Sorbic acid synthetic gene cluster and synthetic method

Abstract

A sorbic acid synthetic gene cluster and synthetic method. Provided is any one of the following substances: A1) a polyketide synthase; A2) a biological material related to the polyketide synthase of A1); A3) a hydrolase; A4) a biological material related to the hydrolase of A3); A5) a combination of A1) and A3); and A6) a combination of A2) and A4). A novel sorbic acid biosynthetic gene cluster, which has not been reported in currently disclosed documents, is found, it is the first time that a biosynthetic gene cluster containing a polyketide synthase gene saA and a hydrolase gene saB is heterologously expressed to synthesize sorbic acid, which has reference significance for green biomanufacturing of sorbic acid.

Description

Synthetic gene cluster and synthetic method of sorbic acid TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a synthetic gene cluster and synthetic method of sorbic acid. BACKGROUND

[0002] Sorbic acid, a natural preservative, was first extracted from the fruit of the white ash in 1859. As an unsaturated fatty acid, sorbic acid has two carbon-carbon double bonds, both of which are in the trans configuration. Studies have shown that sorbic acid has significant antibacterial activity, and it is worth noting that its antibacterial activity increases as the pH value of the culture medium decreases. In a low-pH environment, the acid is completely protonated, and thus more easily absorbed by microorganisms. Therefore, sorbic acid is widely used in the processing of cheese, wine, fruit juice and some baked foods to inhibit the growth of yeast and mold. In addition, sorbic acid is also considered as a substitute for nitrite preservation for meat processing.

[0003] With the continuous maturity of genome sequencing technology and the disclosure of numerous microbial genome sequences, there are a large number of unanalyzed secondary metabolite biosynthetic gene clusters in microbial genomes. The synthesis of microbial secondary metabolites is strictly regulated, and most of the gene clusters are in a silent state under laboratory culture conditions. By using methods such as heterologous expression, these gene clusters are mined, and by forcibly activating the "silent" genes in the gene cluster, combined with traditional separation means, it becomes an important way to develop new natural products.

[0004] SUMMARY

[0005] The technical problem solved by the present application is to provide a sorbic acid synthetic gene cluster and its application.

[0006] To solve the above technical problem, the present application provides the following any protein in the first aspect:

[0007] A1) a polyketide synthase;

[0008] A3) a hydrolase;

[0009] The polyketide synthase is any of the following:

[0010] B1) a protein whose amino acid sequence comprises SEQ ID NO: 3;

[0011] B2) a protein whose amino acid sequence has 95% or more identity with SEQ ID NO: 3 and has the same biological function;

[0012] The hydrolase is any of the following:

[0013] B3) a protein whose amino acid sequence comprises SEQ ID NO: 4;

[0014] B4) a protein having 95% or more identity to SEQ ID NO: 4 and having the same biological function.

[0015] The above protein having 95% or more identity to SEQ ID NO: 3 and having the same biological function is specifically a protein having 95% or 96% or 97% or 98% or 97% identity to SEQ ID NO: 3 and having the same biological function.

[0016] The above protein having 95% or more identity to SEQ ID NO: 4 and having the same biological function is specifically a protein having 95% or 96% or 97% or 98% or 97% identity to SEQ ID NO: 4 and having the same biological function.

[0017] The term "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed by the naked eye or by computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%) which can be used to assess identity between related sequences.

[0018] In a second aspect, the present application provides a protein composition which is a combination of A1) and A3) in the first aspect.

[0019] In the above, the combination of A1) and A3) can be a combination of a polyketide synthase and a hydrolase in the first aspect, or a fusion protein composed of a polyketide synthase and a hydrolase in the first aspect.

[0020] In a third aspect, the present application provides a biological material of A2) or A4) as follows:

[0021] A2) a biological material related to the polyketide synthase of A1);

[0022] The biological material related to the polyketide synthase of A1) is any one of the following:

[0023] C1) a nucleic acid molecule encoding the polyketide synthase in the first aspect;

[0024] C2) an expression cassette containing the nucleic acid molecule of C1);

[0025] C3) a recombinant vector containing the nucleic acid molecule of C1);

[0026] C4) a recombinant vector containing the expression cassette of C2);

[0027] C5) a recombinant microorganism containing the nucleic acid molecule of C1);

[0028] C6) a recombinant microorganism containing the expression cassette of C2);

[0029] C7) a recombinant microorganism containing the recombinant vector of C3);

[0030] C8) a recombinant microorganism containing the recombinant vector of C4);

[0031] A4) a biological material related to the hydrolytic enzyme of A3);

[0032] Said biological material related to the hydrolytic enzyme of A3) is any one of:

[0033] D1) a nucleic acid molecule encoding the hydrolytic enzyme of the first aspect;

[0034] D2) an expression cassette containing the nucleic acid molecule of D1);

[0035] D3) a recombinant vector containing the nucleic acid molecule of D1);

[0036] D4) a recombinant vector containing the expression cassette of D2);

[0037] D5) a recombinant microorganism containing the nucleic acid molecule of D1);

[0038] D6) a recombinant microorganism containing the expression cassette of D2);

[0039] D7) a recombinant microorganism containing the recombinant vector of D3);

[0040] D8) a recombinant microorganism containing the recombinant vector of D4).

[0041] In the above, said nucleic acid molecule encoding a polyketide synthase is any one of:

[0042] C1-1) a DNA molecule whose coding sequence comprises SEQ ID No. 1;

[0043] C1-2) a cDNA molecule or a DNA molecule which hybridizes with the DNA molecule defined in C1-1) and encodes a protein having the same function;

[0044] Or, said nucleic acid molecule encoding a hydrolytic enzyme is any one of:

[0045] D1-1) a DNA molecule whose coding sequence comprises SEQ ID No. 2;

[0046] D1-2) a cDNA molecule or a DNA molecule which hybridizes with the DNA molecule defined in D1-1) and encodes a protein having the same function.

[0047] The terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" as used herein refer to isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), naturally occurring types, mutated types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single- or double-stranded forms. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. A "gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene can include introns and exons in genomic sequence, and / or coding sequences in cDNA, and / or regulatory sequences of cDNA and its control sequences. In particular embodiments, such as with respect to isolated nucleic acid sequences, it is preferred to assume that they are cDNA. The nucleic acid molecules described above can be cDNA.

[0048] In the above, the expression cassette containing the nucleic acid molecule refers to DNA capable of expressing the above-mentioned protein in a host cell. The expression cassette can also include single- or double-stranded nucleic acid molecules of all the regulatory sequences necessary for expression of any of the above-mentioned proteins. The regulatory sequences are capable of directing the expression of the coding sequence in a suitable host cell under compatible conditions to express any of the above-mentioned proteins. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter and transcriptional and translational stop signals. The regulatory sequences can be provided with linkers for attachment to the coding region of the nucleic acid sequence encoding the protein. The regulatory sequences can be a suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by a host cell to initiate transcription of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences that mediate the transcription of the protein. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice including mutated, truncated, and hybrid promoters, and can be derived from genes encoding proteins either homologous or heterologous to the host cell. The regulatory sequences can also be a suitable transcription terminator sequence, i.e., a sequence that is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein. Any terminator that is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a suitable leader sequence, i.e., an mRNA in untranslated region that is vital for translation of the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a signal peptide coding region, which codes for an amino acid sequence that is linked to the amino terminus of the protein and directs the expressed protein into the secretory pathway of the cell. Any signal peptide coding region that is functional in the host cell of choice can be used in the present application. It can also be desirable to add regulatory sequences that allow the regulation of the expression of the protein relative to the growth of the host cell. Examples of regulatory sequences are those that allow expression of the protein to be turned on or off in response to chemical or physical stimulation, including the presence of a regulatory compound. Examples of other regulatory sequences are those that allow gene amplification. In these cases, the nucleic acid sequence encoding the protein is operably linked to the regulatory sequence.

[0049] The recombinant expression vector containing the expression cassette of the protein coding gene can be constructed using existing plant expression vectors.

[0050] In preparing the expression vector, a nucleic acid molecule encoding any of the above proteins can be located in the vector so as to be operably connected to appropriate expression control sequences. The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can conveniently be subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of vector will often depend on the design of the host cell into which the vector is to be introduced. The vector can be linear or closed circular. The vector can be an autonomously replicating vector (i.e., a vector that exists as an episome in a host cell, replicating independent of the chromosomal DNA), e.g., a plasmid, a non-integrating viral vector, a minichromosome or an artificial chromosome. The vector can comprise any mechanism for introduction into the host cell, and for integration by or replication in the chromosome of the host cell. Alternatively, the vector can be one which, when introduced into a host cell, is integrated into the cell chromosome and replicated together with the chromosome. Furthermore, the vector can be a single vector or plasmid, or two or more vectors or plasmids, or a transposon, which together comprise the total DNA to be introduced into the genome of the host cell. The vector will typically contain one or more selectable marker genes to allow selection of host cells into which the vector has been introduced. A selectable marker gene is a gene whose product confers antibiotic or biocide resistance, resistance to heavy metals, or the ability to grow in the absence of a nutritional requirement, among others. Examples of bacterial selectable marker genes are the dal genes of Bacillus subtilis or Bacillus licheniformis, or antibiotic resistance genes such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. The vector will also contain elements that allow it to be stably integrated into the host cell chromosome, or that allow it to replicate autonomously in the cell independent of the chromosome. In the case of autonomous replication, the vector can also contain an origin of replication to allow it to replicate autonomously in the target host cell. The origin of replication can be provided with a mutation that makes it temperature sensitive in the host cell. More than one copy of a nucleic acid sequence encoding any of the above proteins of the present application can be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be brought about by including an additional copy of the nucleic acid sequence, or by including a selectable marker gene with the nucleic acid sequence where the selectable marker gene confers antibiotic resistance or some other trait that can be used to select for cells that have incorporated the nucleic acid sequence. The increase in the copy number of the nucleic acid sequence can be brought about by including an additional copy of the nucleic acid sequence, or by including a selectable marker gene with the nucleic acid sequence where the selectable marker gene confers antibiotic resistance or some other trait that can be used to select for cells that have incorporated the nucleic acid sequence. The procedures used to ligate the elements of the recombinant expression vectors of the present application will be well known to the skilled worker (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0051] The term "operably linked" is defined herein as a conformation wherein the control sequences are positioned in such a way as to direct expression of the coding sequence to which they are operably linked.

[0052] In a fourth aspect, the present application provides a combination of the biological material of A2) and A4) of the third aspect.

[0053] The above-mentioned biological material combination can be a nucleic acid composition or a recombinant microorganism composition, and the nucleic acid composition can be a gene combination, an expression cassette combination or a recombinant vector combination;

[0054] The gene combination is a combination of a nucleic acid molecule (cDNA) encoding the polyketide synthase and a nucleic acid molecule (cDNA) encoding the hydrolase, or a fusion gene composed of the respective nucleic acids (cDNAs);

[0055] The expression cassette combination is a combination of an expression cassette for expressing a nucleic acid molecule (cDNA) encoding the polyketide synthase and an expression cassette for expressing a nucleic acid molecule (cDNA) encoding the hydrolase, or a fusion nucleic acid composed of the respective expression cassettes;

[0056] The recombinant vector combination is a combination of a recombinant vector for expressing a nucleic acid molecule (cDNA) encoding the polyketide synthase and a recombinant vector for expressing a nucleic acid molecule (cDNA) encoding the hydrolase, or a recombinant vector for expressing a fusion gene composed of a nucleic acid molecule (cDNA) encoding the polyketide synthase and a nucleic acid molecule (cDNA) encoding the hydrolase.

[0057] The recombinant microorganism combination is a recombinant microorganism for expressing a nucleic acid molecule (cDNA) encoding the polyketide synthase and a nucleic acid molecule (cDNA) encoding the hydrolase.

[0058] In a fifth aspect, the present application provides a recombinant microorganism containing the gene (cDNA) encoding the polyketide synthase and the gene (cDNA) encoding the hydrolase in the first aspect, and expressing the polyketide synthase and the hydrolase.

[0059] In the above-mentioned recombinant microorganism, the recombinant microorganism is a fungus or a bacterium.

[0060] In the above-mentioned fungus, the fungus is Aspergillus nidulans.

[0061] In a sixth aspect, the present application provides a method for constructing the recombinant microorganism in the fifth aspect, comprising the following steps: introducing the gene encoding the polyketide synthase and the gene encoding the hydrolase in the first aspect into a host microorganism to obtain the recombinant microorganism.

[0062] In the above-mentioned method, the host microorganism is a fungus or a bacterium.

[0063] In the above-mentioned fungus, the fungus is Aspergillus nidulans.

[0064] In the embodiments of the present application, the recombinant vector pJB118, the recombinant vector pJB119 and the empty pYTR can be co-transformed into Aspergillus nidulans A1145 to obtain the recombinant bacteria.

[0065] The recombinant vector pJB118 can be a vector obtained by replacing a fragment between Swal restriction sites of the pYTU vector with the polyketide synthase gene saA, wherein the polyketide synthase gene saA is located downstream of the GlaA promoter (GenBank: EF428455.1) in the vector, and the GlaA promoter drives expression of the polyketide synthase gene saA.

[0066] The recombinant vector pJB119 can be a vector obtained by replacing a fragment between Swal restriction sites of the pYTP vector with the hydrolytic enzyme gene saB, wherein the hydrolytic enzyme gene saB is located downstream of the AmyB promoter (GenBank: CP031434.1) in the vector, and the AmyB promoter drives expression of the hydrolytic enzyme gene saB.

[0067] In a seventh aspect, the present application provides a recombinant microorganism prepared by the method of the sixth aspect.

[0068] In an eighth aspect, the present application provides use of the protein of the first aspect, or the combination of proteins of the second aspect, or the biological material of the third aspect, or the combination of biological materials of the fourth aspect, in the preparation of a recombinant microorganism for synthesizing sorbic acid.

[0069] In a ninth aspect, the present application provides use of the protein of the first aspect, or the combination of proteins of the second aspect, or the biological material of the third aspect, or the combination of biological materials of the fourth aspect, in the synthesis of sorbic acid.

[0070] In a ninth aspect, the present application provides use of the recombinant microorganism of the fifth aspect or the seventh aspect in the synthesis of sorbic acid.

[0071] In a tenth aspect, the present application provides a method for synthesizing sorbic acid, comprising the following steps: fermenting the recombinant microorganism of the fifth aspect or the seventh aspect to obtain sorbic acid.

[0072] In the above method, the fermentation comprises the following steps:

[0073] 1) Seed culture solution:

[0074] The recombinant microorganism is inoculated into the seed culture medium and cultured at 37°C for 3 days until spores are produced by the strain;

[0075] 2) Fermentation

[0076] The spores obtained in 1) above are then spread on the fermentation medium and cultured at 30°C for 3 days to obtain the fermentation product.

[0077] The seed medium is CD medium, which comprises 1% glucose, 2% agar, 5% 20x Nitrate salts (V / V) and 0.1% Trace elements (V / V) respectively, and the rest is water;

[0078] The fermentation medium is CD-ST medium, which comprises 2% starch, 2% agar, 2% Tryptone, 5% 20x Nitrate salts (V / V) and 0.1% Trace elements (V / V) respectively, and the rest is water.

[0079] The 20x Nitrate comprises 120 g / L of NaNO3, 10.4 g / L of KCl, 10.4 g / L of MgSO4·7H2O and 30.4 g / L of KH2PO4 respectively, and the rest is water; the Trace elements comprises 22.0 g / L of ZnSO4·7H2O, 11.0 g / L of H3BO3, 5.0 g / L of MnCl2·4H2O, 1.6 g / L of FeSO4·7H2O, 1.6 g / L of CoCl2·5H2O, 1.6 g / L of CuSO4·5H2O, 1.11 g / L of (NH4)6Mo7O 24 ·4H2O respectively, and the rest is water.

[0080] The structural formula of sorbic acid is as follows: Beneficial effects

[0081] The present application has the beneficial effects that the present application discovers a new sorbic acid biosynthesis gene cluster, which has not been reported in the currently published literature, and is the first time to use the biosynthesis gene cluster comprising polyketide synthase gene saA and hydrolase gene saB to synthesize sorbic acid by heterologous expression, which has reference significance for green biological manufacturing of sorbic acid. BRIEF DESCRIPTION OF DRAWINGS

[0082] Fig. 1 is a schematic diagram of the sorbic acid biosynthesis gene cluster.

[0083] Fig. 2 is a schematic diagram of plasmids pJB118 and pJB119 used for construction of the sorbic acid heterologous expression strain.

[0084] Fig. 3 is an LC-MS detection diagram of fermentation and metabolic products of the heterologous expression strain.

[0085] Figs. 4 to 8 are nuclear magnetic spectra of the synthesized sorbic acid of the present application.

[0086] Fig. 9 is a liquid chromatography detection sorbic acid concentration standard curve. Embodiments of the present application

[0087] The present application will be further described in details with reference to specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0088] The experimental methods in the following examples are all routine methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0089] The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged, unless otherwise specified.

[0090] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clear, the present application will be further described in details with reference to the examples.

[0091] The pYTU vector, the pYTP vector, the pYTR vector and Aspergillus nidulans A1145 (A. nidulans A1145) are all recorded in the following literature: Gao, Y#, Xie M#, Yu C, Zhang M, Huang J, Li Q, Zhang H, Li L*. Heterologous Expression of Macrollins from Phytopathogenic Macrophomina phaseolina Revealed a Cytochrome P450 Monooxygenase in the Biosynthesis of β-Hydroxyl Tetramic Acid, Journal of Agricultural and Food Chemistry 2021, 69(50): 15175-15183.

[0092] Example 1, construction of recombinant AN-sa strain heterologously expressing sorbic acid biosynthesis gene cluster

[0093] Figure 1 is a schematic diagram of sorbic acid biosynthesis gene cluster.

[0094] 1, construction of recombinant vector

[0095] 1) Recombinant vector pJB118

[0096] The recombinant vector pJB118 is a vector obtained by replacing the fragment between the SwaI enzyme cutting sites of the pYTU vector with the polyketide synthase gene saA, wherein the polyketide synthase gene saA is located downstream of the GlaA promoter (GenBank: EF428455.1) in the vector, and the GlaA promoter drives the expression of the polyketide synthase gene saA.

[0097] The nucleotide sequence of the polyketide synthase gene saA is SEQ ID NO: 1, which encodes the protein polyketide synthase saA, and the amino acid sequence of the protein is SEQ ID NO: 3.

[0098] The specific construction method is as follows:

[0099] The Myrothecium sp. FJNU 6 (CGMCC NO. 41255) genome is used as a template, and 118-A-1F / 2R is used as primers to amplify the fragment containing the complete polyketide synthase gene saA gene with 78 base pair overlaps. The PCR product and the SwaI enzyme cutting pYTU vector fragment are collected, and Gibson is used for assembly into the expression plasmid pJB118 (the expression plasmid pJB118 is shown in FIG. 2).

[0100] 2) Recombinant vector pJB119

[0101] The recombinant vector pJB119 is a vector obtained by replacing the fragment between the SwaI enzyme cutting sites of the pYTP vector with the hydrolytic enzyme gene saB, wherein the hydrolytic enzyme gene saB is located downstream of the AmyB promoter (GenBank: CP031434.1) in the vector, and the AmyB promoter drives the expression of the hydrolytic enzyme gene saB.

[0102] The nucleotide sequence of the hydrolytic enzyme saB gene is SEQ ID NO: 2, which encodes the protein hydrolytic enzyme saB, and the amino acid sequence of the protein is SEQ ID NO: 4.

[0103] The specific construction method is as follows:

[0104] The Myrothecium sp. FJNU 6 CGMCC NO. 41255 genome is used as a template, and 119-B-F / R is used as primers to amplify the hydrolytic enzyme saB gene; the PCR product and the SwaI enzyme cutting pYTP vector fragment are collected, and Gibson is used for assembly into the expression plasmid pJB119 (the expression plasmid pJB119 is shown in FIG. 2).

[0105] 2, Construction of recombinant bacteria

[0106] The recombinant vector pJB118, the recombinant vector pJB119 and the blank vector pYTR were co-transformed into Aspergillus nidulans A1145 (A. nidulans A1145) by PEG-mediated method. The host fungus has three nutritional deficiencies, and therefore requires the introduction of pyrG (GenBank: CP084967.1) in the pJB118 vector, pyroA (GenBank: CP097567.1) in the recombinant vector pJB119 and riboB (GenBank: BX649606.1) in the pYTR to supplement the deficient nutrition, thereby constructing the recombinant bacteria AN-sa for heterologous expression of the sorbic acid biosynthesis gene cluster.

[0107] The blank plasmid vectors pYTU, pYTP and pYTR were co-transformed into A. nidulans A1145 by the same method as described above, thereby constructing the control recombinant bacteria AN.

[0108] The primer sequences are shown in Table 1.

[0109] Table 1 is the primer sequence

[0110] 3. Expression of the recombinant bacteria AN-sa

[0111] The recombinant bacteria AN-sa constructed above were inoculated into solid fermentation medium CD-ST (2% Starch (W / W), 2% Tryptone (W / W), 2% Agar (W / W), 5% 20x Nitrate salts (V / V) and 0.1% Trace elements (V / V), and the rest was water), and cultured at 30°C for 3 days. The fermentation product was collected and recorded as the fermentation product.

[0112] About 0.5 cm 3 The fermentation product was placed in a 1.5 mL EP tube, crushed with a pipette tip, extracted with 800 μL of extraction solution (89% ethyl acetate (V / V), 10% methanol (V / V) and 1% acetic acid (V / V)), vortexed for 1 min, ultrasonicated for 10 min, and then centrifuged at 12000 rpm for 5 min. The supernatant was concentrated by reduced pressure to remove the extraction solution, and then the extract was dissolved with 100 μL of methanol and recorded as the fermentation expression sample. After filtration with a 0.22 μm filter membrane, the sample was used for liquid chromatography-mass spectrometry analysis.

[0113] The analysis method is as follows: the instrument used is Thermo U3000 HPLC-LCQ fleet mass spectrometer, equipped with a phenomenex Luna 5 μm C18(2), 150x2.1mm column, the liquid chromatography column temperature is controlled at 30°C, and the detection wavelength is set to 200-800nm. The liquid chromatography flow rate is 0.25mL / min, the mobile phase A is ultrapure water containing 0.1% formic acid, and the mobile phase B is HPLC grade acetonitrile containing 0.1% formic acid, and the B phase is linearly increased from 5% to 95% in 0-30min. The mass spectrometer ion source setting parameters are: the spray pressure is 35psi, the dry gas flow is 35mL / min, the temperature is 350°C, the scanning mode is positive and negative ion continuous full scan, and the scanning range is m / z=50-160.

[0114] From the results of liquid chromatography-mass spectrometry, as shown in Figure 3, it can be seen that, compared with the control recombinant bacteria AN, the fermentation of AN-sa has a compound peak at 13.75min, and the [M+1] ion peak is 113, which proves that the introduction of the polyketide synthase gene saA and the hydrolase gene saB increases a single compound in the fermentation of Aspergillus nidulans.

[0115] The following is the expanded culture to verify the type and composition of the increased single compound.

[0116] Example 2, biosynthesis of sorbic acid

[0117] 1. Fermentation of recombinant bacteria

[0118] 1) Seed culture:

[0119] The recombinant bacteria AN-sa constructed in Example 1 is inoculated into the seed culture medium and cultured at 37°C for 3 days until the strain produces spores;

[0120] 2) Fermentation

[0121] The spores obtained in the above 1) are coated on the fermentation medium and cultured at 30°C for 3 days to obtain the fermentation product.

[0122] The above seed culture medium is CD culture medium, which includes 1% glucose, 2% agar, 5% 20x Nitrate salts (V / V) and 0.1% Trace elements (V / V) by mass fraction, and the balance is water;

[0123] The above fermentation medium is CD-ST culture medium, which includes 2% starch, 2% agar, 2% tryptone, 5% 20x Nitrate salts (V / V) and 0.1% Trace elements (V / V) by mass fraction, and the balance is water.

[0124] The above 20x Nitrate contains NaNO3 at a concentration of 120 g / L, KCl at a concentration of 10.4 g / L, MgSO4-7H2O at a concentration of 10.4 g / L, and KH2PO4 at a concentration of 30.4 g / L, with the balance being water; the above Trace elements contains ZnSO4-7H2O at a concentration of 22.0 g / L, H3BO3 at a concentration of 11.0 g / L, MnCl2-4H2O at a concentration of 5.0 g / L, FeSO4-7H2O at a concentration of 1.6 g / L, CoCl2-5H2O at a concentration of 1.6 g / L, CuSO4-5H2O at a concentration of 1.6 g / L, (NH4)6Mo7O 24 ·4H2O at a concentration of 1.11 g / L, with the balance being water;

[0125] 2. Compound product separation and purification

[0126] The fermentation obtained in the above 1 was extracted with an equal volume of extraction liquid (89% ethyl acetate (V / V), 10% methanol (V / V), and 1% acetic acid (V / V)), soaked for 8 h, with stirring every 3 h, and the extraction was repeated 3 times. The fermentation extraction liquid was filtered using filter paper to obtain a fermentation extraction liquid. The fermentation extraction liquid was concentrated under reduced pressure to remove the extraction liquid to obtain a crude extract as a sample to be separated;

[0127] The crude extract was first separated by Combiflash preparative chromatography (chromatographic column: RediSep Rf, 120 g C18 column). A small amount of methanol was used to dissolve the crude extract, and an appropriate amount of diatomite was added to the sample (the mass ratio of the crude extract to diatomite was 1:2), which was dried under reduced pressure and rotary evaporation. The sample was loaded, and the elution conditions were as follows: during 0-40 min, the B phase was linearly increased from 0% to 100%, and the flow rate was 45 mL / min throughout the process. The collection mode was selected as full collection. After sampling and quality detection by a separation tube, the tubes containing the target product were combined and concentrated under reduced pressure, dissolved in methanol, and then purified on an HPLC by a semi-preparative column phenomenex Luna 5 μm C18(2), 250x 10 mm.

[0128] The purification method is as follows: the instrument used is Thermo U3000 liquid chromatograph, equipped with a phenomenex Luna 5 μm C18(2), 250x 10 mm chromatographic column, and the liquid chromatographic column temperature is controlled at 35°C. The detection wavelength is set to 220 and 260 nm. The liquid chromatography flow rate is 3 mL / min, the mobile phase A is ultrapure water containing 0.1% formic acid, the mobile phase B is HPLC-grade acetonitrile containing 0.1% formic acid, and the sample retention time is 20 min.

[0129] The peaks of the single compound were collected, and the solvent was removed by concentration under reduced pressure to obtain the single compound. The structure of the compound was analyzed by nuclear magnetic resonance using deuterated methanol as the solvent. The compound was identified as sorbic acid (Figures 4-8).

[0130] Accurately weighed 1000 μg of sorbic acid standard (Source Leaf Biotech, B21124) was dissolved in 1 mL of chromatographic grade methanol to obtain a sorbic acid solution with a concentration of 1000 μg / mL. The sorbic acid solution was diluted in a geometric dilution manner to obtain sorbic acid solutions with concentrations of 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, and 3.9062 μg / mL, respectively. Liquid phase detection, according to the peak area to develop a standard curve. The detection method is as follows: the instrument used is Thermo U3000 liquid chromatograph, equipped with a phenomenex Luna 5 μm C18(2), 150 x 3 mm chromatographic column, the liquid chromatographic column temperature is controlled at 35℃, the detection wavelength is set at 220 and 260 nm. The liquid chromatography flow rate is 3 mL / min, the mobile phase A is ultrapure water containing 0.1% formic acid, the mobile phase B is HPLC grade acetonitrile containing 0.1% formic acid, and 25% B isocratic elution is used, and the sample retention time is 20 min.

[0131] The above crude extract was taken 1 mg and analyzed by the above method, and the sample peak area was substituted into the standard curve (Figure 9) to calculate the sorbic acid yield.

[0132] It was calculated that the sorbic acid yield of the recombinant bacteria AN-sa was 142.15 mg / L (calculated based on the volume of the pre-coagulation medium).

[0133] The above describes the present application in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

[0134] Industrial applications

[0135] The present application first uses the biosynthetic gene cluster containing the polyketide synthase gene saA and the hydrolase gene saB to synthesize sorbic acid by heterologous expression, which has reference significance for the green biological manufacturing of sorbic acid.

[0136] Cross Reference to Related Applications

[0137] This application claims priority to Chinese Patent Application No. 202411070679.1, filed August 6, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. Any one of the following proteins: A1) a polyketide synthase; A3) a hydrolase; said polyketide synthase is any one of the following: B1) a protein whose amino acid sequence comprises SEQ ID NO: 3; B2) a protein whose amino acid sequence is 95% or more identical to SEQ ID NO: 3 and has the same biological function; said hydrolase is any one of the following: B3) a protein whose amino acid sequence comprises SEQ ID NO: 4; B4) a protein whose amino acid sequence is 95% or more identical to SEQ ID NO: 4 and has the same biological function.

2. A protein composition which is a combination of A1) and A3) in claim 1.

3. A biological material of A2) or A4) as follows: A2) a biological material related to the polyketide synthase of A1); said biological material related to the polyketide synthase of A1) is any one of the following: C1) a nucleic acid molecule encoding the polyketide synthase as described in claim 1; C2) an expression cassette containing the nucleic acid molecule as described in C1); C3) a recombinant vector containing the nucleic acid molecule as described in C1); C4) a recombinant vector containing the expression cassette as described in C2); C5) a recombinant microorganism containing the nucleic acid molecule as described in C1); C6) a recombinant microorganism containing the expression cassette as described in C2); C7) a recombinant microorganism containing the recombinant vector as described in C3); C8) a recombinant microorganism containing the recombinant vector as described in C4); A4) a biological material related to the hydrolase of A3); said biological material related to the hydrolase of A3) is any one of the following: D1) a nucleic acid molecule encoding the hydrolase as described in claim 1; D2) an expression cassette containing the nucleic acid molecule as described in D1); D3) a recombinant vector containing the nucleic acid molecule as described in D1); D4) a recombinant vector containing the expression cassette as described in D2); D5) a recombinant microorganism containing the nucleic acid molecule as described in D1); D6) a recombinant microorganism containing the expression cassette as described in D2); D7) a recombinant microorganism containing the recombinant vector as described in D3); D8) a recombinant microorganism containing the recombinant vector as described in D4).

4. A combination of the biological materials of A2) and A4) as described in claim 3.

5. A recombinant microorganism containing a gene encoding the polyketide synthase and a gene encoding the hydrolase as described in claim 1 or 2, and expressing the polyketide synthase and the hydrolase.

6. The recombinant microorganism of claim 5, wherein: said recombinant microorganism is a fungus or a bacterium.

7. The recombinant microorganism according to claim 6, wherein: said fungus is Aspergillus nidulans.

8. A method for constructing the recombinant microorganism as described in any one of claims 5 to 7, comprising the step of introducing a gene encoding the polyketide synthase and a gene encoding the hydrolase as described in claim 1 or 2 into a host microorganism to obtain the recombinant microorganism.

9. The method of claim 8, wherein: said host microorganism is a fungus or a bacterium.

10. The method of claim 9, wherein: said fungus is Aspergillus nidulans.

11. The recombinant microorganism prepared by the method as described in any one of claims 8 to 10.

12. Use of the protein as described in claim 1, the protein composition as described in claim 2, the biological material as described in claim 3, or the combination of the biological materials as described in claim 4 in the preparation of a recombinant microorganism for synthesizing sorbic acid.

13. Use of the protein of claim 1 or the combination of proteins of claim 2 or the biomaterial of claim 3 or the combination of biomaterials of claim 4 in the synthesis of sorbic acid.

14. Use of the recombinant microorganism of any one of claims 5-7 or claim 11 in the synthesis of sorbic acid.

15. A method for synthesizing sorbic acid, comprising the step of fermenting the recombinant microorganism of any one of claims 5-7 or claim 11 to obtain sorbic acid.

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