CN115161300B - Aquilaria sinensis-derived III-type polyketide synthase AsPKS, and encoding gene and application thereof - Google Patents

Abstract

The invention discloses aquilaria sinensis-derived III type polyketide synthase AsPKS, and a coding gene and application thereof. The invention screens based on genome and transcriptome data of aquilaria sinensis to obtain new polyketide synthase AsPKS, the amino acid sequence of which is shown as SEQ ID NO.6, and the nucleotide sequence of the encoding gene of which is shown as SEQ ID NO. 5. The recombinant III type polyketide synthase AsPKS3 is obtained through heterologous expression, and an enzyme activity catalysis experiment shows that the recombinant III type polyketide synthase AsPKS has various catalytic activities and can be applied to the production of in-vitro or in-vivo catalytic synthesis of benzyl propyl ketone or quinolone compounds and the like.

Description

Type III polyketide synthase AsPKS3 from Aquilaria sinensis and its encoding gene and application

Technical Field

The present invention relates to a type III polyketide synthase and a gene encoding the same, in particular to a type III polyketide synthase AsPKS3 derived from Aquilaria sinensis and a gene encoding the same. The present invention further relates to their application in catalyzing the synthesis of benzyl acetone or quinolone compounds, belonging to the field of type III polyketide synthase and its application.

Background Art

Polyketides are a class of secondary metabolites containing macrocyclic structures composed of carbon, oxygen and/or nitrogen. They participate in the growth regulation of plants under stress and have pharmacological activities such as antibacterial, anticancer and immunosuppressive activities. Polyketides in plants are synthesized by type III polyketide synthases through condensation reactions of various coenzymes A, which are further modified by different modifying enzymes. A series of type III polyketide synthases with different functions have been found in plants, such as chalcone synthase (CHS) that catalyzes the synthesis of chalcone, stilbene synthase (STS) that catalyzes the synthesis of resveratrol, and curcumin synthase (CUS) that catalyzes the synthesis of curcuminoids.

In vitro catalytic studies have shown that type III polyketide synthase can accept a variety of starting substrates or extension units, thus having functional promiscuity. Quinolone compounds have a wide range of biological activities, such as antibacterial, antitumor and antioxidant activities. The present invention not only provides new ideas for the production of quinolone active compounds using synthetic biology technology, but also provides reference information for the characteristic research and function mining of type III polyketide synthase.

So far, there is no report in the prior art about type III polyketide synthase and its encoding gene isolated from Aquilaria sinensis that catalyzes the synthesis of quinolone compounds.

Summary of the invention

One of the purposes of the present invention is to provide a type III polyketide synthase AsPKS3 derived from Aquilaria sinensis and its encoding gene;

The second purpose of the present invention is to use the type III polyketide synthase AsPKS3 derived from Aquilaria sinensis to catalyze the synthesis of compounds such as benzyl acetones or quinolones.

To achieve the above objectives, the main technical solutions adopted by the present invention include:

One aspect of the present invention discloses a type III polyketide synthase AsPKS3 from Aquilaria sinensis, the amino acid sequence of which is selected from any one of (a) or (b):

(a) the amino acid sequence shown in SEQ ID No. 6; or

(b) a protein variant having the function or activity of plant type III polyketide synthase derived from the amino acid sequence shown in SEQ ID No. 6 by substitution, deletion or/and insertion of one or more amino acid residues;

Another aspect of the present invention discloses a gene encoding type III polyketide synthase AsPKS3 from Aquilaria sinensis, the nucleotide sequence of which is shown in (a), (b) or (c):

(a) the polynucleotide sequence shown in SEQ ID No.5; or

(b) a polynucleotide sequence that can hybridize with the complementary sequence of SEQ ID No. 5 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function or activity of plant type III polyketide synthase; or

(c) a polynucleotide sequence that has at least 85% homology with the polynucleotide sequence of SEQ ID No.5, and the protein encoded by the polynucleotide still has the function or activity of plant type III polyketide synthase; more preferably, a polynucleotide sequence that has at least 90% homology with the polynucleotide sequence of SEQ ID No.5, and the protein encoded by the polynucleotide still has the function or activity of plant type III polyketide synthase.

In another aspect, the present invention provides the use of type III polyketide synthase AsPKS3 derived from Aquilaria sinensis to catalyze the synthesis of compounds such as benzyl acetone or quinolone compounds.

The specific implementation scheme of the use of type III polyketide synthase AsPKS3 derived from white agarwood of the present invention comprises: (1) catalyzing p-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylideneacetone; (2) catalyzing feruloyl-CoA and malonyl-CoA to produce 3-methoxy-4-hydroxybenzylideneacetone; (3) catalyzing 2-(methylamino)benzoyl-CoA and malonyl-CoA to produce 4-hydroxy-1-methyl-2-quinolone; (4) catalyzing 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4(1H)-one.

Those skilled in the art can use conventional technical means in the art to obtain recombinant type III polyketide synthase AsPKS3 from Aquilaria sinensis by conventional prokaryotic expression or eukaryotic expression methods. The recombinant type III polyketide synthase AsPKS3 can be used to catalyze the synthesis of benzyl acetone or quinolone compounds by in vitro conversion or catalytic methods.

The present invention also discloses a recombinant expression vector containing the gene encoding the type III polyketide synthase AsPKS3; preferably, the recombinant expression vector can be a recombinant prokaryotic expression vector and a recombinant eukaryotic expression vector.

The type III polyketide synthase AsPKS3 encoding gene is operably connected to an expression regulatory element to obtain a recombinant expression vector that can express the encoding gene in prokaryotic cells or eukaryotic cells; the recombinant expression vector can be composed of a 5′ non-coding region, a polynucleotide sequence shown in SEQ ID No.5, and a 3′ non-coding region, wherein the 5′ non-coding region can include a promoter sequence, an enhancer sequence and/or a translation enhancing sequence; the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3′ non-coding region can include a terminator sequence, an mRNA cleavage sequence, etc.

The present invention further discloses a recombinant host cell or a recombinant bacterium containing the gene encoding the type III polyketide synthase AsPKS3; wherein the recombinant bacterium includes but is not limited to a recombinant Escherichia coli or a recombinant yeast cell.

In addition, those skilled in the art can optimize the polynucleotide shown in SEQ ID No. 5 to enhance the expression efficiency in the host. For example, the preferred codons of the target host can be used to optimize the synthesis of the polynucleotide to enhance the expression efficiency in the target host.

The chimeric gene or expression cassette obtained by chimerizing or connecting the gene shown in SEQ ID No. 5 of the present invention with other genes belongs to the protection scope of the present invention; the recombinant expression vector containing the chimeric gene or expression cassette also belongs to the protection scope of the present invention.

The aquilinum polyketide synthase AsPKS3 provided by the present invention has multiple enzyme activities and can be used in the catalytic synthesis of benzyl acetone or quinolone compounds and the production of natural products of intermediates of these compounds. It can also be used to guide the molecular breeding of medicinal plants containing related substances and can be used for the in vivo (in vitro) synthesis and preparation of benzyl acetone or quinolone compounds.

Definitions of terms used in this invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described.

The term "homology" refers to sequence similarity to a natural nucleic acid sequence. "Homology" includes nucleotide sequences that are preferably 85% or higher, more preferably 90% or higher, and most preferably 95% or higher identical to the nucleotide sequence of the regulatory fragment of the present invention. Homology can be evaluated by the naked eye or by computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

"Variant" means a substantially similar sequence. For polynucleotides, variants include deletions, insertions, and/or substitutions of one or more nucleotides at one or more sites in natural polynucleotides. For polynucleotides, conservative variants include those that do not change the encoded amino acid sequence due to the degeneracy of the genetic code. Naturally occurring variants such as these can be identified by existing molecular biology techniques. Variant polynucleotides also include polynucleotides of synthetic origin, such as polynucleotide variants that still encode the amino acid sequence shown in SEQ ID No.6 obtained by site-directed mutagenesis or by recombinant methods (e.g., DNA shuffling).

The term "complementary" herein refers to two nucleotide sequences including antiparallel nucleotide sequences that can pair with each other after forming hydrogen bonds between the complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary chains are reverse complementary to each other when both sequences are viewed from the 5' to 3' direction. It is also known in the art that two sequences that can hybridize to each other under a given set of conditions do not necessarily have to be 100% completely complementary.

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times greater than background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. A target sequence that is 100% complementary to the probe can be identified by controlling the stringency of the hybridization or the washing conditions. For detailed guidance on nucleic acid hybridization, reference may be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes," Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a specified ionic strength and pH. Tm is the temperature (under specified ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in a state of equilibrium (because the target sequence is present in excess, 50% of the probes are occupied in a state of equilibrium at Tm). The stringent conditions may be the following conditions: wherein the pH is 7.0 to 8.3, the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salts), and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The wash can be performed for 5, 15, 30, 60, 120 minutes or longer.

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues.

The term "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region can be positioned relative to a nucleic acid sequence encoding an expression product of interest such that transcription of the nucleic acid sequence is directed by the promoter region. Thus, the promoter region is "operably linked to the nucleic acid sequence."

The terms "transformed," "transgenic," and "recombinant" herein refer to a host cell or organism, such as a bacterium or plant cell (e.g., a plant), into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the host's genome, or the nucleic acid molecule may exist as an extrachromosomal molecule. Such an extrachromosomal molecule may be self-replicating. Transformed cells, tissues, or plants are understood to include not only the end product of the transformation process, but also its transgenic progeny. An "untransformed," "untransgenic," or "unrecombined" host refers to a wild-type organism, such as a bacterium or plant, that does not contain the heterologous nucleic acid molecule.

The term "promoter" refers to any nucleic acid sequence (such as a DNA sequence) that is recognized and bound (directly or indirectly) by a DNA-dependent RNA polymerase during the initiation of transcription, resulting in the generation of an RNA molecule complementary to the transcribed DNA; this region may also be referred to as a "5' regulatory region". A promoter is typically located upstream of the 5' untranslated region (UTR) present in front of the coding sequence to be transcribed and has multiple regions that serve as binding sites for RNA polymerase II and other proteins such as transcription factors to initiate transcription of operably linked genes. The promoter itself may contain subelements (i.e., promoter motifs) such as cis-elements or enhancer domains that regulate the transcription of operably linked genes. The promoter and the connected 5'UTR are also referred to as "promoter regions".

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an amino acid sequence comparison diagram of the type III polyketide synthase AsPKS3 of the present invention and several other type III polyketide synthases.

FIG2 is an SDS-PAGE diagram of type III polyketide synthase AsPKS3 of the present invention.

Figure 3 is a reaction formula of the type III polyketide synthase AsPKS3 of the present invention catalyzing p-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylideneacetone or catalyzing feruloyl-CoA and malonyl-CoA to produce 3-methoxy-4-hydroxybenzylideneacetone.

FIG4 is a reaction formula showing the generation of 4-hydroxy-1-methyl-2-quinol by 2-(methylamino)benzoyl-CoA and malonyl-CoA catalyzed by type III polyketide synthase AsPKS3 of the present invention.

Figure 5 is a reaction formula of the type III polyketide synthase AsPKS3 of the present invention catalyzing 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4(1H)-one.

FIG6 is a mass spectrum of various products catalyzed by type III polyketide synthase AsPKS3 of the present invention.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the scope of protection of the present invention.

The analysis or detection methods used in the embodiments of the present invention

1. Agarose Gel Electrophoresis Analysis

The samples were added to an agarose gel containing Polymer M5 HiPure Gelred Plus nucleic acid dye and separated at 120 V for 20 min. The results were then detected using a gel imaging system.

2. SDS-PAGE gel electrophoresis analysis

The electrophoresis tank and electrophoresis power supply of Bio-Rad Company of the United States were used for SDS-PAG (5% concentrated gel, 10% separation gel), the sample volume was 10 μL, and the electrophoresis was performed at 80V. After the sample entered the separation gel, the voltage was adjusted to 100V, and the electrophoresis was stopped until the bromophenol blue just ran out. After Coomassie brilliant blue staining, decolorization and photography were performed.

3. Liquid chromatography mass spectrometry detection

Liquid phase mass spectrometer UPLC-Q-TOF-MS (Waters, USA) was used for detection. The chromatographic column was AcquityUPLC BEH C ₁₈ column (2.1×100 mm, 1.7 μm, Waters), the flow rate was 0.1 mL/min, the DAD detector was scanned at full wavelength, and the gradient elution was acetonitrile-0.1% formic acid water: 0-5 min, 5-30% acetonitrile; 5-11 min, 30-45% acetonitrile; 11-22 min, 45-95% acetonitrile (the above percentage concentrations of acetonitrile are volume concentrations).

Example 1 Cloning and sequence analysis of the gene of polyketide synthase (PKS) AsPKS3 from Aquilaria sinensis

(1) Extraction of total RNA from Aquilaria sinensis callus and synthesis of first-chain cDNA

Take an appropriate amount of wound-induced Aquilaria sinensis callus to extract total RNA, and reverse transcribe it with a primer with a linker to obtain cDNA. Preferably, take 100 mg of Aquilaria sinensis callus induced with 150 mM NaCl for 3 days, grind it in liquid nitrogen, and extract total RNA using the Plant RNA Rapid Extraction Kit RN38 of Adelaide Company according to the instructions. The RNA concentration and quality are detected by Thermo Scientific NanoDrop spectrophotometer, and the RNA quality is detected by agarose gel electrophoresis.

cDNA was synthesized using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit (AE311) from Quanshijin Company according to the product instructions and total RNA as a template.

(2) Cloning of AsPKS3 gene

Design specific primers, the specific primer sequences are as follows:

AsPKS3-F:5’-ATGGCAGCCCAACCTGTG-3’(SEQ ID NO.1)

AsPKS3-R:5’-CTAAGCGTCTGTGAGCGTGAGC-3’(SEQ ID NO.2)

Using cDNA of Aquilaria sinensis callus as a template, the AsPKS3 gene was amplified by PCR and sequenced, and the nucleotide sequence of the AsPKS3 gene was obtained as shown in SEQ ID NO.5, with an initiation codon of ATG and a termination codon of TAG; the translated protein coding sequence was shown in SEQ ID NO.6.

The pMD-19T kit of TaKaRa was used to connect the AsPKS3 gene amplification product obtained by PCR with the pMD-19T vector according to the instructions to construct the pMD-19T-AsPKS3 cloning vector. The constructed recombinant vector was transformed into cloning competent cells E. coli DH5α, and LB solid plates containing ampicillin, IPTG and X-Gal were coated. White single colonies were picked for culture, and colony PCR verification and sequencing verification were performed.

The amino acid sequence of AsPKS3 was compared with the amino acid sequences of the polyketide synthases from four Aquilaria sinensis species reported by previous researchers. The similarity between AsPKS3 and AsPKS1 and AsPKS2 was less than 40%; the similarity between AsPKS3 and AsCHS2 was 65%; and the similarity between AsPKS3 and AsPECPS was 95.24%. The multiple sequence comparison results of AsPKS3 and the four reported Aquilaria sinensis polyketide synthases and the chalcone synthase from alfalfa are shown in Figure 1. The conserved amino acid sites of the catalytic active center of type III polyketide synthase (Cys164, His303, Asn336) and the key amino acid sites that can regulate the size or shape of the active cavity (Thr132, Ser133, Thr197, Phe215, Gly256, Leu263, Phe265, Ser338) are marked in Figure 1, and the site numbers are the corresponding amino acid numbers in alfalfa CHS.

Example 2 Construction of prokaryotic expression vector and heterologous expression of AsPKS3

Design primers without stop codons, the sequences are as follows:

AsPKS3-exp-F:5’-ATGGCAGCCCAACCTGTG-3’(SEQ ID NO.3)

AsPKS3-exp-R:5’-AGCGTCTGTGAGCGTGAGC-3’(SEQ ID NO.4)

The positive colonies obtained in Example 1 were cultured and the recombinant plasmid was extracted and used as a template for PCR amplification to obtain the gene coding sequence without a stop codon. -Blunt E2 Expression Kit, according to the instructions, the AsPKS3 gene coding sequence obtained by PCR was blunt-ended with the pEASY-Blunt-E2 vector to obtain the recombinant expression vector pEASY-Blunt-E2-AsPKS3.

The constructed pEASY-Blunt-E2-AsPKS3 plasmid was transformed into the prokaryotic expression strain E.coli BL21 (DE3), coated with LB solid plates containing ampicillin, picked single colonies for culture, and extracted plasmids for PCR. Screen positive clones to obtain prokaryotic expression engineering bacteria BL21 (DE3) -pEASY-Blunt-E2-AsPKS3. Take 50 μL of bacterial solution and inoculate it into 5mL LB liquid culture medium containing 100 μg/mL ampicillin, and culture it at 37°C, 200rpm overnight. Then inoculate it into 300mL LB liquid culture medium containing ampicillin at a ratio of 1: 100, activate it at 37°C, 200rpm, and add 0.5mM IPTG when OD ₆₀₀ = about 0.6. BL21(DE3)-pEASY-Blunt-E2-AsPKS3 was cultured for 16 hours at 16°C and 180 rpm. The harvested bacterial solution was centrifuged at 5000 rpm and 4°C, and the supernatant was removed. The bacterial pellet was resuspended in lysis buffer, and after ultrasonic disruption, the AsPKS3 protein was purified using a nickel ion affinity chromatography column based on the presence of a His tag at the C-terminus of the recombinant protein. The purified protein was detected by SDS-PAGE electrophoresis. The electrophoresis detection results are shown in Figure 2. The size of the target protein is about 45 kDa. After concentration and desalting through a filter membrane (Millipore, 10 kD), a protein with a concentration of more than 1 mg/mL was obtained.

Experimental Example 1 Experiment on the production of p-hydroxybenzylideneacetone by AsPKS3 enzyme catalysis of p-coumaroyl-CoA and malonyl-CoA

80μM starting substrate (p-coumaryl-CoA), 160μM malonyl-CoA and 20μg purified recombinant protein were added to a total volume of 250μL of 100mM potassium phosphate buffer (KPB buffer) (pH 7.0), reacted at 32°C in a constant temperature water bath for 6h, and then 20μL of 20% HCl was added to terminate the enzyme reaction. The reaction solution was extracted 3 times with 500μL ethyl acetate, the extracts were combined, drained by a low-temperature centrifugal concentrator, and then re-dissolved with 100μL methanol and detected by liquid phase mass spectrometer UPLC-Q-TOF-MS. The molecular formula was predicted by analyzing the high-resolution mass spectrometry information, and compared with the standard or literature reported information to identify the reaction product.

According to the measured products and the principle of chemical reaction, the reaction in this experimental example is: Aquilaria sinensis polyketide synthase AsPKS3 catalyzes the condensation of one molecule of p-coumaroyl-CoA and one molecule of malonyl-CoA to produce p-hydroxybenzylideneacetone. The relevant reaction formula is shown in FIG3 , and the mass spectrometry identification of the generated product p-hydroxybenzylideneacetone is shown in FIG6 .

Experimental Example 2 Experiment on the production of 3-methoxy-4-hydroxybenzylideneacetone by AsPKS3 enzyme catalysis of feruloyl-CoA and malonyl-CoA

The specific operation steps of Experimental Example 1 were followed, and the starting substrate was replaced with feruloyl-CoA. The other conditions and operation methods were the same as those of Experimental Example 1. According to the measured products and the chemical reaction principle, the reaction formula of this experimental example is shown in FIG3 , that is, the Aquilaria sinensis polyketide synthase AsPKS3 catalyzes the condensation of feruloyl-CoA and malonyl-CoA to generate 3-methoxy-4-hydroxybenzylideneacetone. The mass spectrometry identification of the generated product 3-methoxy-4-hydroxybenzylideneacetone is shown in FIG6 .

Experimental Example 3 Experiment on the production of 4-hydroxy-1-methyl-2-quinol by AsPKS3 enzyme catalysis of 2-(methylamino)benzoyl-CoA and malonyl-CoA

The specific operation steps of Experimental Example 1 were followed, and the starting substrate was replaced with 2-(methylamino)benzoyl-CoA. Other conditions and operation methods were the same as those of Experimental Example 1. After detection, the relevant reaction formula in this embodiment is shown in FIG5 , and the Aquilaria sinensis polyketide synthase AsPKS3 catalyzes the condensation of one molecule of 2-(methylamino)benzoyl-CoA and one molecule of malonyl-CoA to produce 4-hydroxy-1-methyl-2-quinolone, and the mass spectrometry identification of the produced product 4-hydroxy-1-methyl-2-quinolone is shown in FIG6 .

Experimental Example 4 Experiment on the reaction of 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenylethylquinolin-4(1H)-one by AsPKS3 enzyme

The specific operation steps of Experimental Example 1 were followed, and the reaction substrates were replaced with 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid. The other conditions and operation methods were the same as those of Experimental Example 1. According to the measured products and the chemical reaction principle, the relevant reaction formula in this experimental example is shown in FIG6 , and the Aquilaria sinensis polyketide synthase AsPKS3 catalyzes 1 molecule of 2-(methylamino)benzoyl-CoA and 1 molecule of 3-oxo-5-phenylpentanoic acid to generate 1-methyl-2-phenethylquinoline-4(1H)-one, and the mass spectrum identification of the produced product 1-methyl-2-phenethylquinoline-4(1H)-one is shown in FIG6 .

SEQUENCE LISTING

<110> Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences

<120> Type III polyketide synthase AsPKS3 from Aquilaria sinensis and its encoding gene and application

<130> BJ-2004-220303A

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<400> 1

atggcagccc aacctgtg 18

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence

<400> 2

ctaagcgtct gtgagcgtga gc 22

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence

<400> 3

atggcagccc aacctgtg 18

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence

<400> 4

agcgtctgtg agcgtgagc 19

<210> 5

<211> 1200

<212> DNA

<213> Aquilaria sinensis

<400> 5

atggcagccc aacctgtgga gtgggtgagg aatgcagaga gggctgccgg accggcaacc 60

gttctcgcca tcgccactgc caatccctcc aacttcttcc tacagtcgga cttccccgac 120

ttctatttcc gggtcactag gagcgatcac atgtccgacc tgaaggaaaa attcaagcga 180

atttgcaaga agacgacggt caggaagaga cacatgatcc tgacggagga gatcctaaac 240

aagaatcccg ccattgctga ctactggtcg ccttcgctgg ccgcccgcca cgacctcggg 300

ctggccaaca tcccccagct aggaaaggaa gccgccgaca aggccatcaa ggagtggggc 360

cagcccaagt ccaaaatcac ccaccttgtc ttttgcactt ccgccggcgt ccacatgcct 420

ggcgccgact accagctcac catgctcctc ggccttaacc cctccatcag ccgcctcatg 480

ctccataacc ttggctgcta cgccggagga accgccctcc gagtcgccaa agacctcgcc 540

gagaacaacg gtggtgccag ggtacttgtc gtctgctcgg aggccaacct actcaacttc 600

cggggcccgt cggagaccca catcgacgcg ctcagaactc aatcactctt cgcggacgga 660

gcggccgcgc tcattgtagg ttctgatccc gatccccata ccgagagtcc gctgtacgaa 720

ctcatctcgg cgtcgcagag gatactcccg gagtcggaag atgcgattgt gggacgcttg 780

accgaagcag gtctagtccc ctatttgcct aaagacctcc ccaaattggt ctcgacgaac 840

atcagaagca tcttggagga tgccttggcg ccgaccggag tccaagactg gaactctatc 900

ttctggattg ttcaccctgg catgccggcg attctggacc aaacagagaa gctgctccag 960

ctcgataaag agaaactcaa agcaactcga cacgtgctca gtgagtttgg aaatatgttt 1020

agtgccaccg tactgtttat cttggaccag atgaggaaag gtgctgtggc ggaagggaag 1080

tccaccaccg gggaaggctg cgagtggggt gtccttctct cgttcgggcc aggcttcacc 1140

gttgagacag tgttgctacg tagtgtcact aatggtgcgc tcacgctcac agacgcttag 1200

<210> 6

<211> 399

<212> PRT

<213> Aquilaria sinensis

<400> 6

Met Ala Ala Gln Pro Val Glu Trp Val Arg Asn Ala Glu Arg Ala Ala

1 5 10 15

Gly Pro Ala Thr Val Leu Ala Ile Ala Thr Ala Asn Pro Ser Asn Phe

20 25 30

Phe Leu Gln Ser Asp Phe Pro Asp Phe Tyr Phe Arg Val Thr Arg Ser

35 40 45

Asp His Met Ser Asp Leu Lys Glu Lys Phe Lys Arg Ile Cys Lys Lys

50 55 60

Thr Thr Val Arg Lys Arg His Met Ile Leu Thr Glu Glu Ile Leu Asn

65 70 75 80

Lys Asn Pro Ala Ile Ala Asp Tyr Trp Ser Pro Ser Leu Ala Ala Arg

85 90 95

His Asp Leu Gly Leu Ala Asn Ile Pro Gln Leu Gly Lys Glu Ala Ala

100 105 110

Asp Lys Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His

115 120 125

Leu Val Phe Cys Thr Ser Ala Gly Val His Met Pro Gly Ala Asp Tyr

130 135 140

Gln Leu Thr Met Leu Leu Gly Leu Asn Pro Ser Ile Ser Arg Leu Met

145 150 155 160

Leu His Asn Leu Gly Cys Tyr Ala Gly Gly Thr Ala Leu Arg Val Ala

165 170 175

Lys Asp Leu Ala Glu Asn Asn Gly Gly Ala Arg Val Leu Val Val Cys

180 185 190

Ser Glu Ala Asn Leu Leu Asn Phe Arg Gly Pro Ser Glu Thr His Ile

195 200 205

Asp Ala Leu Arg Thr Gln Ser Leu Phe Ala Asp Gly Ala Ala Ala Leu

210 215 220

Ile Val Gly Ser Asp Pro Asp Pro His Thr Glu Ser Pro Leu Tyr Glu

225 230 235 240

Leu Ile Ser Ala Ser Gln Arg Ile Leu Pro Glu Ser Glu Asp Ala Ile

245 250 255

Val Gly Arg Leu Thr Glu Ala Gly Leu Val Pro Tyr Leu Pro Lys Asp

260 265 270

Leu Pro Lys Leu Val Ser Thr Asn Ile Arg Ser Ile Leu Glu Asp Ala

275 280 285

Leu Ala Pro Thr Gly Val Gln Asp Trp Asn Ser Ile Phe Trp Ile Val

290 295 300

His Pro Gly Met Pro Ala Ile Leu Asp Gln Thr Glu Lys Leu Leu Gln

305 310 315 320

Leu Asp Lys Glu Lys Leu Lys Ala Thr Arg His Val Leu Ser Glu Phe

325 330 335

Gly Asn Met Phe Ser Ala Thr Val Leu Phe Ile Leu Asp Gln Met Arg

340 345 350

Lys Gly Ala Val Ala Glu Gly Lys Ser Thr Thr Gly Glu Gly Cys Glu

355 360 365

Trp Gly Val Leu Leu Ser Phe Gly Pro Gly Phe Thr Val Glu Thr Val

370 375 380

Leu Leu Arg Ser Val Thr Asn Gly Ala Leu Thr Leu Thr Asp Ala

385 390 395

Claims (9)

1. A type III polyketide synthase AsPKS3 derived from Aquilaria sinensis , characterized in that its amino acid sequence is the amino acid sequence shown in SEQ ID No.6. 2. A gene encoding type III polyketide synthase AsPKS3 from Aquilaria sinensis , characterized in that its nucleotide sequence is the polynucleotide sequence shown in SEQ ID No.5. 3. An expression cassette containing the coding gene according to claim 2. 4. A recombinant expression vector containing the coding gene according to claim 2. 5. A host cell containing the recombinant expression vector according to claim 4, wherein the host cell is Escherichia coli or a yeast cell. 6. The use of the type III polyketide synthase AsPKS3 of claim 1 in catalyzing the synthesis of benzyl acetone or quinolone compounds, comprising: (1) catalyzing the reaction of p-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylidene acetone; or (2) catalyzing the reaction of feruloyl-CoA and malonyl-CoA to produce 3-methoxy-4-hydroxybenzylidene acetone; or (3) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and malonyl-CoA to produce 4-hydroxy-1-methyl-2-quinolone; or (4) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4(1H)-one. 7. Use of the gene encoding the type III polyketide synthase AsPKS3 of claim 2 or the expression cassette of claim 3 in catalyzing the synthesis of benzyl acetones or quinolone compounds, comprising: (1) catalyzing the reaction of p-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylideneacetone; or (2) catalyzing the reaction of feruloyl-CoA and malonyl-CoA to produce 3-methoxy-4-hydroxybenzylideneacetone; or (3) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and malonyl-CoA to produce 4-hydroxy-1-methyl-2-quinolone; or (4) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4(1H)-one. 8. Use of the recombinant expression vector of claim 4 in catalyzing the synthesis of benzyl acetone or quinolone compounds, comprising: (1) catalyzing the reaction of p-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylidene acetone; or (2) catalyzing the reaction of feruloyl-CoA and malonyl-CoA to produce 3-methoxy-4-hydroxybenzylidene acetone; or (3) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and malonyl-CoA to produce 4-hydroxy-1-methyl-2-quinolone; or (4) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4(1H)-one. 9. Use of the host cell of claim 5 in catalytic synthesis of benzyl acetone or quinolone compounds, comprising: (1) catalyzing the reaction of p-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylidene acetone; or (2) catalyzing the reaction of feruloyl-CoA and malonyl-CoA to produce 3-methoxy-4-hydroxybenzylidene acetone; or (3) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and malonyl-CoA to produce 4-hydroxy-1-methyl-2-quinolone; or (4) catalyzing the reaction of 2-(methylamino)benzoyl-CoA and 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4(1H)-one.