CN115011576A

CN115011576A - Aquilaria sinensis-derived III-type polyketide synthase AsPKS5, and coding gene and application thereof

Info

Publication number: CN115011576A
Application number: CN202210516709.1A
Authority: CN
Inventors: 魏建和; 高志晖; 肖梦君; 王彬彬; 孙佩文; 吕菲菲; 徐艳红; 刘洋洋; 陈德力; 杨云; 冯雅楠
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2022-09-06

Abstract

The invention discloses an aquilaria sinensis-derived III-type polyketide synthase AspKS5 and a coding gene and application thereof. The invention screens and obtains a novel polyketone synthase AspKS5 based on genome and transcriptome data of aquilaria sinensis, wherein the amino acid sequence of the polyketone synthase AspKS5 is shown as SEQ ID NO.4, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3. The invention obtains the recombinant III type polyketide synthase AspKS5 through heterologous expression, and enzyme activity catalysis experiments show that the recombinant III type polyketide synthase AspKS5 has multiple catalytic activities, can be applied to the production of in vitro or in vivo catalytic synthesis of benzyl acetone, pyrone or quinolone compounds and the like, and can also be applied to the guidance of molecular breeding of medicinal plants containing related substances.

Description

Aquilaria sinensis-derived III-type polyketide synthase AsPKS5, and coding gene and application thereof

Technical Field

The invention relates to a type III polyketide synthase and a coding gene thereof, in particular to a type III polyketide synthase AspKS5 derived from Aquilaria sinensis (Aquilaria sinensis) and a coding gene thereof, and further relates to application of the polyketide synthase AspKS5 in catalyzing synthesis of benzyl acetone, pyrone or quinolone compounds, belonging to the field of type III polyketide synthase and application thereof.

Background

Polyketides are secondary metabolites containing macrocyclic structures formed by atoms such as carbon atoms and oxygen atoms, participate in growth regulation of plants in a stress environment, and have pharmacological activities such as antibiosis, anticancer and immunosuppression. Polyketides in plants are produced by catalyzing various coenzyme A by type III polyketide synthase, synthesizing a skeleton structure through a condensation reaction, and further modifying through different modifying enzymes. A diverse range of functionally distinct type III polyketide synthases have been found in plants, such as chalcone synthase (CHS) which catalyzes the synthesis of chalcone, stilbene synthase (STS) which catalyzes the synthesis of resveratrol, and curcumin synthase (CUS) which catalyzes the synthesis of curcuminoids.

In vitro catalytic studies have shown that type III polyketides synthase can accept a variety of starting substrates or extension units and thus have functional heteroleptics. The quinolone compound has wide biological activity, such as antibacterial, antitumor and antioxidant activity. The invention not only provides a new thought for producing quinolone active compounds by using synthetic biology technology, but also provides reference information for characteristic research and function mining of type III polyketide synthase.

To date, there is no report in the prior art about multifunctional type III polyketide synthase and its coding gene for catalyzing the synthesis of pyrones and quinolones compounds separated from aquilaria sinensis.

Disclosure of Invention

One of the purposes of the invention is to provide an Aquilaria sinensis (Aquilaria sinensis) derived type III polyketide synthase AspKS5 and a coding gene thereof;

the second purpose of the invention is to apply the Aquilaria sinensis III type polyketide synthase AspKS5 to catalyze the synthesis of compounds such as benzyl propyl ketone, pyrone or quinolone compounds.

In order to achieve the purpose, the invention adopts the main technical scheme that:

one aspect of the invention discloses an Aquilaria sinensis (Aquilaria sinensis) derived type III polyketide synthase AspKS5, the amino acid sequence of which is selected from any one of (a) or (b):

(a) an amino acid sequence shown as SEQ ID No. 4; or

(b) Protein variants derived from the amino acid sequence shown in SEQ ID No.4 by substitution, deletion or/and insertion of one or more amino acid residues and still having plant type III polyketide synthase function or activity.

On the other hand, the invention discloses a coding gene of Aquilaria sinensis (Aquilaria sinensis) derived type III polyketide synthase AsPKS5, the nucleotide sequence of which is shown as (a), (b) or (c):

(a) a polynucleotide sequence shown as SEQ ID No. 3; or

(b) A polynucleotide sequence which hybridizes under stringent hybridization conditions to the complement of SEQ ID No.3 and which encodes a protein which still functions or is active as a plant type iii polyketide synthase; or

(c) A polynucleotide sequence having at least 85% homology with the polynucleotide sequence of SEQ ID No.3 and encoding a protein which still has the function or activity of plant type III polyketide synthase; preferably, the polynucleotide sequence has at least 90% homology with the polynucleotide sequence of SEQ ID No.3, and the protein encoded by the polynucleotide still has the function or activity of plant type III polyketide synthase.

In another aspect, the invention provides application of Aquilaria sinensis III polyketide synthase AsPKS5 in catalysis of synthesis of compounds such as benzyl acetone, pyrone or quinolone compounds.

A particular embodiment of the use of the Aquilaria sinensis-derived type III polyketide synthase AsPKS5 as per the invention comprises: (1) catalyzing p-coumaroyl-coenzyme A and malonyl-coenzyme A to generate p-hydroxybenzylideneacetone; (2) catalyzing feruloyl coenzyme A and malonyl coenzyme A to generate 3-methoxy-4-hydroxybenzylideneacetone; (3) catalyzing propionyl coenzyme A and malonyl coenzyme A to generate 4-hydroxy-6-phenethyl pyran-2-ketone; (4) catalyzing 2- (methylamino) benzoyl coenzyme A with malonyl coenzyme A to form 4-hydroxy-1-methyl-2-quina lone; (5) catalyzing the reaction of 2- (methylamino) benzoyl coenzyme A with 3-oxo-5-phenylpentanoic acid to form 1-methyl-2-phenethylquinolin-4 (1H) -one.

The skilled person can obtain recombinant polyketide synthase AspKS5 by conventional prokaryotic expression or eukaryotic expression of the coding gene of Aquilaria sinensis derived type III polyketide synthase AspKS5 by adopting the conventional technical means in the field, and can catalyze the synthesis of benzyl propiophenone, pyrone or quinolone compounds by adopting the recombinant polyketide synthase AspKS5 through in vitro transformation or catalysis.

The invention also discloses a recombinant expression vector containing the encoding gene of the type III polyketide synthase AspKS 5; preferably, the recombinant expression vector can be a recombinant prokaryotic expression vector and a recombinant eukaryotic expression vector.

Operably connecting the III type polyketide synthase AsPKS5 encoding gene with an expression regulation element to obtain a recombinant expression vector capable of expressing the encoding gene in prokaryotic cells or eukaryotic cells; the recombinant expression vector can consist of a 5' end non-coding region, a polynucleotide sequence shown in SEQ ID No.3 and a 3 ' non-coding region, wherein the 5' end non-coding region can comprise a promoter sequence, an enhancer sequence or/and 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 may comprise a terminator sequence, an mRNA cleavage sequence, and the like.

The invention further discloses a recombinant host cell or a recombinant bacterium containing the coding gene of the type III polyketide synthase AspKS 5; wherein, the recombinant bacteria include but are not limited to recombinant Escherichia coli or recombinant yeast cells.

In addition, the polynucleotide of the AspKS5 encoding gene shown in SEQ ID No.3 may be optimized by one skilled in the art to enhance expression efficiency in a host. For example, polynucleotides may be synthesized using optimization of preferred codons of the target host to enhance expression efficiency in the target host.

The chimeric gene or the expression cassette obtained by the chimeric or connected gene shown in SEQ ID No.3 of the invention and other genes belongs to the protection scope of the invention; the recombinant expression vector containing the chimeric gene or the expression cassette also belongs to the protection scope of the invention.

The aquilaria sinensis polyketide synthase AsPKS5 provided by the invention has multiple enzyme activities, can be applied to the aspects of catalytic synthesis of benzyl propiophenone, pyrone or quinolone compounds and production of natural products of intermediates of the compounds, and the like, can also be applied to guidance of molecular breeding of medicinal plants containing related substances, and can be applied to in vivo (in vitro) synthesis preparation of benzyl propiophenone, pyrone or quinolone compounds.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, the preferred methods, devices, and materials are now described.

The term "homology" refers to sequence similarity to a native nucleic acid sequence. "homology" includes a nucleotide sequence having preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity to the nucleotide sequence of the regulatory fragment of the present invention. Homology can be assessed visually or by computer software. Using computer software, homology between two or more sequences can be expressed as a percentage (%), which can be used to assess homology between related sequences.

By "variant" is meant a substantially similar sequence, and for polynucleotides, a variant comprises a deletion, insertion, or/and substitution of one or more nucleotides at one or more sites in the native polynucleotide. For polynucleotides, conservative variants include those that do not alter 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, for example, variants of polynucleotides which still encode the amino acid sequence shown in SEQ ID No.4 by site-directed mutagenesis or by recombinant means (e.g., DNA shuffling).

The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other upon hydrogen bonding between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are reverse complementary to each other when the sequences are viewed in both 5 'to 3' directions. It is also known in the art that two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% perfectly complementary.

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

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

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

The term "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region may 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, a promoter region is "operably linked" to the nucleic acid sequence.

The terms "transformation", "transgene", and "recombinant" herein refer to a host cell or organism, such as a bacterial 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 genome of the host, or the nucleic acid molecule may also be present 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 transgenic progeny thereof. A "untransformed", or "non-recombinant" host refers to a wild-type organism, such as a bacterium or a plant, which does not comprise a heterologous nucleic acid molecule.

The term "promoter" refers to any of the following nucleic acid sequences (e.g., DNA sequences): such sequences are recognized by DNA-dependent RNA polymerase during transcription initiation and bind (directly or indirectly) resulting in the production of RNA molecules complementary to the transcribed DNA; such regions may also be referred to as "5' regulatory regions". Promoters are generally located upstream of the 5' untranslated region (UTR) that is present in front of the coding sequence to be transcribed and have regions that serve as binding sites for RNA polymerase II and other proteins such as transcription factors to initiate transcription of an operably linked gene. The promoter itself may contain sub-elements (i.e., promoter motifs) such as cis-elements or enhancer domains that regulate transcription of an operably linked gene. The promoter and the linked 5' UTR are also referred to as "promoter regions".

Drawings

FIG. 1 is a diagram showing the alignment of the amino acid sequences of the type III polyketide synthase AsPKS5 of the present invention and several other type III polyketide synthases.

FIG. 2 is a SDS-PAGE pattern of type III polyketide synthase AsPKS5 of the present invention.

FIG. 3 is a reaction scheme of the type III polyketide synthase AsPKS5 of the present invention catalyzing p-coumaroyl-CoA with malonyl-CoA to produce p-hydroxybenzylideneacetone or ferulic acid-acyl-CoA with malonyl-CoA to produce 3-methoxy-4-hydroxybenzylideneacetone.

FIG. 4 is a diagram of the reaction of type III polyketide synthase AsPKS5 of the present invention to catalyze the reaction of propionyl-CoA with malonyl-CoA to produce 4-hydroxy-6-phenethylpyran-2-one.

FIG. 5 is a reaction scheme of type III polyketide synthase AsPKS5 catalyzing the reaction of 2- (methylamino) benzoyl coenzyme A with malonyl coenzyme A to produce 4-hydroxy-1-methyl-2-quinanone.

FIG. 6 is a reaction scheme of type III polyketide synthase AsPKS5 catalyzing the reaction of 2- (methylamino) benzoyl coenzyme A with 3-oxo-5-phenylpentanoic acid to form 1-methyl-2-phenethylquinolin-4 (1H) -one according to the present invention.

FIG. 7 is a mass spectrum of various products catalysed by the type III polyketide synthase AsPKS5 of the present invention.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Analysis or detection methods used in embodiments of the invention

1. Agarose gel electrophoresis analysis

The sample was added to agarose gel containing polymeric M5 HiPure Gelred Plus nucleic acid dye, separated at 120V for 20min and the results were detected using a gel imaging system.

SDS-PAGE gel electrophoretic analysis

SDS-PAG (5% concentrated gel, 10% separation gel) was applied using the electrophoresis tank and electrophoresis apparatus power supply of the American Bio-Rad company, the sample loading volume was 10. mu.L, 80V electrophoresis was performed first, and after the sample entered the separation gel, the voltage was adjusted to 100V until bromophenol blue just exited, and the electrophoresis was stopped. After staining with Coomassie brilliant blue, decolorized and photographed.

3. Liquid phase mass spectrometry detection

Detection was performed using a liquid phase mass spectrometer UPLC-Q-TOF-MS (Waters, USA). Using chromatographic column Acquity UPLC BEH C ₁₈ Column (2.1X 100mm,1.7 μm, Waters), flow rate 0.1mL/min, full wavelength scan of DAD detector, gradient elution with acetonitrile-0.1% formic acid water: 0-5min, 5-30% acetonitrile; 5-11min, 30-45% acetonitrile; 11-22min, and 45-95% acetonitrile (the percentage concentration of the acetonitrile is volume concentration).

Example 1 cloning and sequence analysis of Aquilaria sinensis Polyketide synthases (PKS) AspKS5 Gene

(1) Extraction of total RNA of aquilaria sinensis callus and synthesis of cDNA first chain

Taking a proper amount of injury-induced aquilaria sinensis callus to extract total RNA, and carrying out reverse transcription by a primer with a joint to obtain cDNA. Preferably, 100mg of Aquilaria sinensis callus induced with 150mM NaCl for 3 days is ground in liquid nitrogen, and total RNA is extracted using Ether company plant RNA rapid extraction kit RN38 according to the instruction. RNA concentration and quality were determined using a Thermo Scientific NanoDrop spectrophotometer and RNA quality was determined by agarose gel electrophoresis.

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

(2) Cloning of AspKS5 Gene

Designing specific primers, wherein specific primer sequences are as follows:

AsPKS5-F:5’-ATGGCAGCCCAACCTGT-3’(SEQ ID NO.1)

AsPKS5-R:5’-TTACAGTGAAGCCCTAAGCGC-3’(SEQ ID NO.2)

taking cDNA of the aquilaria sinensis callus as a template, amplifying genes of AsPKS3 and AsPKS5 by PCR and sequencing to obtain a nucleotide sequence of the AsPKS5 gene as shown in SEQ ID NO.3, wherein an initiation codon is ATG and a termination codon is TAA; the translated protein coding sequence is shown in SEQ ID NO. 4.

The amplification product of the AspKS5 gene obtained by PCR was ligated with the pMD-19T vector using the pMD-19T kit of TaKaRa company according to the instructions to construct the pMD-19T-AspKS5 cloning vector. The constructed recombinant vector is transformed into a clone competent cell E.coli DH5 alpha, an LB solid plate containing ampicillin, IPTG and X-Gal is coated, a white single colony is selected for culture, and colony PCR verification and sequencing verification are carried out.

The AspKS5 amino acid sequence was compared with the previously reported amino acid sequence of polyketide synthase in 4 Aquilaria sinensis. The similarity of the AspKS5 to both AspKS1 and AspKS2 is less than 40%; the similarity of AspKS5 and AsCHS2 is 62.92%; the similarity of AsPKS5 to AsPECPS was 83.73%. The results of multiple sequence comparisons of AspKS5 with 4 reported Aquilaria sinensis polyketide synthases and chalcone synthase in alfalfa are shown in FIG. 1. In FIG. 1, the conserved catalytic activity central amino acid sites (Cys164, His303, Asn336) of type III polyketide synthase and the key amino acid sites (Thr132, Ser133, Thr197, Phe215, Gly256, Leu263, Phe265, Ser338) capable of regulating the size or shape of the active cavity are marked, and the site numbers are the corresponding amino acid numbers in alfalfa CHS.

Example 2 prokaryotic expression vector construction and heterologous expression of AspKS5

Primers were designed without stop codons and the sequence was as follows:

AsPKS5-exp-F:5’-ATGGCAGCCCAACCTGT-3’(SEQ ID NO.5)

AsPKS5-exp-R:5’-CAGTGAAGCCCTAAGCGC-3’(SEQ ID NO.6)

the positive colonies obtained in example 1 were cultured to extract recombinant plasmids as templates for PCR amplification, and the gene coding sequences without stop codons were obtained. All-purpose gold corporation

The Expression Kit of Blunt E2, the coding sequence of AspKS5 gene obtained by PCR was Blunt-end ligated with pEASY-Blunt-E2 vector according to the instructions to obtain recombinant Expression vector pEASY-Blunt-E2-AspKS 5.

The constructed plasmid pEASY-Blunt-E2-AspKS5 is transformed into a prokaryotic expression strain E.coli BL21(DE3), an LB solid plate containing ampicillin is coated, a single colony is selected for culture, and plasmids are extracted for PCR. Screening positive clones to obtain prokaryotic expression engineering bacteria BL21(DE3) -pEASY-Blunt-E2-AspKS 5. 50. mu.L of the culture broth was inoculated into 5mL of LB liquid medium containing 100. mu.g/mL ampicillin, and cultured overnight at 37 ℃ and 200 rpm. Then according to the following steps of 1: inoculating 100-proportion inoculum size into 300mL LB liquid culture medium containing ampicillin, activating at 37 deg.C and 200rpm, and culturing to 0D ₆₀₀ When the concentration was about 0.6, 0.5mM IPTG was added. BL21(DE3) -pEASY-Blunt-E2-AspKS5 was cultured at 180rpm at 24 ℃ for 12 h. The harvested bacterial liquid was centrifuged at 5000rpm and 4 ℃ to remove the supernatant. Then, the lysate is used for resuspending the thallus precipitate, after ultrasonication, the AsPK5 protein is purified by using a nickel ion affinity chromatographic column according to the fact that the recombinant protein contains a His label at the C end, the purified protein is detected by SDS-PAGE electrophoresis, and electricity is applied toThe electrophoresis detection result is shown in FIG. 2, and the size of the target protein is about 45 kDa. Concentration and desalination by a filter membrane (Millipore, 10kD) can obtain protein with the concentration of more than 1 mg/mL.

Experimental example 1 enzyme catalysis of AspKS5 on P-coumaroyl-CoA and malonyl-CoA to produce p-hydroxybenzylideneacetone

mu.M of the starting substrate (p-coumaroyl-CoA), 160. mu.M of malonyl-CoA and 20. mu.g of the purified recombinant protein were added together to a total volume of 250. mu.L of 100mM potassium phosphate buffer (KPB buffer) (pH 7.0), reacted at 32 ℃ in a thermostatic water bath for 6 hours, and then 20. mu.L of 20% HCl was added to terminate the enzyme reaction. Extracting the reaction solution with 500 μ L ethyl acetate for 3 times, mixing the extractive solutions, centrifuging at low temperature, concentrating, drying, re-dissolving with 100 μ L methanol, and detecting with liquid phase mass spectrometer UPLC-Q-TOF-MS. And (3) analyzing the high-resolution mass spectrum information, predicting the molecular formula, and comparing with standard products or literature report information to identify reaction products.

According to the measured product and the chemical reaction principle, the reaction of the experimental example is that the Aquilaria sinensis polyketide synthase AsPKS5 catalyzes 1 molecule of p-coumaroyl coenzyme A to be condensed with 1 molecule of malonyl coenzyme A to generate p-hydroxybenzylideneacetone, the related reaction formula is shown in figure 3, and the mass spectrometric identification of the generated product p-hydroxybenzylideneacetone is shown in figure 7.

EXAMPLE 2 AsPKS5 enzyme catalysis of Feruloyl-CoA with malonyl-CoA to generate 3-methoxy-4-hydroxybenzylideneacetone

The procedure was carried out in accordance with the specific procedures of Experimental example 1, replacing the starting substrate with feruloyl-CoA, and the other conditions and procedures were the same as those of Experimental example 1. According to the measured product and the principle of chemical reaction, the reaction formula of the experimental example is shown in FIG. 3, i.e., Aquilaria sinensis polyketide synthase AsPKS5 catalyzes the condensation of feruloyl-CoA with malonyl-CoA to generate 3-methoxy-4-hydroxybenzylideneacetone. The mass spectrometric identification of the product 3-methoxy-4-hydroxybenzylideneacetone formed is shown in FIG. 7.

EXAMPLE 3 AsPKS5 enzyme catalysis of benzoylcoenzyme A and malonyl coenzyme A to produce 4-hydroxy-6-phenethylpyran-2-one

The procedure was carried out in accordance with the specific procedures of example 1 except that the starting substrate was replaced with phenylpropanoyl coenzyme A and the other conditions and the procedure were the same as those of example 1. According to the measured products and the principle of chemical reaction, the reaction formula of the embodiment is shown in FIG. 4, Aquilaria sinensis polyketide synthase AsPKS5 catalyzes 1 molecule of propionyl-CoA to condense with 2 molecules of malonyl-CoA to produce 4-hydroxy-6-phenethylpyran-2-one, and the mass spectrometric identification of the produced product 4-hydroxy-6-phenethylpyran-2-one is shown in FIG. 7.

EXAMPLE 4 AsPKS5 enzyme catalysis of 2- (methylamino) benzoyl-CoA with malonyl-CoA to produce 4-hydroxy-1-methyl-2-quino-none

The procedure was carried out in accordance with the specific procedures of Experimental example 1, replacing the starting substrate with 2- (methylamino) benzoyl coenzyme A, and the other conditions and procedures were the same as those of Experimental example 1. Through detection, the relevant reaction formula in this example is shown in fig. 5, aquilaria sinensis polyketide synthase AsPKS5 catalyzes 1 molecule of 2- (methylamino) benzoyl coenzyme a to condense with 1 molecule of malonyl coenzyme a to generate 4-hydroxy-1-methyl-2-quina-none, and the mass spectrometric identification of the produced product 4-hydroxy-1-methyl-2-quina-none is shown in fig. 7.

EXAMPLE 5 AsPKS5 enzyme catalysis of 2- (methylamino) benzoyl coenzyme A with 3-oxo-5-phenylpentanoic acid to produce 1-methyl-2-phenethylquinolin-4 (1H) -one

The procedure was carried out in accordance with the specific procedures of Experimental example 1 except that the reaction substrates were replaced with 2- (methylamino) benzoyl-CoA and 3-oxo-5-phenylpentanoic acid, and the other conditions and procedures were the same as those of Experimental example 1. According to the measured products and the principle of chemical reaction, the relevant reaction formula in this experimental example is shown in FIG. 6, Aquilaria sinensis polyketide synthase AsPKS5 catalyzes 1 molecule of 2- (methylamino) benzoyl coenzyme A and 1 molecule of 3-oxo-5-phenylpentanoic acid to generate 1-methyl-2-phenethylquinolin-4 (1H) -one, and the mass spectrometric identification of the produced product 1-methyl-2-phenethylquinolin-4 (1H) -one is shown in FIG. 7.

SEQUENCE LISTING

<110> institute of medicinal plants of academy of Chinese medical science

<120> Aquilaria sinensis (lour.) Kuntze-derived type III polyketide synthase AspKS5, and coding gene and application thereof

<130> BJ-2004-220314A

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 17

<212> DNA

<213> Artifical sequence

<400> 1

atggcagccc aacctgt 17

<210> 2

<211> 21

<212> DNA

<213> Artifical sequence

<400> 2

ttacagtgaa gccctaagcg c 21

<210> 3

<211> 1257

<212> DNA

<213> Aquilaria sinensis

<400> 3

atggcagccc aacctgtgga ggggatgacc aaggcagaga gggccaccgg accagcaacc 60

gtcctcgcca ccgccactgc cacgcccccc aacgtcttcc tacagtcgga gttccccgac 120

ttctatttcc gggtcactag gagcgagcac atgtccgacc tcaaggaaaa attcaagcga 180

atgtgcgtga ggacgacggt caggaagaga cacatgatcc tgacggagga gatcttaaac 240

aagaactccg ccattgctga ctattggtcg ccgtcgctgg ccgcccggca caacctcgcg 300

ctggccaaca tcccccagct cggaaaggaa gccgctgaca aggccatcaa ggagtggggc 360

cagcccaagt ccaaaatcac ccaccttatc ttctgcacct ccgctggcgt ccacatgcct 420

ggcgccgact accagctcac caagctcctc ggcctcaacc cctccatcag ccgcgtcatg 480

ctctataacc ttggctgcta cgccggtgga accgcactcc gagtcgccaa agacctcgcc 540

gagaacaact gtggcgccag ggtccttgtc gtctgctcgg agaccaacct actccacttc 600

cggggcccgt cggagaccca catcgactcg ctcataactc aatctctctt cggggacgga 660

gcggccgctc tcattgtagg ttctgatccc gatctccaga ccgagcgtcc gctgtacgaa 720

ctcatctcgg cgtcgcagag gatactcccg gagtcagagg atgcgattgt gggacgcttg 780

accgaagtag gtctagccgc ctatttgcct aaaaacctcc ccaaactgat ctcgacaaac 840

atcagaagca tcttggagga ggccttggcg ccgaccgggg tccaagactg gaactctatc 900

ttctggattc ttcaccctgg catgccggcg attctggacc aaacagagaa gctgctccag 960

ctcgataaaa agaaactcaa ggcaactcga cacgtgctca gtgaatttgg gaatatgttt 1020

ggtgccaccg tacttttcat cttggaccag atgaggaaag gcgcagtggc ggaagggaag 1080

tccaccaccg gggaaggctg cgagtggggc gttcttttcg cgttcgggcc aggcctcacc 1140

gttgagaccg tgttgctacg tagtgtcact actggttgcc tcactaacgg tatgaaagtt 1200

tatcaccatg agaaagcaac tagaagcaag ccaccagcgc ttagggcttc actgtaa 1257

<210> 4

<211> 418

<212> PRT

<213> Aquilaria sinensis

<400> 4

Met Ala Ala Gln Pro Val Glu Gly Met Thr Lys Ala Glu Arg Ala Thr

1 5 10 15

Gly Pro Ala Thr Val Leu Ala Thr Ala Thr Ala Thr Pro Pro Asn Val

20 25 30

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

35 40 45

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

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

85 90 95

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

260 265 270

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

275 280 285

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

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

325 330 335

Gly Asn Met Phe Gly 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 Phe Ala Phe Gly Pro Gly Leu Thr Val Glu Thr Val

370 375 380

Leu Leu Arg Ser Val Thr Thr Gly Cys Leu Thr Asn Gly Met Lys Val

385 390 395 400

Tyr His His Glu Lys Ala Thr Arg Ser Lys Pro Pro Ala Leu Arg Ala

405 410 415

Ser Leu

<210> 5

<211> 17

<212> DNA

<213> Artifical sequence

<400> 5

atggcagccc aacctgt 17

<210> 6

<211> 18

<212> DNA

<213> Artifical sequence

<400> 6

cagtgaagcc ctaagcgc 18

Claims

1. An Aquilaria sinensis (Aquilaria sinensis) derived type III polyketide synthase AsPKS5, characterized in that the amino acid sequence thereof is selected from any one of (a) or (b):

(a) an amino acid sequence shown as SEQ ID No. 4;

or (b) a protein variant derived from the amino acid sequence shown in SEQ ID No.4 by substitution, deletion or/and insertion of one or more amino acid residues and still having the function or activity of a plant type III polyketide synthase.

2. An encoding gene of Aquilaria sinensis (Aquilaria sinensis) derived type III polyketide synthase AspKS5, wherein the nucleotide sequence is shown as (a), (b) or (c):

(a) a polynucleotide sequence shown as SEQ ID No. 3;

or (b) a polynucleotide sequence capable of hybridising under stringent hybridisation conditions to the complement of SEQ ID No.3 and which encodes a protein which still has the function or activity of a plant type III polyketide synthase;

or (c) a polynucleotide sequence which has at least more than 85% homology with the polynucleotide sequence of SEQ ID No.3, and the protein encoded by the polynucleotide still has the function or activity of plant type III polyketide synthase; preferably, the polynucleotide sequence has at least 90% homology with the polynucleotide sequence of SEQ ID No.3 and encodes a protein which still has the function or activity of a plant type III polyketide synthase.

3. An expression cassette comprising the coding gene of claim 2.

4. A recombinant expression vector comprising the coding gene of claim 2.

5. A host cell comprising the recombinant expression vector of claim 4.

6. Use of the type iii polyketide synthase AsPKS5 of claim 1 for the catalytic synthesis of benzyl propiones, pyrones, or quinolones.

7. Use of the gene encoding the type iii polyketide synthase AsPKS5 of claim 2 or the expression cassette of claim 3 for the catalytic synthesis of benzyl propiophenones, pyrones or quinolones.

8. The recombinant expression vector of claim 4, for use in catalytic synthesis of benzyl propiophenone, pyrone or quinolone compounds.

9. Use of the host cell of claim 5 for the catalytic synthesis of benzyl propiophenone, pyrone or quinolone compounds.

10. Use according to any one of claims 6 to 9, comprising: (1) catalyzing p-coumaroyl-coenzyme A and malonyl-coenzyme A to generate p-hydroxybenzylideneacetone; or (2) catalyzing feruloyl coenzyme A and malonyl coenzyme A to generate 3-methoxy-4-hydroxybenzylideneacetone; or (3) catalyzing the reaction of propionyl-CoA and malonyl-CoA to produce 4-hydroxy-6-phenethylpyran-2-one; or (4) catalyzing the reaction of 2- (methylamino) benzoyl coenzyme A with malonyl coenzyme A to produce 4-hydroxy-1-methyl-2-quina lone; or (5) catalyzing the reaction of 2- (methylamino) benzoyl-coenzyme A with 3-oxo-5-phenylpentanoic acid to form 1-methyl-2-phenethylquinolin-4 (1H) -one.