WO2023241224A1 - Method for biosynthesizing nootkatone and carrier - Google Patents

Abstract

Disclosed in the present application are a method for biosynthesizing nootkatone and a carrier. The method comprises using recombinant bacteria, which is capable of expressing valentene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase, to synthesize nootkatone; the valentene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase are derived from Alpinia oxyphylla Miq. The present application identifies a complete set of enzymes and encoding genes for synthesizing nootkatone from farnesyl pyrophosphate, and provides a method for biosynthesizing nootkatone by using said enzymes. Employing the enzymes and biosynthesis method of the present application is beneficial to obtaining a high-yield strain of nootkatone and increasing the yield of biosynthetic nootkatone.

Description

A method and carrier for biosynthesizing nokatone

Cross-references to related applications

This application claims the priority rights and interests of Chinese patent application CN202210671358.1, which was submitted to the China Intellectual Property Office on June 14, 2022 and is titled "A method and carrier for biosynthesizing nokalone". The entire contents of which are hereby incorporated by reference into this application in their entirety.

Technical field

This application belongs to the field of nokatone biosynthesis, and specifically relates to a method and carrier for biosynthesizing nokatone.

Background technique

Nokatone is found in Alaskan cypress, grapefruit and other plants. It is a natural sesquiterpene ketone with a pleasant grapefruit aroma, so nokatone can be used to prepare spices or as a grapefruit flavoring. Agent use. In addition, people have also found that nokalone has the effect of repelling mosquitoes. In August 2020, the U.S. Environmental Protection Agency approved the use of nokalone as an insect repellent or insecticide. Therefore, nokalone can also be made into mosquito repellent water and tick repellent. When used as an insect repellent, there are also relevant studies showing that nokatone can stimulate the human body's sympathetic nerves to secrete related hormones and play a role in burning fat and losing weight. The above shows that nokatone has high application value.

Nokatone can be extracted from plants, but the low content of nokalone in plants limits its application. Nokatone can also be obtained by chemical synthesis of valensene, but the reaction process involves toxic heavy metals, which also affects the application of this method. The use of microbial cell factories to fully synthesize nokatone is a promising approach.

Contents of the invention

The purpose of this application is to provide a method and carrier for biosynthesizing nokatone.

In order to solve the above technical problems, this application proposes the following technical solutions:

The first aspect of the present application provides a method for biosynthesizing nokatone, which includes using a recombinant bacterium capable of expressing valentene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase to synthesize nokatone. Kadone; wherein, the valenene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase come from Puzzle.

The second aspect of the present application provides an enzyme for the synthesis of nokatone, which has the same characteristics as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 Or the amino acid sequence shown in SEQ ID NO. 6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. sequence.

The third aspect of the present application provides a polynucleotide molecule, which includes at least one of the nucleotide sequence encoding the enzyme provided in the second aspect of the present application or its complementary sequence.

The fourth aspect of the present application provides a nucleic acid construct, which contains at least one of the polynucleotide molecules provided by the third aspect of the present application.

The fifth aspect of the present application provides a recombinant bacterium, which contains the polynucleotide molecule of the third aspect of the present application, or the nucleic acid construct of the fourth aspect of the present application; the recombinant bacterium is obtained by combining the polynucleotide molecule or the nucleic acid construct of the fourth aspect of the present application. The nucleic acid construct is introduced into a host cell to obtain it; preferably, the host cell is a eukaryotic cell; more preferably, it is Saccharomyces cerevisiae.

The sixth aspect of this application provides the enzyme of the second aspect of this application, the polynucleotide molecule of the third aspect of this application, the nucleic acid construct of the fourth aspect of this application or the recombinant bacteria of the fifth aspect of this application in producing valene, Uses in nocanol and/or nocanone.

This application identifies a complete set of enzymes and encoding genes for synthesizing nokatone from farnesyl pyrophosphate, and provides a method for biosynthesizing nokatone using the enzyme. Using the enzyme and biosynthetic method of this application is beneficial to obtain Nokatone high-yield strain increases the production of biosynthetic nokatone.

Description of the drawings

Figure 1 is a schematic diagram of the construction of plasmid pZY900;

Figure 2 is a schematic diagram of the construction of plasmid pDXYZ3;

Figure 3 is a schematic diagram of the construction of plasmids pDXVS1 and pDXVS2;

Figure 4 is a schematic diagram of the construction of plasmids pDXNL1, pDXNL2, pDXNL3 and pCK;

Figure 5 is a schematic diagram of the construction of plasmid pDXNT1;

Figure 6 shows the extracted ion chromatogram of valentene and valentene standard in the fermentation product of JDXYZ3 strain;

Figure 7 shows the shake flask fermentation products of CK strain, JDXNL1 strain (marked as CYP6 in the figure), JDXNL2 strain (marked as CYP9 in the figure) and JDXNL3 strain (marked as AoKo in the figure), and the extracted ion chromatography of the nocanol standard. picture;

Figure 8 shows the shake flask fermentation products of JDXNT1 strain (marked as CYP6 in the figure), JDXNT2 strain (marked as CYP9 in the figure) and JDXNT3 strain (marked as AoKo in the figure), and the extracted ion chromatogram of the nokatone standard.

Detailed ways

The terminology and descriptions used herein are for the purpose of describing particular embodiments only and are not intended to limit the application. 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 this disclosure belongs. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

definition

As used herein, the terms "a" and "an" as well as "the" and similar referents refer to the singular and the plural unless otherwise indicated herein or otherwise clearly contradicted by context.

As used herein, the terms "about" and "similar to" mean that a particular value is likely to be as determined by one of ordinary skill in the art. A margin of error is accepted, which may depend in part on how the value is measured or determined, or on limitations of the measurement system.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form. Unless expressly limited, the term "nucleic acid" or "polynucleotide" also includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and behave in a manner similar to naturally occurring nucleosides. Acid is metabolized in a similar manner (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo Methods). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs) and complementary sequences as well as clearly indicated sequences.

"Construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, linear or circular single- or double-stranded DNA or RNA polynucleotide molecule) that can Derived from any source, capable of integrating into the genome or replicating autonomously, it can be operably linked to one or more polynucleotide molecules. In the present application, a construct generally includes a polynucleotide molecule of the present application operably linked to transcription initiation regulatory sequences that direct the transcription of the polynucleotide molecule of the present application in a host cell. Heterologous promoters or endogenous promoters may be used to direct expression of the nucleic acids of the present application.

"Vector" refers to any recombinant nucleic acid construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell). The vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of a protein of interest in an organism. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, vectors with an origin of replication functioning in the host cell). Other vectors can be introduced into a host cell, integrated into the host cell's genome, and thus replicated with the host genome. In addition, certain preferred vectors are capable of directing the expression of foreign genes to which they are linked. One type of vector is a "plasmid," which generally refers to a circular double-stranded DNA circle into which additional DNA segments (foreign genes) can be ligated, but may also include linear double-stranded molecules, such as those derived from polymerase chains. Amplification by reaction (PCR) or treatment of circular plasmid with restriction enzymes yields linear double-stranded molecules.

Plasmid vectors include vector backbone (i.e. empty vector) and expression framework.

The term "expression framework" refers to a sequence with the potential to encode a protein.

The term "host cell" refers to a cell, such as a microorganism, that can introduce a gene of interest and provide conditions for cloning and/or expression of the gene of interest.

The term "recombinant bacteria" refers to bacteria that have been genetically modified (such as bacteria, yeast, actinomycetes, etc.), which means that foreign gene fragments have been introduced into their bacteria. One way of modification includes bacteria. The bacterial genome is changed after new DNA fragments are introduced. Another way includes the introduction of artificially constructed or modified plasmids into the bacterial cells, so that the bacterial cells gain the ability to express the target genes.

The first aspect of the present application provides a method for biosynthesizing nokatone, which includes using a recombinant bacterium capable of expressing valentene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase to synthesize nokatone. Cataone; wherein, the valentene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase are from Puzzle.

Alpinia oxyphylla Miq. (Latin scientific name: Alpinia oxyphylla Miq.), alias: Alpinia oxyphylla Miq. Zingiberaceae, Alpinia genus is a perennial herbaceous plant. Through in-depth research on Puzzle, the inventor identified a complete set of enzymes and coding genes involved in the synthesis of nokatone from Puzzle, and based on this, provided a method for biosynthesizing nokatone. Among them, the implementation of valencene synthase can synthesize valencene using farnesyl pyrophosphate as a substrate; valencene generates nocanol under the joint action of cytochrome P450 oxidase and cytochrome P450 oxidoreductase, and then nocanol is synthesized. Under the action of alcohol dehydrogenase, nokatone is obtained.

In some embodiments, the valentene synthase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO. , an amino acid sequence with 98%, 99% or 100% sequence identity;

The cytochrome P450 oxidase is selected from at least one of cytochrome P450 oxidase CYP6, cytochrome P450 oxidase CYP9 and cytochrome P450 oxidase AoKo; wherein, the cytochrome P450 oxidase CYP6 has the same properties as SEQ ID NO.2 The amino acid sequence shown has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; cytochrome P450 oxidase CYP9 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.3 The amino acid sequence; cytochrome P450 oxidase AoKo has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO.4 , an amino acid sequence with 99% or 100% sequence identity;

The cytochrome P450 oxidoreductase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 of the amino acid sequence shown in SEQ ID NO.5 % or 100% sequence identity of the amino acid sequence;

The alcohol dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or Amino acid sequence with 100% sequence identity.

In some embodiments, the amino acid sequence of the valencene synthase YZT3 is as follows (amino terminus to carboxyl terminus):

In some embodiments, the amino acid sequence of the cytochrome P450 oxidase CYP6 is as follows (amino terminus to carboxyl terminus):

In some embodiments, the amino acid sequence of the cytochrome P450 oxidase CYP9 is as follows (amino terminus to carboxyl terminus):

In some embodiments, the amino acid sequence of the cytochrome P450 oxidase AoKo is as follows (amino terminus to carboxyl terminus):

In some embodiments, the amino acid sequence of the cytochrome P450 oxidoreductase AoCPR is as follows (amino terminus to carboxyl terminus):

In some embodiments, the amino acid sequence of the alcohol dehydrogenase AoADH is as follows (amino terminus to carboxyl terminus):

In some embodiments, the recombinant bacteria are capable of synthesizing farnesyl pyrophosphate.

In some embodiments, the recombinant bacterium is capable of expressing acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, methanol At least one of valonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesyl pyrophosphate synthase.

In some embodiments, the hydroxymethylglutaryl-CoA reductase is a truncated hydroxymethylglutaryl-CoA reductase, which truncates the endoplasmic reticulum localization sequence and enhances the enzyme's location in the cytoplasm. stability.

The inventor found that acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, Mevalonate pyrophosphate decarboxylase and isoprene pyrophosphate isomerase are enzymes in the mevalonate pathway, which can synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), both of which can be used as precursors to synthesize farnesyl pyrophosphate (FPP) under the catalysis of farnesyl pyrophosphate synthase, and FPP is the substrate for the biosynthesis of nokatone. Therefore, when When the recombinant bacterium can express at least one of the enzymes in the mevalonate pathway and farnesyl pyrophosphate synthase, it is beneficial to the synthesis of FPP and further to the biosynthesis of nokatone.

The second aspect of this application provides an enzyme for the synthesis of nokatone, which has the characteristics of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 Or the amino acid sequence shown in SEQ ID NO.6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of amino acids. sequence.

The third aspect of the present application provides a polynucleotide molecule, which includes at least one of the nucleotide sequence encoding the enzyme of the second aspect of the present application or its complementary sequence.

In some embodiments, the polynucleotide molecule comprises SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or The nucleotide sequence shown in SEQ ID NO.13 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Nucleotide sequence.

In some embodiments, the nucleotide sequence encoding valencene synthase YZT3 (wild type) is as follows (5' end to 3' end):

In some embodiments, by optimizing the nucleotide sequence of the wild-type valencene synthase YZT3 according to the codon preference of Saccharomyces cerevisiae, the nucleotide sequence of the encoding gene AoVS of valencene synthase is obtained as follows ( 5' end to 3' end):

In some embodiments, the nucleotide sequence encoding cytochrome P450 oxidase CYP6 (wild type) is as follows (5' end to 3' end):

In some embodiments, the nucleotide sequence encoding cytochrome P450 oxidase CYP9 (wild type) is as follows (5' end to 3' end):

In some embodiments, the nucleotide sequence encoding cytochrome P450 oxidase AoKo (wild type) is as follows (5' end to 3' end):

In some embodiments, the nucleotide sequence encoding cytochrome P450 oxidoreductase AoCPR (wild type) is as follows (5' end to 3' end):

In some embodiments, the nucleotide sequence encoding alcohol dehydrogenase AoADH (wild type) is as follows (5' end to 3' end):

The fourth aspect of the present application provides a nucleic acid construct comprising at least one polynucleotide molecule of the third aspect of the present application. In this application, the polynucleotide molecule connected to the nucleic acid construct is called the target gene, and the enzyme encoded by the polynucleotide molecule is called the target protein.

In some embodiments, the nucleic acid construct also contains regulatory elements that regulate the expression of the gene encoding the target, such as a promoter, a terminator, etc. Exemplarily, the promoter can be a constitutive promoter such as P _TEF1 , P _TDH3 , _PGPM1 , _PTPI1 , etc., inducible promoters such as _PHXT1 (high concentration glucose induction), _PCUP1 (copper ion induction), _PGAL1 , _PGAL2 , _PGAL7 , _PGAL10 (galactose induction), etc., this Those skilled in the art can choose according to needs, and this application does not limit it here.

In some embodiments, the nucleic acid construct also contains a marker gene for screening recombinant bacteria containing the target gene or target protein, such as a leucine screening marker, a histidine screening marker, a tryptophan screening marker, a urine screening marker, and a leucine screening marker. Pyrimidine screening markers, etc., can be specifically selected by those skilled in the art according to needs, and are not limited in this application.

In some embodiments, the nucleotide sequence is located between two insertion elements used to integrate the nucleotide sequence into the genome of the host cell.

In some embodiments, a nucleotide sequence with insertion elements connected at both ends is connected to a nucleic acid construct, such as a plasmid backbone of a plasmid vector. When the nucleic acid construct is used to introduce a target gene into a host cell, it can be restricted by restriction. Endonuclease and other tools are used to digest the nucleic acid construct to obtain a linearized target gene fragment with insertion elements connected at both ends. The linearized target gene fragment is introduced into the host cell and allowed to pass through both ends. The end insertion element is inserted into the corresponding position of the host cell genome, thereby obtaining the recombinant bacterium of the present application.

Those skilled in the art can use conventional methods to introduce linearized target gene fragments into host cells. For example, for yeast, the lithium acetate method can be used, and for E. coli, the calcium transfer method can be used. This is a routine operation in this field. , this application is not limited here.

In some embodiments, the two insertion elements appear in pairs, for example, they can be the left and right homology arms of leu2, the left and right homology arms of Ura3, the left and right homology arms of YPRCdelta15, etc. The homology arms of different genes can combine the target genes. Integration into different positions of the host cell genome. Those skilled in the art can specifically select the type of homology arm according to the position where they wish to be integrated into the host cell genome. This application is not limited here.

In some embodiments, regulatory elements such as promoters and terminators for regulating the expression of the target gene are also included between the two inserted elements. This application does not limit the types of promoters and terminators.

In some embodiments, the nucleic acid construct further comprises encoding acetoacetyl-CoA thiolase (ERG10), hydroxymethylglutaryl-CoA synthase (ERG13), hydroxymethylglutaryl-CoA reductase ( HMG1), truncated hydroxymethylglutaryl-CoA reductase (tHMG1), mevalonate kinase (ERG12), mevalonate-5-phosphate kinase (ERG8), mevalonate pyrophosphate decarboxylase (MVD1), isoprene pyrophosphate isomerase (IDI1), and farnesyl pyrophosphate synthase (ERG20); the names of the genes encoding these enzymes are shown in brackets.

Exemplary, non-limiting disclosure of genes encoding the above enzymes is as follows:

ERG10(Accession/GENE ID:856079), ERG13(Accession/GENE ID:854913), tHMG1(Accession/GENE ID:854900, truncated 4-1659bp), ERG12(Accession/GENE ID:NM_001182715.1), ERG8( Accession/GENE ID: CP046093.1, 689693..691048), MVD1 (Accession/GENE ID: NM_001183220.1), IDI1 (Accession/GENE ID: NM_001183931.1), ERG20 (Accession/GENE ID: 853272).

In some embodiments, the nucleic acid construct is a plasmid vector; preferably, the plasmid vector is a eukaryotic expression vector.

In some embodiments, the nucleic acid construct includes a pRS426 plasmid backbone. The inventor found that the pRS426 plasmid backbone contains the AmpR screening marker suitable for E. coli, the URA3 screening marker suitable for S. cerevisiae, and the replicon suitable for E. coli and the multi-copy replicon of S. cerevisiae. Using the pRS426 plasmid backbone, It is beneficial to maintain a high copy of the plasmid containing the target gene after being introduced into Saccharomyces cerevisiae.

In some embodiments, the mutation present in the pRS426 plasmid backbone eliminates the restriction site BsaI in the pRS426 plasmid backbone, so that BsaI can be used as a restriction endonuclease when constructing the vector using the Goldengate method. .

In some embodiments, the nucleic acid construct includes a pESC-TRP plasmid backbone. The inventor found that the pESC-TRP plasmid backbone contains an AmpR selection marker suitable for E. coli, a TRP1 selection marker suitable for S. cerevisiae, and a replicon suitable for E. coli and a multi-copy replicon of S. cerevisiae. Using the pESC -The TRP plasmid backbone is conducive to maintaining a high copy of the plasmid containing the target gene after being introduced into Saccharomyces cerevisiae.

In some embodiments, the plasmid vector is at least one of pDXYZ3, pDXVS1, pDXVS2, pDXNL1, pDXNL2, pDXNL3, and pDXNT1. The construction schematic diagram of the plasmid vector is as shown in Figure 2, Figure 3, Figure 4 or Figure 5 ; Among them, the construction schematic diagram of plasmid pDXYZ3 is shown in Figure 2, the construction schematic diagram of plasmids pDXVS1 and pDXVS2 is shown in Figure 3, the construction schematic diagram of plasmids pDXNL1, pDXNL2, and pDXNL3 is shown in Figure 4, and the construction schematic diagram of plasmid pDXNT1 is shown in Figure 5. shown.

In some embodiments, the plasmid vector can be directly introduced into the host cell, or the plasmid vector can be digested with enzymes to obtain the target gene fragment containing the insertion element, and the gene fragment can be further integrated into the host cell. in the genome.

The fifth aspect of the present application provides a recombinant bacterium, which contains the polynucleotide molecule of the third aspect of the present application, or the nucleic acid construct of the fourth aspect of the present application; the recombinant bacterium is obtained by combining the polynucleotide molecule or the nucleic acid construct of the fourth aspect of the present application. The nucleic acid construct is introduced into a host cell to obtain it; preferably, the host cell is a eukaryotic cell; more preferably, it is Saccharomyces cerevisiae.

In some embodiments, the nucleic acid construct can be directly included in the recombinant bacterium. For example, the nucleic acid construct exists alone in the host cell in the form of a plasmid and expresses the enzyme required for the synthesis of nokatone.

In other embodiments, the polynucleotide molecule is integrated into the genome of the host cell. The polynucleotide molecule is integrated into the genome of the host cell, which is beneficial to the long-term stable expression of the target gene, thereby obtaining a recombinant bacterium capable of stable inheritance.

Those skilled in the art can use conventional methods to integrate polynucleotide molecules into the genome of the host cell. This application is not limited here. For example, the target gene can be connected between two insertion elements, and the insertion element can be used to integrate the polynucleotide molecules into the genome of the host cell. The target gene is inserted into the genome of the host cell. For example, the insertion element can be the left and right homology arms of leu2, the left and right homology arms of Ura3, and the left and right homology arms of YPRCdelta15. The homology arms of different genes are used to The target gene is inserted into different positions of the host cell genome. The inventor found that the recombinant bacteria of the present application can be obtained by inserting the target gene into a site that does not interfere with the normal physiological metabolism of the host cell.

In some embodiments, the copy number of the polynucleotide molecule in the genome of the recombinant bacterium is at least 1, preferably at least 2, and more preferably at least 3. Increasing the copy number of the polynucleotide molecule is beneficial to the high expression of the enzyme encoded by it.

In some embodiments, the recombinant bacterium is capable of expressing acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, methanol At least one of valonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesyl pyrophosphate synthase.

The inventor found that Saccharomyces cerevisiae can synthesize FPP endogenously. Therefore, in some preferred embodiments, using Saccharomyces cerevisiae as a host cell is beneficial to obtain recombinant bacteria that can efficiently synthesize nokatone.

The sixth aspect of this application provides the enzyme of the second aspect of this application, the polynucleotide molecule of the third aspect of this application, the nucleic acid construct of the fourth aspect of this application or the recombinant bacteria of the fifth aspect of this application in producing valene, Uses in nocanol and/or nocanone.

The method and carrier for biosynthesizing nokatone of the present application will be described below through specific examples. The following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. The plasmids involved in the following examples are all plasmids well known to those skilled in the art. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1 Expression vector and strain construction

1.1 Construction of universal vector for yeast expression

The specific construction process of plasmid pZY900: use Saccharomyces cerevisiae S288c genome (extraction method, see: Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform of Saccharomyces cerevisiae [D]. Wuhan University, 2015.2.3.6 Yeast genomic DNA extraction method) as a template, using primers 900-1F/1R, 900-2F/2R, 900-6F/6R, and 900-7F/7R were amplified respectively to obtain fragments 9001 (left homology arm of Leu2), 9002 (terminator tTDH2), and 9006 (gene ERG20 and Terminator tERG20), 9007 (Leu2 right homology arm); based on the genome of Saccharomyces cerevisiae CEN.PK2-1D (extraction method, see: Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform of Saccharomyces cerevisiae [D]. Wuhan University, 2015.2 .3.6 Yeast genomic DNA extraction method) as a template, use primers 900-3F/3R and 900-5F/5R to amplify fragments 9003 (terminator tCYC1) and 9005 (promoters pGAL1 and pGAL10) respectively; use primer 900-4F /4R with pCAS (see Zhang, Yueping et al. "A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae." Nature communications vol.10,1 1053.5 Mar.2019,doi:10.1038/s41467 -019-09005- 3) Obtain fragment 9004 (nonsense gene lacZ, used for replacement of the target gene) for template amplification; use pRS426 as a template and amplify it with primers 900-8F/8R, 900-9F/9R, and 900-10F/10R. Plasmid backbone (introduction of MssI restriction site, screening markers (AmpR, URA3, etc.)). The above fragments were recombined in Saccharomyces cerevisiae to construct pZY900 through DNA assemble (also known as yeast assembly, Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University, 2015.), and then in Escherichia coli After amplification, enzyme digestion verification and sequencing, pZY900 was obtained. The schematic diagram of the construction of plasmid pZY900 is shown in Figure 1, in which fragments 9001(HA), 9002(T), 9003(T), 9004, 9005, 9006, and 9007(HA) are connected in sequence from left to right, and the remaining parts are from the plasmid backbone of pRS426 .

The sequences of the primers used to construct plasmid pZY900 are shown in Table 1 below.

Table 1

1.2 Construction of different gene expression vectors

The cDNA of Pleurotus lucidum was extracted from the Pleurotus chinensis fruit tissue (using TIANGEN's RNAprep Pure Plant Plus Kit (Cat. No. DP441)), and Vazyme's HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit (Cat. No. R212) was used to RNA was reverse transcribed to obtain cDNA) as a template, and the primer pair P5/P6 was used as a primer. Takara's Prime STAR high-fidelity enzyme was used to amplify the cDNA of Yizhi through PCR to obtain a gene fragment named YZT3. After using Tiangen glue recovery reagent After the box gel was recovered, the Yisheng Company homologous recombination kit was used to connect it to the BsaI-cut yeast expression universal vector pZY900 using homologous recombination. After sequencing and confirmation, the yeast expression vector containing the YZT3 gene was obtained and named is pDXYZ3, the schematic diagram of its plasmid construction is shown in Figure 2, in which The YZT3 gene replaces the lacZ gene in pZY900.

The sequence of primer pair P5/P6 is shown in Table 2 below:

Table 2

Using the primer pair P7/P8, the AoVS gene fragment was amplified using the synthesized AoVS gene (the nucleotide sequence is shown in SEQ ID NO. 13) as a template. Using Yisheng Company's homologous recombination kit, the amplified AoVS gene fragment was connected to the BsaI-cut pZY900. After sequencing and confirmation, a yeast expression vector containing the AoVS gene was obtained, named pDXVS1. Schematic diagram of its plasmid construction. See pDXVS1 in Figure 3, in which the lacZ gene in pZY900 is replaced by the AoVS gene.

The sequence of primer pair P7/P8 is shown in Table 3 below:

table 3

Using primer pair P9/P10, the synthesized AoVS gene (the nucleotide sequence is shown in SEQ ID NO. 13) was used as a template to amplify the AoVS gene fragment. Use primers P11/P12 to plasmid pHM001 (for the construction of pHM001, please refer to the literature Deng et al. "Systematic identification of Ocimum sanctum sesquiterpenoid synthases and (-)-eremophilene overproduction in engineered yeast". Metabolic Engineering, 2022, 69: 122-133) As a template, amplify the vector containing PKG1 terminator (T), URA homology arm (HA), vector backbone, HIS3 tag, CYC1 terminator (T), tHMG1 and pGAL1-pGAL10 promoter (P _GAL10 and P _GAL1 ) fragment. Use the Yisheng Company homologous recombination kit to connect the AoVS gene fragment and the vector fragment through homologous recombination to obtain plasmid pDXVS2. The schematic diagram of the plasmid construction is shown in pDXVS2 in Figure 3.

The primer sequences are shown in Table 4 below:

Table 4

Using the primer pair P13/P14, Prime STAR high-fidelity enzyme was used to clone CYP6 from the cDNA template of Pleurotus lucidum. Gene; use primer pair P15/P16, use Prime STAR high-fidelity enzyme to clone the AoCPR gene from the cDNA template of Pleurotus lucidum; use primer pair P17/P18, use Prime STAR high-fidelity enzyme to clone pGAL1- from pESC-TRP plasmid pGAL10 promoter fragment; use SacI/XhoI double enzyme digestion of pESC-TRP plasmid to obtain the vector backbone. Use the homologous recombination kit of Yisheng Company to connect the above four fragments to obtain plasmid pDXNL1. The schematic diagram of the plasmid construction is shown in pDXNL1 in Figure 4.

The primer sequences are shown in Table 5 below:

table 5

Use the primer pair P19/P20 and Prime STAR high-fidelity enzyme to clone the CYP9 gene from the Pleurotus cDNA template; use the primer pair P15/P16 to clone the AoCPR gene from the Pleurotus cDNA template using Prime STAR high-fidelity enzyme; use Primer pair P17/P18 uses Prime STAR high-fidelity enzyme to clone the pGAL1-pGAL10 promoter fragment from pESC-TRP plasmid; use SacI/XhoI double enzyme to digest pESC-TRP plasmid to obtain the vector backbone. Use the homologous recombination kit of Yisheng Company to connect the above four fragments to obtain plasmid pDXNL2. The schematic diagram of the plasmid construction is shown in pDXNL2 in Figure 4.

The primer sequences are shown in Table 6 below:

Table 6

Using the primer pair P21/P22, use Prime STAR high-fidelity enzyme to clone the AoKo gene from the Pleurotus cDNA template (the nucleotide sequence is shown in SEQ ID NO.10); use the primer pair P15/P16, use Prime STAR high-fidelity enzyme Zhenzyme cloned the AoCPR gene from the Pleurotus cDNA template; used primer pair P17/P18 and Prime STAR high-fidelity enzyme to clone the pGAL1-pGAL10 promoter fragment from the pESC-TRP plasmid; used SacI/XhoI double enzyme digestion The pESC-TRP plasmid was used to obtain the vector backbone. Use the homologous recombination kit of Yisheng Company to connect the above four fragments to obtain plasmid pDXNL3. The schematic diagram of the plasmid construction is shown in pDXNL3 in Figure 4.

The primer sequences are shown in Table 7 below:

Table 7

Using the primer pair P23/P24, the AoADH gene fragment was cloned from the cDNA template of Pleurotus lucidum. The cDNA fragment was obtained through BamHI/XhoI double enzyme digestion, and then ligated with the BamHI/XhoI double enzyme digestion pESC-URA plasmid vector using T4DNA. Enzyme was used for ligation, and the obtained plasmid was named pDXNT1. The schematic diagram of its plasmid construction is shown in Figure 5.

The primer sequences are shown in Table 8 below:

Table 8

Homologous recombination was performed between the AoCPR gene fragment cloned using the primer pair P15/P16 and the vector fragment recovered from the pESC-TRP plasmid after EcoRI/SacI double enzyme digestion to obtain the pCK plasmid. The schematic diagram of the plasmid construction is shown in pCK in Figure 4.

1.3 Strain construction

The pDXYZ3 plasmid was transformed into yeast YZL141 strain by lithium acetate transformation method (for the construction of YZL141 strain, see Bian, G., Hou, A., Yuan, Y., Hu, B., Cheng, S., Ye, Z. ,Di,Y.,Deng,Z.,&Liu,T.(2018).Metabolic Engineering-Based Rapid Characterization of a Sesquiterpene Cyclase and the Skeletons of Fusariumdiene and Fusagramineol from Fusarium grinearum.Organic letters,20(6),1626– 1629. https://doi.org/10.1021/acs.orglett.8b00366), spread it on the SD-URA screening plate, pick a single clone, and obtain a mutant strain named JDXYZ3.

The pDXVS1 plasmid was digested with MssI endonuclease to recover the gene fragment containing AoVS, and the fragment was introduced into the yeast strain JCR27 using the lithium acetate method (for the construction of JCR27, see Siemon, T., Wang, Z., Bian, G., Seitz , T., Ye, Z., Lu, Y., Cheng, S., Ding, Y., Huang, Y., Deng, Z., Liu, T., & Christmann, M. (2020). Semisynthesis of Plant -Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.Journal of the American Chemical Society,142(6),2760–2765.https://doi.org/10.1021/jacs .9b12940), after performing yeast colony PCR verification, the positive bacteria were named JDXVS1. Furthermore, PmeI endonuclease was used to digest the pDXVS2 plasmid, and the gene fragment containing AoVS was recovered. The fragment was introduced into JDXVS1 using the lithium acetate method. After verification by yeast colony PCR, the positive bacteria were named JDXVS2.

The pCK, pDXNL1, pDXNL2 and pDXNL3 plasmids were transformed into JDXVS2 using the lithium acetate method to obtain the CK control strain, JDXNL1, JDXNL2 and JDXNL3 mutant strains. The pDXNT1 plasmid was co-transformed with pDXNL1, pDXNL2 and pDXNL3 plasmids into JDXVS2 to obtain JDXNT1, JDXNT2 and JDXNT3 mutant strains respectively.

Example 2 Gene Function Identification

2.1 Functional identification of valentene synthesis genes

The JDXYZ3 strain was inoculated into Sc-Ura liquid medium and cultured overnight at 30°C on a 200rpm shaker; the next day, it was transferred to 45 ml YPDHG liquid medium (20g/L peptone, 10g/L yeast powder, 10g) according to the initial OD600 = 0.1. /L glucose, 10g/L galactose), add 5 ml of isopropyl myristate, and incubate on a shaking table at 30°C and 200rpm for 72 hours. Collect the oil layer, dilute it to an appropriate concentration with n-hexane, and use GC-MS to detect the product. , the detection conditions are as follows:

Thermo Fisher Scientific TRACE GC ULTRA gas chromatograph with AS 3000 automatic injection, split/splitless injector, and TSQ QUANTUM XLS MS with triple quadrupole detector.

The chromatographic column is TR-5MS column (30m×0.25mm×0.25um). The carrier gas is high-purity helium with a flow rate of 1mL/min. Acetone is used as a needle cleanser. The injection volume is 1uL, and the split ratio is 50. The injection port temperature is 240°C, and the ion transfer tube temperature is 270°C.

Detection procedure: The initial column temperature is 50°C and maintained for 1 min; the temperature is increased to 280°C at 15°C/min and maintained for 1 min; the temperature is increased to 300°C at 20°C/min and maintained for 2 minutes.

The extracted ion chromatograms of JDXYZ3 fermentation product (labeled YZT3 in the figure) and valencene standard (Valencene, Sigma (#75056, CAS: 4630-07-3)) are shown in Figure 6. By comparing the retention times of the chromatograms of the products, it can be determined that the strain JDXYZ3 can synthesize valentene. This result indicates that the protein encoded by the YZT3 gene is valentene synthase.

2.2 Functional identification of cytochrome P450 oxidase and cytochrome P450 oxidoreductase

The strain CK containing only the AoCPR gene vector, and the strains JDXNL1, JDXNL2 and JDXNL3 containing both cytochrome P450 oxidase and cytochrome P450 oxidoreductase gene vectors were respectively inoculated into Sc-Trp liquid medium at 30°C, 200rpm. Culture on a shaking table overnight; the next day, transfer to 50 ml of Sc-Trp liquid culture medium containing 10g/L glucose and 10g/L galactose according to the initial OD600 = 0.1, and culture on a shaking table at 30°C and 200rpm for 72 hours to collect the cells. , add 10mL n-hexane to extract the bacteria. The extracted solution is tested by GC-MS. The detection instrument is the same as 2.1. The detection procedure is as follows: the starting column temperature is 80°C and maintained for 1 min; the temperature is increased to 280°C at 8°C/min and maintained for 5 minutes; the temperature is increased to 20°C/min. 300℃, keep for 2min.

Shake flask fermentation products of CK strain, JDXNL1 strain (marked as CYP6 in the figure), JDXNL2 strain (marked as CYP9 in the figure) and JDXNL3 strain (marked as AoKo in the figure), and Nootkatol standard (Nootkatol uses Nootkatol from source leaves). The extracted ion chromatogram of katone (obtained by reduction of lithium aluminum hydride) is shown in Figure 7. Performed with and nocanol standards It can be seen from the comparison of the retention times of the chromatograms that the control strain CK cannot synthesize nocanol, while the strains JDXNL1, JDXNL2 and JDXNL3 can synthesize nocanol. This result shows that when the cytochrome P450 oxidoreductase AoCPR and at least one of the cytochrome P450 oxidases CYP6, CYP9, and AoKO exist simultaneously, valenene can be oxidized to produce nocanol.

2.3 Functional identification of alcohol dehydrogenase

JDXNT1, JDXNT2 and JDXNT3 strains were inoculated into Sc-Ura-Trp liquid medium respectively, and cultured overnight at 30°C and 200rpm shaker; the next day, they were transferred to 50 ml of Sc-Ura-Trp liquid medium (containing 10g/L glucose, 10g/L galactose), 30°C, 200rpm shaker culture for 72 hours, collect the cells, add 10mL n-hexane to extract the cells. The extraction solution is tested by GC-MS, and the detection conditions are the same as 2.2.

Shake flask fermentation products of JDXNT1 strain (marked as CYP6 in the figure), JDXNT2 strain (marked as CYP9 in the figure) and JDXNT3 strain (marked as AoKo in the figure), and Nootkatone standard (Nootkatone, source leaf B20925) extracted ion current The chromatogram is shown in Figure 8. By comparing the retention time of the chromatogram with the nokatone standard, it can be determined that strains JDXNT1, JDXNT2 and JDXNT3 can synthesize nokatone. This result indicates that the alcohol dehydrogenase encoded by AoADH can further oxidize nocanol to generate the final product nocanone.

Example 3 Fermentation tank fermentation to synthesize nokatone

Reference literature (SIEMON T, WANG Z, BIAN G, et al. 2020. Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block. Journal of the American Chemical Society[ J], 142:2760-2765.), the constructed strains JDXNT1, JDXNT2, and JDXNT3 are subjected to fed-batch fermentation, and a covering agent is added during the fermentation process to achieve in-situ Extraction, covering agent is isopropyl myristate. During the fermentation process, the dissolved oxygen is controlled to be above 20%, the pH is 5, the glucose concentration is 1-2g/L, and the ethanol concentration is below 5g/L. Finally, on the 7L fermentation tank, the production of valentene, nocanol, and nocanone in strain JDXNT1 reached 500mg/L, 30mg/L, and 1.2g/L respectively; in strain JDXNT2, the production of valencene, nocanol, and The production of nocanone reached 350mg/L, 25mg/L, and 1.5g/L respectively; the production of valenene, nocanol, and nocanone in strain JDXNT3 reached 500mg/L, 60mg/L, and 1.9g respectively. /L.

Claims (15)

1. A method for biosynthesizing nokatone, which includes using a recombinant bacterium capable of expressing valentene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase to synthesize nokatone; wherein, the Valenene synthase, cytochrome P450 oxidase, cytochrome P450 oxidoreductase and alcohol dehydrogenase come from nootropics.
2. The method according to claim 1, wherein the valentene synthase has at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity;

   The cytochrome P450 oxidase is selected from at least one of cytochrome P450 oxidase CYP6, cytochrome P450 oxidase CYP9 and cytochrome P450 oxidase AoKo; wherein, the cytochrome P450 oxidase CYP6 has the same properties as SEQ ID NO.2 The amino acid sequence shown has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; cytochrome P450 oxidase CYP9 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.3 The amino acid sequence; cytochrome P450 oxidase AoKo has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO.4 , an amino acid sequence with 99% or 100% sequence identity;

   The cytochrome P450 oxidoreductase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 of the amino acid sequence shown in SEQ ID NO.5 % or 100% sequence identity of the amino acid sequence;

   The alcohol dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or Amino acid sequence with 100% sequence identity.
3. The method according to claim 1 or 2, wherein the recombinant bacterium is capable of synthesizing farnesyl pyrophosphate.
4. The method according to claim 3, wherein the recombinant bacterium is capable of expressing acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate At least one of acid kinase, mevalonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesyl pyrophosphate synthase.
5. An enzyme for the synthesis of nokatone, which has the same properties as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 The amino acid sequences shown are amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
6. A polynucleotide molecule comprising at least one of the nucleotide sequence encoding the enzyme of claim 5 or its complementary sequence.
7. The polynucleotide molecule according to claim 6, which includes SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or the nucleotide sequence shown in SEQ ID NO.13 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity nucleotide sequence.
8. A nucleic acid construct comprising at least one of the polynucleotide molecules of claim 6 or 7.
9. The nucleic acid construct according to claim 8, further comprising encoding acetoacetyl-CoA thiolase, hydroxymethylpentanol Diacyl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isophosphate At least one nucleotide sequence of constructase and farnesyl pyrophosphate synthase.
10. The nucleic acid construct according to claim 8 or 9, which is a plasmid vector; preferably, the plasmid vector is a eukaryotic expression vector; preferably, the plasmid vector is pDXYZ3, pDXVS1, pDXVS2, pDXNL1, pDXNL2, pDXNL3 , at least one of pDXNT1, wherein the construction schematic diagram of the plasmid vector is shown in Figure 2, Figure 3, Figure 4 or Figure 5.
11. A recombinant bacterium comprising the polynucleotide molecule according to claim 6 or 7, or the nucleic acid construct according to any one of claims 8-10; the recombinant bacterium is obtained by converting the polynucleotide molecule into Or the nucleic acid construct is introduced into a host cell to obtain it; preferably, the host cell is a eukaryotic cell; more preferably, it is Saccharomyces cerevisiae.
12. The recombinant bacterium according to claim 11, wherein the polynucleotide molecule is integrated into the genome of the host cell.
13. The recombinant bacterium according to claim 12, wherein the copy number of the polynucleotide molecule in the genome of the recombinant bacterium is at least 1, preferably at least 2, and more preferably at least 3.
14. The recombinant bacterium according to claim 11, which is capable of expressing acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, and At least one of valonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesyl pyrophosphate synthase.
15. The enzyme of claim 5, the polynucleotide molecule of claims 6 or 7, the nucleic acid construct of any one of claims 8-10 or the recombinant of any one of claims 11-14 Use of bacteria in the production of valenene, nocanol and/or nocanone.