus aculeatus (a. aculeatus), and the results of annotation and alignment of the ORFs involved in aspeproperanate synthesis on the apm gene cluster are shown in table 1 and fig. 2.
TABLE 1
Figure BDA0000987520710000051
From this, it was concluded that ATEG _09068 gene of Aspergillus terreus (A.terreus) (shown in SEQ ID NO: 7) and ORF10 gene of Aspergillus aculeatus (A.aculeatus) (shown in SEQ ID NO: 11) had the same or similar functions as the apmA gene of NRPS, and ATEG _09064 gene of Aspergillus terreus (A.terreus) (shown in SEQ ID NO: 8) and ORF14 gene of Aspergillus aculeatus (A.aculeatus) (shown in SEQ ID NO: 12) had the same or similar functions as the apmB gene of NRPS.
Example 3 construction and validation of genetically engineered bacteria for asperphamate biosynthesis of Key genes
Cloning apmA gene and apmB gene respectively by using Penicillium brevicompactum genome DNA as a template, and based on pYH-gpdA-pyrG and pYWB2-Ribo constructed pYH-gpdA-apmA, pYH-gpdA-apmB and pYWB2-apmA respectively from pYH-WA-pyrG (Yin W.B.et al.discovery of Crytic polykeyplates from genetic polymorphisms using heterologous expression in Aspergillus nidulans. ACS Synthbiol.2013,2(11):629 634.), aspergillus nidulans (A. nidulans LO8030) was then transformed by protoplast transformation, transformants were selected based on the auxotrophic markers pyrG and Riboflavin on plasmids pYH-gpdA-pyrG and pYWB2-Ribo, respectively, and carrying out molecular verification on the transformant to obtain three Aspergillus nidulans genetically engineered bacteria AN-apmA, AN-apmB and AN-apmA + apmB which respectively contain apmA or apmB genes and apmA and apmB genes.
The vector construction method used in the invention is as follows: (1) plasmid pYH-WA-pyrG WAs modified by the Quick-Change method (Nancy P.K. and Geoffrey T. (eds.), Fungal Secondary diagnosis Methods: Methods and Protocols, Methods in Molecular Biology,2012,944:163-174), and plasmid pYH-gpdA-pyrG having gpdA promoter and auxotrophic marker gene pyrG and plasmid pYWB2-Ribo having auxotrophic marker gene Riboflavin were obtained on the basis of PCR amplification of DNA fragments of Aspergillus nidulans gpdA promoter sequence (720bp) and Aspergillus fumigatus auxotrophic marker gene Riboflavin expression cassette (2300bp), respectively. (2) Using Penicillium notatum genome DNA as a template, cloning apmA gene (7094bp, shown as SEQ ID NO: 3) and apmB gene (7121bp, shown as SEQ ID NO: 4) by using Q5 high fidelity polymerase of NEB company, respectively, and constructing apmA gene and apmB gene on pYH-gpdA-apmA and pYH-gpdA-apmB by using yeast assembly technology (Yin W.B.et al.discovery of Crytic polygenetic plasmids from deletion expression in Aspergillus nidulans. ACS Synth biol.2013,2(11):629 and 634.) respectively; the apmA gene is constructed on pYWB2-Ribo to obtain an expression vector pYWB2-apmA which is used for transformation and construction of Aspergillus nidulans genetic engineering bacteria.
The transformation and screening method of the aspergillus nidulans genetic engineering bacteria used by the invention is as follows: (1) germinating Aspergillus nidulans spores in LMM medium at 37 deg.C for 4-6 hr, collecting germinated spores, and culturing in hypertonic medium (containing 1.2M MgSO containing lyase such as lysine enzyme and 2mg/mL Yatalase) to obtain final concentration4Buffer, ph5.8) for 3-5 hours, and collecting the protoplasts for transformation. (2) Mu.g of plasmid was added to 100. mu.L of a hypertonic buffer containing protoplasts (containing 1.2M sorbitol and 10mM CaCl)2) After 50 min incubation on ice, 1.25mL 60% PEG buffer was added, mixed well and incubated at room temperature for 20min, 5mL hypertonic buffer (containing 1.2M sorbitol and 10mM CaCl) was added2) Mixing, spreading on corresponding SMM medium, and culturing at 37 deg.C for 3-4 days. (3) And (3) selecting a monoclonal colony on the screened SMM medium plate for further isolated culture, extracting genome DNA, and performing PCR identification by using a primer in a target gene, wherein the positive colony is the genetically engineered bacterium.
The Aspergillus nidulans strain activation culture medium used in the invention is GMM culture medium, and each 1L of the solid culture medium is prepared according to the following method: 10g of glucose, 50mL of nitrate mother liquor, 1mL of trace element mother liquor, 1g of yeast extract and 15-20g of agar, wherein the components are sequentially added, and finally distilled water is added to the mixture until the final volume is 1L, the pH value is adjusted to 6.5, and the mixture is sterilized by moist heat under high pressure at 121 ℃ for 20-30 minutes. Pouring into a 90mm culture dish, streaking and inoculating, standing and culturing at 37 ℃ for 4 days, and storing at 4 ℃ for culture and inoculation. LMM culture medium, the preparation method of every 1L of the liquid culture medium is the same as that of GMM culture medium except that agar is not added.
Wherein, the preparation method (1L) of the nitrate mother liquor is as follows: sodium nitrate NaNO3120g, potassium chloride KCl 10.4g, magnesium sulfate MgSO4·7H2O10.4 g, dipotassium hydrogen phosphate K2HPO4·3H2O30.4 g, adding the above components in sequence, adding distilled water to a final volume of 1L, sterilizing at 121 deg.C for 20-30 min, and storing at room temperature;
the preparation method of the microelement mother liquor (100mL) comprises the following steps: zinc sulfate ZnSO4·7H2O2.2 g, boric acid H3BO31.1g of manganese chloride MnCl2·4H20.5g of O, ferrous sulfate FeSO4·7H2O0.16 g, CoCl2·5H20.16g of O, copper sulfate CuSO4·5H2O0.16 g, ammonium molybdate (NH4)6Mo7O24·4H2O0.11 g, EDTA sodium salt Na2EDTA5.0g, adding above components in sequence, adding distilled water to final volume of 100mL, sterilizing at 121 deg.C under high pressure and moist heat for 20-30 min, and storing at room temperature.
The transformation screening culture medium used by the invention is an SMM culture medium, and each 1L of the solid culture medium is prepared according to the following method: 218.6g of sorbitol is added to the GMM culture medium, and 1mL/L of 1% Pyridoxin mother liquor and 5mL/L of 0.05% Riboflavin mother liquor (AN-apmA and AN-apmB) or 1mL/L of 1% Pyridoxin mother liquor, 0.5g/L of uridine and 0.5g/L of uracil (AN-apmA + apmB) are added to the screening culture medium.
Example 4 precursor feeding and HPLC analysis of genetically engineered bacteria
To further verify the function of asperphyramate biosynthesis-related genes in Penicillium brevicompactum, genetically engineered bacteria AN-apmA, AN-apmB and AN-apmA + apmB were cultured in a plate containing 10mL of GMM medium at 25 ℃ for 6 days, while Benzoic Acid (BA) and phenylalanine (Phe) or N-benzoylphenylalaninol (N-nol) and N-Benzoylphenylalanine or its analog N-acid-S-N-acetylcysteine (SNAC-acid) dissolved in dimethyl sulfoxide were added to the medium as needed to feed the precursor.
The specific operation and results are as follows:
the genetically engineered bacterium (AN-apmA) fed benzoic acid with a final concentration of 60 μ g/mL and phenylalanine with a final concentration of 60 μ g/mL can generate N-Benzoylphenylalaninol (FIG. 4-Ai and FIG. 4-Aii); no target compound was produced after the control bacteria were fed benzoic acid at a final concentration of 60. mu.g/mL and phenylalanine at a final concentration of 60. mu.g/mL (FIG. 4-Aiii), and no target compound was produced after the control bacteria were fed N-Benzoylphenylalaninol at a final concentration of 30. mu.g/mL (FIG. 4-Aiv). Simultaneously feeding genetic engineering bacteria (AN-apmB) to obtain final concentrationBenzoic acid at a concentration of 60. mu.g/mL, phenylalanine at a concentration of 60. mu.g/mL, and N-Benzoylphenylalaninol at a concentration of 30. mu.g/mL, produced asperiphenate (FIG. 4-Bi), with no target compound produced in control bacteria (FIG. 4-Bii); genetically engineered bacteria (AN-apmB) are fed with N-benzol phenyl alaninol at a final concentration of 30 mu g/mL and N-benzol phenyl alaninol at a final concentration of 30 mu g/mL13C-labeled N-Benzoylphenylalanine can be produced13C-labeled asperphenamate (FIG. 4-Ci), control bacteria were sterile and free of target compound (FIG. 4-Cii).
Asperiphenamide was produced by feeding N-acid-S-N-acetylcysteine (SNAC-acid) at a final concentration of 30. mu.g/mL and N-benzoylalaninol at a final concentration of 30. mu.g/mL (FIG. 7).
The genetically engineered bacteria (AN-apmA + apmB) can generate precursor N-benzol alaninol and final product asperiphenamide after being fed with benzoic acid with a final concentration of 60 mug/mL and phenylalanine with a final concentration of 60 mug/mL (figure 5-i), and no target compound is generated when not being fed (figure 5-ii); the genetically engineered bacterium (AN-apmA) fed benzoic acid at a final concentration of 60. mu.g/mL as a control and phenylalanine at a final concentration of 60. mu.g/mL produced only the precursor N-benzoylalaninol (FIG. 5-iii), the genetically engineered bacterium (AN-apmB) fed benzoic acid at a final concentration of 60. mu.g/mL and phenylalanine at a final concentration of 60. mu.g/mL did not produce the target compound (FIG. 5-iv), and asperiphenate was produced after N-benzoylalaninol at a final concentration of 30. mu.g/mL (FIG. 5-v).
The results of FIGS. 4, 5 and 7 show that the apmA gene has a function of synthesizing an amino acid substrate into a linear dipeptide, the apmB gene has a function of coupling the linear dipeptide substrate into an amino acid ester compound, and the apmA and apmB genes have a function of synthesizing the amino acid substrate into the amino acid ester compound.
The condition that different genetically engineered bacteria produce asperphenamate and precursors thereof is detected by a High Performance Liquid Chromatography (HPLC) analysis method. And the biosynthesis pathway of asperphenamate is deduced from the experimental results, as shown in FIG. 6. The cultured cells and the culture medium were extracted under the following conditions: mycelia and the culture medium obtained after 48 hours of culture were subjected to compound extraction. 1 plate of the culture was divided into small pieces and placed in a triangular flask, and 20-40mL of MEA (ethyl acetate: methanol: glacial acetic acid: 89:10:1) was added for ultrasonic extraction (ultrasonic frequency 80Hz, time 1 hour); collecting extractive solution, and performing rotary evaporation (temperature 30 deg.C, rotation speed 100rpm, vacuum degree less than 3 mmHg); evaporating to dryness, dissolving with 0.30-0.60mL methanol, diluting with appropriate concentration, injecting 20-40ul, washing column with 40-100% methanol, and performing HPLC analysis.
HPLC analysis was performed under the following conditions: the device comprises the following steps: waters e2695 system detector: waters 2998(200-600 nm); a chromatographic column: 38020-41COSMOSIL 5C18-MS-II Packed Column, 4.6mm I.D.x250mm; elution speed: 1mL/min, eluent: water and methanol increased the methanol content from 40% methanol to 100% methanol within 20 min.
EXAMPLE 5 obtaining amino acid lipid Compounds Using genetically engineered bacteria
To further verify the additional functions of the asperphyramate biosynthesis-related genes apmA and apmB in Penicillium brevicompactum, the genetically engineered bacterium AN-apmA + apmB was cultured in a plate containing 10mL of GMM medium, while feeding product analysis was performed by adding Benzoic Acid (BA) dissolved in dimethyl sulfoxide to a final concentration of 60. mu.g/mL and Phenylisoleucine (PIS) to a final concentration of 60. mu.g/mL to the medium, as shown in FIG. 8. After culturing for 6 days at 25 ℃, compound extraction separation is carried out, and the product change after the genetically engineered bacteria are fed with 2 substrates is detected by mass spectrum HPLC and LC-MS analysis.
The extraction was carried out under the following conditions: mycelia and the culture medium obtained after 48 hours of culture were subjected to compound extraction. 1 plate of the culture was divided into small pieces and placed in a triangular flask, and 20-40mL of MEA (ethyl acetate: methanol: glacial acetic acid: 89:10:1) was added for ultrasonic extraction (ultrasonic frequency 80Hz, time 1 hour); collecting extractive solution, and performing rotary evaporation (temperature 30 deg.C, rotation speed 100rpm, vacuum degree less than 3 mmHg); evaporating to dryness, dissolving with 0.30-0.60mL methanol, diluting with appropriate concentration, injecting 20-40ul, washing column with 40-100% methanol, and performing HPLC analysis and LC-MS analysis.
HPLC analysis was performed under the following conditions: the device comprises the following steps: waters e2695 system detector: waters 2998(200-600 nm); a chromatographic column: 38020-41COSMOSIL 5C18-MS-II Packed Column, 4.6mm I.D.x250 mm; elution speed: 1mL/min, eluent: water and methanol increased the methanol content from 40% methanol to 100% methanol within 20 min.
LC-MS analysis was performed under the following conditions: the device comprises the following steps: QTOF LC/MS 6520 systems (Agilent Corp.); RP C18 column: kromasil 100-5C18 column; 4.6x 250 mm; 25 ℃, electrospray ionization source, elution rate: 1 mL/min.
As can be seen from FIG. 8, different products with molecular weights of 256.1361, 507.2305, 523.2250 and 539.2182 are produced when the genetically engineered bacterium AN-apmA + apmB is fed with Benzoic Acid (BA) and Phenylisoline (PIS), which indicates that the apmA and apmB genes have the function of coupling single amino acid substrates to generate new amino acid ester compounds.
Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Figure IDA0000987520810000011
Figure IDA0000987520810000021
Figure IDA0000987520810000031
Figure IDA0000987520810000041
Figure IDA0000987520810000051
Figure IDA0000987520810000061
Figure IDA0000987520810000071
Figure IDA0000987520810000081
Figure IDA0000987520810000091
Figure IDA0000987520810000101
Figure IDA0000987520810000111
Figure IDA0000987520810000121
Figure IDA0000987520810000131
Figure IDA0000987520810000141
Figure IDA0000987520810000151
Figure IDA0000987520810000161
Figure IDA0000987520810000171
Figure IDA0000987520810000181
Figure IDA0000987520810000191
Figure IDA0000987520810000201
Figure IDA0000987520810000211
Figure IDA0000987520810000221
Figure IDA0000987520810000231
Figure IDA0000987520810000241
Figure IDA0000987520810000251
Figure IDA0000987520810000261
Figure IDA0000987520810000271
Figure IDA0000987520810000281
Figure IDA0000987520810000291
Figure IDA0000987520810000301
Figure IDA0000987520810000311
Figure IDA0000987520810000321
Figure IDA0000987520810000331
Figure IDA0000987520810000341
Figure IDA0000987520810000351

Claims (9)

1. A protein for synthesizing asperphenamate, comprising the following proteins:
the amino acid sequence is shown in a sequence table SEQ ID NO: 1 and SEQ ID NO: 2.
2. A gene encoding a protein which synthesizes asperphenamate, comprising the following genes:
the nucleotide sequence is shown in a sequence table SEQ ID NO: 3 and SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.
3. A gene cluster for synthesizing asperphyrate comprising the coding gene of claim 2.
4. A recombinant vector comprising the coding gene of claim 2.
5. An engineered bacterium producing asperphyramate comprising the coding gene of claim 2.
6. A method for preparing gene engineering bacteria for producing asperphamate comprises the following steps:
(1) the cloning sequences are shown in sequence table SEQ ID NO: 3 and SEQ ID NO: 4;
(2) introducing the gene in the step (1) into a vector to obtain a recombinant vector;
(3) and (3) introducing the recombinant vector in the step (2) into a receptor strain, and obtaining the asperphenamate-producing genetic engineering bacteria by precursor feeding after screening and verification.
7. The method for preparing amino acid ester compounds or linear dipeptide compounds by feeding amino acid precursors using the genetically engineered bacteria of claim 6, wherein the genetically engineered bacteria catalyze the synthesis of N-Benzoylphenylalaninol from benzoic acid and phenylalanine substrates after feeding benzoic acid and phenylalanine; after feeding benzoic acid and benzyl isoleucine, the gene engineering bacteria catalyze benzoic acid and benzyl isoleucine to synthesize the amino acid ester compound.
8. The method for preparing amino acid ester compounds by feeding linear dipeptide precursors using genetically engineered bacteria as set forth in claim 6, wherein the genetically engineered bacteria synthesize the amino acid ester compounds asperiphenamide by receiving a linear dipeptide as a substrate and catalyzing an esterification reaction to release an end product after feeding the linear dipeptide, wherein the linear dipeptide is N-benzoylphenylamine and N-benzoylphenylanol, or wherein the linear dipeptide is N-acid-S-N-acetylcysteimine and N-benzoylphenylanol.
9. Use of the genetically engineered bacterium of claim 6 in synthesizing an asperphenamate compound or in preparing an amino acid ester compound synthesized by feeding benzoic acid and benzylic isoleucine to the genetically engineered bacterium of claim 6.
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