Expression cassette, expression vector, recombinant microorganism, heterologous expression method of non-ribosomal peptide synthetic gene cluster, and use thereof

Abstract

Provided are an expression cassette, an expression vector, a recombinant microorganism, a heterologous expression method of a non-ribosomal peptide synthetic gene cluster, and the use thereof. The expression cassette comprises a promoter, the non-ribosomal peptide synthetic gene cluster and a terminator which are linked tandemly in sequence, the non-ribosomal peptide synthetic gene cluster containing N synthetic genes linked tandemly in sequence, and N being a positive integer greater than or equal to 2. At least one ribosome binding site is provided between each two adjacent synthetic genes. The promoter, the terminator and the ribosome binding site are derived from Bacillus subtilis, thus reducing the difficulty in expressing the non-ribosomal peptide synthetic gene cluster in a heterologous expression host (Bacillus subtilis).

Description

Expression cassette, expression vector, recombinant microorganism, heterologous expression method of non-ribosomal peptide synthesis gene cluster and application thereof Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to an expression cassette, an expression vector, a recombinant microorganism, and a heterologous expression method and application of a non-ribosomal peptide synthesis gene cluster. Background Art

[0002] Nonribosamal peptides (NRPs) refer to peptide compounds that are synthesized independently of ribosomes by nonribosomal peptide synthetases (NRPSs). They have complex structures and a wide range of activities. Nonribosomal peptides are an important source of antibiotics and can be used to prepare antibacterial drugs, antitumor drugs, immunosuppressants, antiviral drugs, etc.

[0003] To address the growing problem of antibiotic resistance, the development of non-ribosomal peptide antibiotics is a key area of ​​research. Microorganisms possess abundant non-ribosomal peptide synthesis gene clusters, but most remain silent under laboratory culture conditions, making their development challenging. Heterologous expression is a key strategy for developing silent natural products, but non-ribosomal peptide synthesis gene clusters often fail to express or express at very low levels in heterologous expression hosts. This may be due to the incompatibility of the non-ribosomal peptide synthesis gene cluster's expression elements with the heterologous expression host, hindering their expression. Therefore, optimizing non-ribosomal peptide synthesis gene clusters to reduce their expression challenges is crucial for the application and development of non-ribosomal peptides.

[0004] Summary of the Invention

[0005] The present application provides an expression cassette, an expression vector, a recombinant microorganism, a heterologous expression method of a non-ribosomal peptide synthesis gene cluster, and applications thereof, to reduce the difficulty of expressing the non-ribosomal peptide synthesis gene cluster in a heterologous expression host.

[0006] The technical solution of this application is as follows:

[0007] In a first aspect, the present application provides an expression cassette, comprising a promoter, a non-ribosomal peptide synthesis gene cluster and a terminator connected in series, wherein the non-ribosomal peptide synthesis gene cluster comprises N synthetic genes connected in series, N is a positive integer greater than or equal to 2, and at least one ribosome binding site is provided between two adjacent synthetic genes; the promoter, the terminator and the ribosome binding site are derived from Bacillus subtilis.

[0008] Optionally, a ribosome binding site is provided between two adjacent synthetic genes; and / or

[0009] When the expression cassette comprises two or more ribosome binding sites, the two or more ribosome binding sites are different from each other.

[0010] Optionally, the promoter is selected from one or more of PymdA, PahpC, PsodA, Phbs, PrpsO, PrplM, PrpsU, Pveg, PrplGB, Ppta, Pcdd, PyukE, PserA, Picd, PrpoA, PrpsL, PrplS and PtufA; and / or

[0011] The terminator is selected from one or more of TrrnB, TrpsI and TtufA; and / or

[0012] The ribosome binding site is selected from one or more of RBStsf, RBSsodA, RBSfbaA, RBSaspB, RBSthiC, RBSacpA, RBSSpdhC, RBSStufA, RBSgroEL, RBShag, RBSmdh, RBSaroA, RBSicd, RBSrplL, RBSahpC, RBS0, RBSfusA, RBSpdhD, RBSrpsG, RBSrpsE, RBSpdhB, RBSatpD, RBSrpsP, RBSymdA, RBSmtnA, RBSrpmC, RBSrplk, RBSglyA, RBShbs, RBSgndA, RBSypfD and RBSpgk.

[0013] Optionally, the expression cassette further comprises a resistance gene, and the resistance gene is located at the 3' end of the terminator; optionally, the resistance gene is a chloramphenicol resistance gene.

[0014] Optionally, the non-ribosomal peptide synthesis gene cluster is used to synthesize tyrocryptides, and the N synthesis genes are tyrA gene, tyrB gene, tyrC gene, tyrD gene and tyrE gene.

[0015] Optionally, the expression cassette comprises PymdA, tyrA gene, ribosome binding site RBSsodA, tyrB gene, ribosome binding site RBSfbaA, tyrC gene, ribosome binding site RBSaspB, tyrD gene, ribosome binding site RBSthiC, tyrE gene, terminator TrrnB, terminator TrpsI and terminator TtufA connected in series.

[0016] Optionally, the non-ribosomal peptide synthesis gene cluster is used to synthesize tauramamide, and the N synthesis genes are tauB gene, tauC gene and tauA gene respectively.

[0017] Optionally, the expression cassette comprises a promoter PymdA, a tauB gene, a ribosome binding site RBStsf, a tauC gene, a ribosome binding site RBSsodA, a tauA gene, a terminator TrrnB, a terminator TrpsI and a terminator TtufA connected in series.

[0018] Optionally, the non-ribosomal peptide synthesis gene cluster is used to synthesize kurstakin, and the N synthesis genes are kurA gene, kurB gene and kurC gene respectively.

[0019] Optionally, the expression cassette comprises a promoter PymdA, a kurA gene, a ribosome binding site RBStsf, a kurB gene, a ribosome binding site RBSsodA, a kurC gene, a terminator TrrnB, a terminator TrpsI, and a terminator TtufA connected in series.

[0020] In a second aspect, the present application provides an expression vector comprising any one of the expression cassettes described in the first aspect.

[0021] Optionally, the expression vector is a recombinant plasmid obtained by replacing the fragment with the 2235th site as the starting point and the 4422nd site as the end point in the yeast plasmid pESC-Leu with the amyE gene derived from Bacillus subtilis, and replacing the fragment with the 904th site as the starting point and the 1048th site as the end point within the amyE gene with the expression cassette.

[0022] In a third aspect, the present application provides a recombinant microorganism comprising a host, and an expression cassette as described in any one of the first aspect or an expression vector as described in any one of the second aspect located in the body of the host.

[0023] Optionally, the host is yeast or Bacillus subtilis.

[0024] In a fourth aspect, the present application provides a method for heterologous expression of a non-ribosomal peptide synthesis gene cluster, comprising the following steps:

[0025] Providing a first recombinant microorganism, comprising yeast, and an expression cassette according to any one of the first aspect or an expression vector according to any one of the second aspect located in the yeast;

[0026] extracting the expression cassette or the expression vector, and introducing the expression cassette or the expression vector into Bacillus subtilis to obtain a second recombinant microorganism; and

[0027] The second recombinant microorganism is fermented and cultured to express the non-ribosomal peptide synthesis gene cluster.

[0028] Optionally, the yeast is Saccharomyces cerevisiae BY4742; and / or

[0029] The Bacillus subtilis is Bacillus subtilis ZL01.

[0030] The present application also provides the use of the expression cassette as described in any one of the first aspects, or the expression vector as described in any one of the second aspects, or the recombinant microorganism as described in any one of the third aspects, or the heterologous expression method as described in any one of the fourth aspects in synthesizing non-ribosomal peptides.

[0031] The present application provides an expression cassette, an expression vector, a recombinant microorganism, a heterologous expression method of a non-ribosomal peptide synthesis gene cluster, and its application, which has the following technical effects:

[0032] In the expression cassette of the present application, the elements used to express the non-ribosomal peptide synthesis gene cluster are derived from Bacillus subtilis, that is, the expression elements of Bacillus subtilis are used to replace the expression elements of the non-ribosomal peptide synthesis gene cluster itself, which can reduce the difficulty of expressing the non-ribosomal peptide synthesis gene cluster in a heterologous expression host (such as Bacillus subtilis).

[0033] In the heterologous expression method of the non-ribosomal peptide synthesis gene cluster of the present application, on the one hand, the expression elements of the non-ribosomal peptide synthesis gene cluster itself are replaced with the expression elements of the expression host, and the cloning and reconstruction of the non-ribosomal peptide synthesis gene cluster are achieved in yeast in one step, which fully utilizes the cloning advantages of the yeast homologous recombination-based cloning method for large non-ribosomal peptide synthesis gene clusters. Compared with conventional methods, the cloning and reconstruction time of the non-ribosomal peptide synthesis gene cluster is effectively shortened, and the efficiency is improved; on the other hand, Bacillus subtilis with natural transformation ability is used as a heterologous expression host, which can efficiently transform large plasmids, thereby eliminating the need to use Escherichia coli as an intermediate host, effectively simplifying the operational process of heterologous expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The following detailed description of the specific embodiments of the present application in conjunction with the accompanying drawings will make the technical solutions and beneficial effects of the present application apparent.

[0035] Figure 1 is a schematic structural diagram of the recombinant plasmid pESC_Ppta;

[0036] Figure 2 is a graph showing the intensity detection data of different promoters;

[0037] FIG3 is a graph showing the intensity detection data of different ribosome binding sites;

[0038] FIG4 is an LC-MS detection diagram of the product obtained after Bacillus subtilis ZL01 expresses the exogenous tyrocryopeptide synthesis gene cluster;

[0039] FIG5 is an LC-MS detection diagram of the product obtained by heterologous expression of the first control sample;

[0040] FIG6 is a graph showing LC-MS detection of the product obtained by heterologous expression of the second control sample;

[0041] FIG7 is an LC-MS detection diagram of the product obtained after Bacillus subtilis ZL01 expresses the exogenous tauramamide synthesis gene cluster;

[0042] FIG8 is a graph showing LC-MS detection of the product obtained by heterologous expression of the third control sample;

[0043] FIG9 is an LC-MS detection diagram of the product obtained after Bacillus subtilis ZL01 expresses the exogenous kurstakin synthetic gene cluster. DETAILED DESCRIPTION

[0044] The terms used in this application are defined as follows:

[0045] As used in this application, "plurality" refers to two or more, for example, two, three, four, five, six, seven, eight, nine, ten, etc.

[0046] As used in this application, "and / or" means that the selection range includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, a combination of any two or any three of A, B, C, and D, and also includes a four-item combination of A, B, C, and D (that is, a technical solution connected by "logical AND").

[0047] As used in this application, "homology" refers to the "identity" between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared obtained after the best alignment (best alignment), and the differences between the two sequences are randomly distributed over their entire length. The percentage of identity or consistency means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared obtained after the best alignment (best alignment), which is purely statistical, and the differences between the two sequences are randomly distributed and distributed over their entire length. Usually, the best alignment (best alignment) is the alignment with the highest percentage of identity between the two sequences to be compared, which can be performed manually or using a sequence alignment tool (such as Blast or other online sequence alignment software).

[0048] In the present application embodiment, at least 80% homology can be understood as at least greater than 81%, or at least greater than 82%, or at least greater than 83%, or at least greater than 84%, or at least greater than 84%, or at least greater than 85%, or at least greater than 86%, or at least greater than 87%, or at least greater than 88%, or at least greater than 89%, or at least greater than 90%, or at least greater than 91%, or at least greater than 92%, or at least greater than 93%, or at least greater than 94%, or at least greater than 95%, or at least greater than 96%, or at least greater than 97%, or at least greater than 98%, or at least greater than 99% sequence homology, examples of which are 80%, 81%, 82%, 83%, 84%, or at least greater than 96%. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, the corresponding homology value is an integer; it can also be further understood as 80.1%, 81.2%, 82.3%, 83.4%, 84.5%, 85.6%, 86.7%, 87.8%, 88.9%, 89.8%, 90.3%, 91.7%, 92.2%, 93.5%, 94.8%, 95.9%, 96.6%, 97.5%, 98.4% or 99.9%, but the sequence identity is less than 100%, the corresponding homology value is a decimal.

[0049] As used herein, a "synthetic gene cluster" refers to a group of genes or variants thereof involved in the biosynthesis of non-ribosomal peptides. A synthetic gene cluster may be unmodified or modified, and the modification of a synthetic gene cluster may be by any genetic recombination technique known in the art, including but not limited to mutagenesis, inactivation, or replacement. Those skilled in the art are aware of methods for obtaining synthetic gene clusters for known non-ribosomal peptides, for example, by consulting literature and databases.

[0050] As used herein, an "expression cassette" refers to a set of DNA sequences comprising a non-ribosomal peptide synthesis gene cluster and all elements for expressing the non-ribosomal peptide synthesis gene cluster. For example, an expression cassette includes one or more of a promoter, a terminator, an intron, an exon, an enhancer, and a ribosome binding site. To facilitate detection, the expression cassette may also include a selectable marker gene.

[0051] As used in this application, "recombinant expression vector" refers to a nucleic acid molecule construct that contains an exogenous nucleic acid fragment operably linked to an appropriate control sequence that can achieve expression of the exogenous nucleic acid fragment in a suitable host. Recombinant expression vectors include, for example: (1) a collection of genetic elements that regulate the expression of exogenous nucleic acid fragments, such as promoters, enhancers, enhancers, silencers, etc.; (2) a structure or coding sequence that is transcribed into mRNA and translated into protein. Those skilled in the art can use widely known methods to construct recombinant expression vectors, including but not limited to double enzyme digestion construction methods or homologous recombination construction methods. The expression vector may contain one or more selectable marker genes.

[0052] As used herein, "host" refers to a type of recipient microorganism capable of accepting exogenous nucleic acids (e.g., the expression cassette of this application) through a specific method. The specific method includes, but is not limited to, at least one of conjugation, electroporation, chemical transformation, transduction, transfection, or ultrasonic transformation. The appropriate method can be selected based on the selected host; for example, when the host is Saccharomyces cerevisiae, lithium acetate transformation can be selected to accept exogenous nucleic acids. In the embodiments of this application, the host can be a wild-type microorganism or a microorganism obtained by natural mutation or artificial modification of the wild-type.

[0053] An embodiment of the present application provides an expression cassette, which comprises a promoter, a non-ribosomal peptide synthesis gene cluster, and a terminator connected in series, wherein the non-ribosomal peptide synthesis gene cluster comprises N synthetic genes connected in series, where N is a positive integer greater than or equal to 2, and at least one ribosome binding site is provided between two adjacent synthetic genes; the promoter, terminator, and ribosome binding site are derived from Bacillus subtilis.

[0054] In the expression cassette of the embodiment of the present application, the elements used to express the non-ribosomal peptide synthesis gene cluster are derived from Bacillus subtilis, that is, the expression elements of Bacillus subtilis are used to replace the expression elements of the non-ribosomal peptide synthesis gene cluster itself, which can reduce the difficulty of expressing the non-ribosomal peptide synthesis gene cluster in a heterologous expression host (such as Bacillus subtilis).

[0055] It is understandable that the number of promoters located at the 5' end of the entire non-ribosomal peptide synthesis gene cluster can be one or more, and the number of terminators located at the 3' end of the entire non-ribosomal peptide synthesis gene cluster can also be one or more. For a single synthetic gene in a non-ribosomal peptide synthesis gene cluster, one or more promoters can be set at the 5' end of the single synthetic gene, and / or one or more terminators can be set at the 3' end of the single synthetic gene. In addition, in the expression cassette of the embodiment of the present application, a ribosome binding site may or may not be set between the promoter and the non-ribosomal peptide synthesis gene cluster. The serial connection order of the N synthetic genes in the non-ribosomal peptide synthesis gene cluster is not specifically limited. The order of serial connection mainly affects the construction method and does not have a significant impact on the expression effect. In addition, each synthetic gene in the expression cassette can be a single copy or multiple copies.

[0056] In the expression cassette of the embodiment of the present application, the non-ribosomal peptide synthesis gene cluster includes but is not limited to a tyrosinase synthesis gene cluster, a tauramamide synthesis gene cluster, a kurstakin synthesis gene cluster, a polymyxin synthesis gene cluster, a daptomycin synthesis gene cluster, a bleomycin synthesis gene cluster, auriporcine synthesis gene cluster, a tridecaptin synthesis gene cluster, a bacitracin synthesis gene cluster, a vancomycin synthesis gene cluster, a cyclosporin synthesis gene cluster, an isoniazid synthesis gene cluster, a bouvardin synthesis gene cluster or an epothilone synthesis gene cluster.

[0057] In some embodiments of the present application, the promoter is selected from one or more of PymdA, PahpC, PsodA, Phbs, PrpsO, PrplM, PrpsU, Pveg, PrplGB, Ppta, Pcdd, PyukE, PserA, Picd, PrpoA, PrpsL, PrplS, and PtufA. It is understood that the promoter located at the 5' end of the entire non-ribosomal peptide synthesis gene cluster can be a promoter with the highest strength (e.g., PymdA), or the promoter located at the 5' end of the entire non-ribosomal peptide synthesis gene cluster can be formed by a series of multiple promoters with higher strengths.

[0058] In the expression cassettes of the embodiments of the present application, the type and number of terminators are not specifically limited, as long as the terminators are derived from Bacillus subtilis and are sufficient to terminate transcription. In some embodiments of the present application, the terminators are selected from one or more of TrrnB, TrpsI, and TtufA, and an exemplary terminator is formed in tandem by TrrnB, TrpsI, and TtufA.

[0059] In some embodiments of the present application, the ribosome binding site is selected from one or more of RBStsf, RBSsodA, RBSfbaA, RBSaspB, RBSthiC, RBSacpA, RBSSpdhC, RBSStufA, RBSgroEL, RBShag, RBSmdh, RBSaroA, RBSicd, RBSrplL, RBSahpC, RBS0, RBSfusA, RBSpdhD, RBSrpsG, RBSrpsE, RBSpdhB, RBSatpD, RBSrpsP, RBSymdA, RBSmtnA, RBSrpmC, RBSrplk, RBSglyA, RBShbs, RBSgndA, RBSypfD and RBSpgk. It is understandable that the ribosome binding site between two adjacent synthetic genes is selected from any one or more of the above-mentioned ribosome binding sites; when a ribosome binding site is provided between the promoter and the non-ribosomal peptide synthetic gene cluster, the ribosome binding site is selected from any one or more of the above-mentioned ribosome binding sites.

[0060] In order to improve assembly efficiency, in some embodiments of the present application, a ribosome binding site is provided between two adjacent synthetic genes.

[0061] In order to reduce the mismatch rate and improve the accuracy of assembly, in some embodiments of the present application, when the expression cassette comprises two or more ribosome binding sites, the two or more ribosome binding sites are different from each other.

[0062] In order to facilitate the detection of the expression effect of the non-ribosomal peptide synthesis gene cluster, in some embodiments of the present application, the expression cassette further comprises a resistance gene, which is located at the 3' end of the terminator; optionally, the resistance gene is a chloramphenicol resistance gene.

[0063] In some embodiments of the present application, a non-ribosomal peptide synthesis gene cluster is used to synthesize tyroin from Tyr. The N synthesis genes in the non-ribosomal peptide synthesis gene cluster are tyrA gene, tyrB gene, tyrC gene, tyrD gene, and tyrE gene, respectively. Correspondingly, the expression cassette comprises a promoter, tyrA gene, a first ribosome binding site, tyrB gene, a second ribosome binding site, tyrC gene, a third ribosome binding site, tyrD gene, a fourth ribosome binding site, tyrE gene, and a terminator in series, wherein the first to fourth ribosome binding sites are independently selected from one or more of the aforementioned ribosome binding sites. When the first to fourth ribosome binding sites are each one ribosome binding site, the order of strength of the first to fourth ribosome binding sites is not specifically limited (the order of strength does not significantly affect the expression effect). For example, the strength of the first to fourth ribosome binding sites decreases or increases in sequence. For example, the strength of the first to fourth ribosome binding sites is similar. Preferably, the first to fourth ribosome binding sites are different from each other. When the first to fourth ribosome binding sites are each multiple ribosome binding sites, the order of strength of each ribosome binding site is not specifically limited.

[0064] As an example, the expression cassette comprises, in sequence, a promoter PymdA, a tyrA gene, a ribosome binding site RBSsodA, a tyrB gene, a ribosome binding site RBSfbaA, a tyrC gene, a ribosome binding site RBSaspB, a tyrD gene, a ribosome binding site RBSSthiC, a tyrE gene, a terminator TrrnB, a terminator TrpsI, and a terminator TtufA. As another example, the expression cassette comprises, in sequence, a promoter PymdA, a tyrA gene, a ribosome binding site RBSStsf, a tyrB gene, a ribosome binding site RBSsodA, a tyrC gene, a ribosome binding site RBSfbaA, a tyrD gene, a ribosome binding site RBSaspB, a tyrE gene, a terminator TrrnB, a terminator TrpsI, and a terminator TtufA.

[0065] In some embodiments of the present application, a non-ribosomal peptide synthesis gene cluster is used to synthesize tauramamide, and the N synthetic genes in the non-ribosomal peptide synthesis gene cluster are tauB gene, tauC gene, and tauA gene, respectively. The expression cassette comprises a promoter, a tauB gene, a first ribosome binding site, a tauC gene, a second ribosome binding site, a tauA gene, and a terminator connected in series, wherein the first ribosome binding site and the second ribosome binding site are independently selected from one or more of the aforementioned ribosome binding sites, and the strength relationship between the first ribosome binding site and the second ribosome binding site is not specifically limited. Preferably, the first ribosome binding site and the second ribosome binding site are different from each other.

[0066] As an example, the expression cassette comprises the promoter PymdA, the tauB gene, the ribosome binding site RBStsf, the tauC gene, the ribosome binding site RBSsodA, the tauA gene, the terminator TrrnB, the terminator TrpsI, and the terminator TtufA in series.

[0067] In some embodiments of the present application, a non-ribosomal peptide synthesis gene cluster is used to synthesize kurstakin, and the N synthesis genes in the non-ribosomal peptide synthesis gene cluster are kurA gene, kurB gene, and kurC gene, respectively. The expression cassette comprises a promoter, kurA gene, a first ribosome binding site, kurB gene, a second ribosome binding site, kurC gene, and a terminator connected in series, wherein the first ribosome binding site and the second ribosome binding site are independently selected from one or more of the aforementioned ribosome binding sites, and the strength relationship between the first ribosome binding site and the second ribosome binding site is not specifically limited. Preferably, the first ribosome binding site and the second ribosome binding site are different from each other.

[0068] As an example, the expression cassette comprises the promoter PymdA, the kurA gene, the ribosome binding site RBStsf, the kurB gene, the ribosome binding site RBSsodA, the kurC gene, the terminator TrrnB, the terminator TrpsI, and the terminator TtufA in tandem.

[0069] The embodiment of the present application also provides a recombinant expression vector, which comprises any one of the expression cassettes described above.

[0070] In order to further improve the expression effect, in some embodiments of the present application, the expression vector is a recombinant plasmid obtained by replacing the fragment with the 2235th site as the starting point and the 4422th site as the end point in the yeast plasmid pESC-Leu with the amyE gene derived from Bacillus subtilis, and replacing the fragment with the 904th site as the starting point and the 1048th site within the amyE gene with the expression cassette.

[0071] In some embodiments of the present application, the amyE gene is derived from Bacillus subtilis ZL01.

[0072] The embodiments of the present application also provide a recombinant microorganism, including a host, and an expression cassette as described above, or an expression vector as described above, located in the body of the host.

[0073] In some embodiments of the present application, the host is yeast or Bacillus subtilis.

[0074] Yeasts include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces pastoris, Saccharomyces boulardii, Saccharomyces fermentans, Saccharomyces paradoxus, Saccharomyces vitis, Saccharomyces bayanus, Schizosaccharomyces, Kluyveromyces, Torulospora, Pichia, Zygosaccharomyces, Brettanomyces, or Metschnikova. Schizosaccharomyces includes, but is not limited to, Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octospora, or Schizosaccharomyces psychrophilus; Torulospora includes, but is not limited to, Deboispora; Kluyveromyces includes, but is not limited to, Kluyveromyces marxianus; Pichia includes, but is not limited to, Pichia stipitis, Pichia pastoris, or Pichia angus; Zygosaccharomyces includes, but is not limited to, Zygosaccharomyces bailii; Brettanomyces includes, but is not limited to, Brettanomyces intermedius, Brettanomyces bruxellensis, Brettanomyces heterotypicus, Brettanomyces bantuensis, Brettanomyces natterii, Brettanomyces nanus, Dekkera bruxellensis, or Dekkera heterotypicus; and Metschnikova includes, but is not limited to, Issaffine yeast or Klerkera. As an example, the yeast is selected from Saccharomyces cerevisiae BY4742.

[0075] In some embodiments of the present application, the host is Bacillus subtilis ZL01.

[0076] It is understandable that heterologous expression of a non-ribosomal peptide synthesis gene cluster involves three key steps: cloning the non-ribosomal peptide synthesis gene cluster, reconstructing the non-ribosomal peptide synthesis gene cluster, and introducing it into a heterologous host for expression. Due to the large size of the non-ribosomal peptide synthesis gene cluster, all three steps present varying degrees of difficulty.

[0077] First, as described above, the expression effect of the expression elements of the non-ribosomal peptide synthesis gene cluster itself in the heterologous expression host is usually not ideal. In order to improve the heterologous expression effect, it is necessary to use expression elements derived from the expression host to replace the expression elements of the non-ribosomal peptide synthesis gene cluster itself to achieve the reconstruction of the non-ribosomal peptide synthesis gene cluster. However, some expression elements of many expression hosts are currently unknown and / or lack sources. This application overcomes this difficulty by screening the expression elements (such as endogenous promoters and ribosome binding sites) of the model strain Bacillus subtilis.

[0078] Secondly, the applicant found that the cloning method based on yeast homologous recombination has great advantages for cloning large non-ribosomal peptide synthesis gene clusters, but it is difficult to extract large plasmids from yeast, and due to current technical limitations, the concentration of plasmids extracted from yeast is usually low, and it is generally difficult to directly transform the expression host (the transformation efficiency of general microorganisms is difficult to transform low-concentration large plasmids). Therefore, it is often necessary to use Escherichia coli to amplify the plasmid or to perform conjugation transfer through Escherichia coli, that is, the heterologous expression of non-ribosomal peptides usually depends on the intermediate host Escherichia coli. Conventional heterologous expression methods for non-ribosomal peptides include, for example, the following steps: first, cloning the gene sequence carrying the non-ribosomal peptide synthesis gene cluster using yeast homologous recombination cloning methods, a step that takes approximately five days; then, extracting the yeast plasmid containing the non-ribosomal peptide synthesis gene cluster and transforming it into Escherichia coli, where the expression elements of the non-ribosomal peptide synthesis gene cluster are replaced in the E. coli to achieve reconstruction of the non-ribosomal peptide synthesis gene cluster; this step takes more than six days; and finally, introducing the plasmid carrying the reconstructed non-ribosomal peptide synthesis gene cluster into a suitable host for expression, a step that takes at least two days. This method has the disadvantages of cumbersome procedures and low efficiency.

[0079] Based on this, the present invention also provides a method for heterologous expression of a non-ribosomal peptide synthesis gene cluster, comprising the following steps:

[0080] S1. Providing a first recombinant microorganism, wherein the first recombinant microorganism comprises yeast, and an expression cassette as described above or an expression vector as described above located in the yeast;

[0081] S2. extracting the expression cassette or the expression vector, and introducing the expression cassette or the expression vector into Bacillus subtilis to obtain a second recombinant microorganism;

[0082] S3. Fermenting and culturing the second recombinant microorganism to express the non-ribosomal peptide synthesis gene cluster.

[0083] In the heterologous expression method of the non-ribosomal peptide synthesis gene cluster of the embodiment of the present application, on the one hand, the expression elements of the non-ribosomal peptide synthesis gene cluster itself are replaced with the expression elements of the expression host, and the cloning and reconstruction of the non-ribosomal peptide synthesis gene cluster are achieved in yeast in one step, which fully utilizes the cloning advantages of the yeast homologous recombination-based cloning method for large non-ribosomal peptide synthesis gene clusters. Compared with conventional methods, the cloning and reconstruction time of the non-ribosomal peptide synthesis gene cluster is effectively shortened, and the efficiency is improved; on the other hand, Bacillus subtilis (such as Bacillus subtilis ZL01) with natural transformation ability is used as a heterologous expression host, which can efficiently transform large plasmids, thereby eliminating the need for Escherichia coli as an intermediate host, effectively simplifying the operational process of heterologous expression.

[0084] In some embodiments of the present application, the yeast in step S1 is Saccharomyces cerevisiae BY4742.

[0085] In some embodiments of the present application, the Bacillus subtilis in step S2 is Bacillus subtilis ZL01.

[0086] In step S3, the culture medium used in the fermentation culture can be selected from various culture media suitable for the growth of Bacillus subtilis, including but not limited to beef extract peptone culture medium, LB culture medium, etc. The culture conditions can be, for example, 30° C. and aerobic culture.

[0087] The embodiments of the present application also provide an expression cassette as described above, or an expression vector as described above, or a recombinant microorganism as described above, or a heterologous expression method as described above in the synthesis of non-ribosomal peptides.

[0088] Specifically, the expression cassette described above or the expression vector described above is expressed using any of the heterologous expression methods described above to synthesize a non-ribosomal peptide, wherein the non-ribosomal peptide includes, but is not limited to, tyrocidine, tauramamide, kurstakin, auriporcine, daptomycin, bleomycin, auriporcine, auriporcine, tridecaptin, bacitracin, vancomycin, cyclosporin, roquefortine, bouvardin, or epothilone.

[0089] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0090] Unless otherwise defined or indicated, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art, and the materials or reagents used in the examples and comparative examples of the present application are commercially available. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only and do not limit the content of this application. The terms "first," "second," "third," or similar expressions are used for descriptive purposes only and are not to be construed as indicating or implying relative importance, nor as indicating or implying a sequential order.

[0091] I. Description of reagents and materials involved in the examples of this application:

[0092] The primers and gene fragments involved in the examples of this application were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0093] The PCR reaction reagents involved in the examples of this application were purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0094] The yeast plasmid pESC-Leu involved in the examples of this application was purchased from Agilent Technologies with the serial number #217452.

[0095] The pNW-sfGFP (SPS6) plasmid involved in the examples of this application was purchased from Addgene with the number 133814.

[0096] The pJMP2 plasmid involved in the examples of this application was purchased from Solarbio, with the number VT002678.

[0097] The cerevisiae BY4742 involved in the examples of this application is derived from the Biobw strain library and is numbered bio-74224.

[0098] The Bacillus subtilis ZL01 involved in the examples of the present application has a deposit number of GDMCC No: 63698, and the deposit center is Guangdong Provincial Microbial Culture Collection Center.

[0099] The LB medium used in the examples of this application was prepared independently. The formula of LB liquid medium is as follows: per 100 mL of LB liquid medium, 1.0 g of peptone, 0.5 g of yeast extract, and 1.0 g of NaCl, with a pH of 7.0. For LB solid medium, 15 g / L of agar was added to the LB liquid medium formula.

[0100] The formula of the 1×MC culture medium involved in the examples of this application is derived from the document Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights, Nature Communiacation, 2021, 12: 5429.

[0101] The Sc-Leu culture medium involved in the examples of this application was purchased from Beijing Coolaibo Technology Co., Ltd., model number PM2203.

[0102] III. Non-ribosomal peptide detection methods involved in the experimental examples of this application

[0103] The present invention uses liquid chromatography-mass spectrometry (LC-MS) to detect non-ribosomal peptides. The specific detection method is as follows:

[0104] (1) Preparation of injection solution: 1 mL of fermentation broth was lyophilized, then extracted with 1 mL of methanol. The organic phase formed by the extraction was collected and filtered through a 0.45 μm filter membrane. The filtrate obtained was the injection solution, and 5 μL was injected each time.

[0105] (2) Chromatographic column: Poroshell 120 EC-C18, 2.1 mm × 50 mm × 1.9 μm;

[0106] (3) Preparation of mobile phase: Phase A was prepared by adding 0.1% trifluoroacetic acid to ultrapure water; Phase B was prepared by adding 0.1% trifluoroacetic acid to anhydrous acetonitrile; gradient elution was performed at a flow rate of 0.3 mL / min; the gradient elution procedure is shown in Table 1 below:

[0107] Table 1 Gradient elution program list

[0108] (4) Column temperature: 30°C;

[0109] (5) DAD detection wavelength: 214 nm;

[0110] (6) Mass spectrometry conditions: quadrupole mass spectrometry, ion source: electrospray ionization (ESI); scan mode: positive ion mode; ion scan range: 100 m / z–1400 m / z; collision-induced dissociation (Fragmentor): 135 V; nebulizer pressure: 40 psi; drying gas temperature: 350 °C; drying gas flow rate: 10 L / min; capillary voltage: 4000 V.

[0111] (7) Component analysis: Determine the specific ion peak, and then use the molecular weight to determine whether it is equivalent to the molecular weight of the non-ribosomal peptide family reported in the literature, or equivalent to the molecular weight of the non-ribosomal peptide obtained by bioinformatics analysis. In summary, it can be determined whether the product is the corresponding expressed non-ribosomal peptide.

[0112] The technical solutions and beneficial effects of the present invention are further illustrated below with reference to embodiments, comparative examples and experimental examples.

[0113] Example 1

[0114] Since the expression elements of the non-ribosomal peptide synthesis gene cluster itself are usually not expressed well in heterologous expression hosts, it is necessary to replace the expression elements of the non-ribosomal peptide synthesis gene cluster itself (such as promoters, terminators, and ribosome binding sites) according to the expression host to achieve the reconstruction of the non-ribosomal peptide synthesis gene cluster. In addition, the synthetic genes based on non-ribosomal peptides are relatively large, and the promoter of the non-ribosomal peptide synthesis gene cluster itself can be replaced with a strong promoter derived from the expression host.

[0115] In this example, 18 constitutive promoters were obtained through transcriptome data of Bacillus subtilis 168 and literature screening, namely PymdA, PahpC, PsodA, Phbs, PrpsO, PrplM, PrpsU, Pveg, PrplGB, Ppta, Pcdd, PyukE, PserA, Picd, PrpoA, PrpsL, PrplS and PtufA, among which PymdA, Pcdd, PserA and Pveg were derived from the literature. Among them, the method for screening promoters using the transcriptome data of Bacillus subtilis 168 is as follows: the mean and deviation values ​​of each treatment in the transcriptome data of Bacillus subtilis 168 (Pierre Nicolas et al. Science 335, 1103 (2012), DOI: 10.1126 / science.1206848) are calculated, and genes that simultaneously meet the three conditions of mean value higher than 15.0, deviation value less than 1.0, and transcript number higher than 15.0 under BT1 treatment conditions (30°C shaking culture for 24 hours) are selected. Promoter prediction is performed using the BPROM website, and finally the top 15 promoters with the highest mean values ​​that meet the conditions are obtained as candidate promoters for testing.

[0116] 1.1 Construction of recombinant plasmid pESC_Ppta

[0117] The construction method of the recombinant plasmid pESC_Ppta includes the following steps:

[0118] S1.11. Using the yeast plasmid pESC-Leu as a template, perform PCR amplification using primers pESC_For (nucleotide sequence shown in SEQ ID NO. 1) and pESC_Rev (nucleotide sequence shown in SEQ ID NO. 2) to obtain a linearized pESC-Leu fragment;

[0119] S1.12. Using the Bacillus subtilis ZL01 genome as a template, PCR amplification was performed using primers ZL01amyE_For (nucleotide sequence shown in SEQ ID NO. 3) and ZL01amyE_Rev (nucleotide sequence shown in SEQ ID NO. 4) to obtain the amyE fragment;

[0120] S1.13. Ligate the amyE fragment and the pESC-Leu fragment using the Gibson reaction to obtain plasmid pESC_amyE.

[0121] S1.14. Using the Bacillus subtilis ZL01 genome as a template, PCR amplification was performed using the primer pair TserA_For (nucleotide sequence shown in SEQ ID NO.5) and TserA_Rev (nucleotide sequence shown in SEQ ID NO.6) to obtain the TserA fragment; and, using the Bacillus subtilis ZL01 genome as a template, PCR amplification was performed using the primer pair tyrA_For (nucleotide sequence shown in SEQ ID NO.7) and tyrA_Rev (nucleotide sequence shown in SEQ ID NO.8) to obtain the tyrA fragment; and, using the pNW-sfGFP (SPS6) plasmid as a template, PCR amplification was performed using the primer pair Ppta_For (nucleotide sequence shown in SEQ ID NO.9) and Ppta_Rev (nucleotide sequence shown in SEQ ID NO.10) to obtain the promoter Ppta; and, using the pNW-sfGFP (SPS6) plasmid as a template, PCR amplification was performed using the primer pair RBS0-sfGFP-TER_For (nucleotide sequence shown in SEQ ID NO.11) to obtain the promoter Ppta. PCR amplification was performed using primers CmR_For (nucleotide sequence shown in SEQ ID NO. 13) and CmR_Rev (nucleotide sequence shown in SEQ ID NO. 14) to obtain a CmR resistance fragment.

[0122] S1.15. Perform overlap extension PCR on the fragments synthesized in step S1.14 in the order of TserA-Ppta-tyrA-RBS0-sfGFP-TER-CmR to obtain the TserA-Ppta-tyrA-RBS0-sfGFP-TER-CmR expression cassette;

[0123] S1.16. Use primer pairs pESC_amyE_For (nucleotide sequence as shown in SEQ ID NO.15) and pESC_amyE_Rev (nucleotide sequence as shown in SEQ ID NO.16) to linearize the plasmid pESC_amyE prepared in step S1.13, and then use the Gibson reaction to connect the TserA-Ppta-tyrA-RBS0-sfGFP-TER-CmR expression frame prepared in step S1.15 to the plasmid pESC_amyE to obtain the recombinant plasmid pESC_Ppta.

[0124] Among them, the structure of the recombinant plasmid pESC_Ppta is shown in Figure 1. The plasmid pESC_amyE is obtained by replacing the fragment with the 2235th site as the starting point and the 4422th site as the end point in the yeast plasmid pESC-Leu with the amyE fragment; the recombinant plasmid pESC_Ppta is obtained by replacing the fragment with the 904th site as the starting point and the 1048th site as the end point within the amyE gene in the plasmid pESC_amyE with the TserA-Ppta-tyrA-RBS0-sfGFP-TER-CmR expression cassette.

[0125] The details of the primers involved in constructing the recombinant plasmid pESC_Ppta are shown in Table 2 below:

[0126] Table 2 List of primers involved in the construction of recombinant plasmid pESC_Ppta

[0127] 1.2 Construction of recombinant plasmids with other promoters

[0128] Using the recombinant plasmid pESC_Ppta as the starting plasmid, recombinant plasmids (promoter detection vectors) containing a promoter obtained by screening were constructed. The construction method of each recombinant plasmid includes the following steps:

[0129] S1.21. Using the recombinant plasmid pESC_Ppta as a template, perform PCR amplification using primers pESC_Ppta__For (nucleotide sequence shown in SEQ ID NO. 17) and pESC_Ppta__Rev (nucleotide sequence shown in SEQ ID NO. 18) to obtain a linearized vector backbone;

[0130] S1.22. Using the Bacillus subtilis ZL01 genome as a template, perform PCR amplification using the primer pairs corresponding to each promoter in Table 2 to obtain a single promoter sequence;

[0131] S1.23. Use the Gibson reaction to connect the promoter sequence prepared in step S1.22 with the single promoter sequence prepared in step S1.21 to obtain a recombinant plasmid containing the target promoter.

[0132] According to the methods of steps S1.21 to S1.23, recombinant plasmids pESC_PymdA, recombinant plasmid pESC_PahpC, recombinant plasmid pESC_PsodA, recombinant plasmid pESC_Phbs, recombinant plasmid pESC_PrpsO, recombinant plasmid pESC_PrplM, recombinant plasmid pESC_PrpsU, recombinant plasmid pESC_Pveg, recombinant plasmid pESC_PrplGB, recombinant plasmid pESC_Pcdd, recombinant plasmid pESC_PyukE, recombinant plasmid pESC_PserA, recombinant plasmid pESC_Picd, recombinant plasmid pESC_PrpoA, recombinant plasmid pESC_PrpsL, recombinant plasmid pESC_PrplS and recombinant plasmid pESC_PtufA were successfully constructed respectively.

[0133] Among them, the primers involved are shown in Table 3 below:

[0134] Table 3 Primer information list

[0135] 1.3 Detecting the strength of each promoter

[0136] The recombinant plasmids containing the target promoter are respectively transformed into Bacillus subtilis, comprising the steps of: picking a fresh single colony of Bacillus subtilis ZL01 and inoculating it into 2 mL of 1×MC medium, culturing it at 37°C overnight, taking 40 μL of the culture solution and inoculating it into 2 mL of fresh 1×MC medium, and then culturing it at 37°C for 3 to 4 hours to obtain a bacterial solution; then taking 400 μL of the bacterial solution and placing it in a shaking tube, adding 1 μg of a single recombinant plasmid containing the target promoter, and then continuing to culture it at 37°C for 2 hours to obtain a transformation solution; then, taking 100 μL of the transformation solution and applying it to an LB plate supplemented with 5 mg / mL chloramphenicol, culturing it at 37°C overnight, and picking clones for colony PCR and / or sequencing to obtain transformants.

[0137] The method for detecting promoter strength comprises the following steps: a single transformant of a recombinant plasmid containing a target promoter is inoculated into LB medium, cultured at 30°C with shaking for 24 hours to obtain a fermentation broth; then, 100 μL of the fermentation broth is placed in a 96-well plate (Costar 3904), and the OD is measured using a microplate reader (Infinite 200Pro). 600The values ​​and fluorescence signals were obtained, wherein the excitation wavelength of the fluorescence signal was 485 nm and the emission wavelength was 512 nm, and three biological replicates were used for each sample. The negative control was a fermentation broth sample of a transformant containing the recombinant plasmid pESC_Pfree. The construction method of the recombinant plasmid pESC_Pfree was carried out according to S1.21 to S1.23 in 1.2, except that the PCR amplification primer pair in step S1.22 was replaced with "Pfree_For (nucleotide sequence shown in SEQ ID NO. 53) and Pfree_Rev (nucleotide sequence shown in SEQ ID NO. 54)".

[0138] The test results are shown in Table 4 and Figure 2 below:

[0139] Table 4 Summary of the strength of each promoter

[0140] As shown in Table 4 and Figure 2, except for PtufA, the other promoters all have good strength. Among them, PymdA is the strongest, and the strengths of PahpC, PahpC, PsodA, Phbs, PrpsO, PrplM, PrpsU, Pveg, PrplGB, Ppta, Pcdd and PyukE are also relatively ideal.

[0141] Example 2

[0142] In this example, 29 ribosome binding sites with a length of 20 bp were screened through the proteomic data of Bacillus subtilis 168, namely RBStsf, RBSfbaA, RBSaspB, RBSthiC, RBSacpA, RBSpdhC, RBSStufA, RBSgroEL, RBShag, RBSmdh, RBSaroA, RBSicd, RBSrplL, RBSahpC, RBSfusA, RBSpdhD, RBSrpsG, RBSrpsE, RBSpdhB, RBSatpD, RBSrpsP, RBSmtnA, RBSrpmC, RBSrplk, RBSglyA, RBShbs, RBSgndA, RBSypfD and RBSpgk. Among them, the method for screening ribosome binding sites through the proteomic data of Bacillus subtilis 168 is specifically as follows: the proteomic data of Bacillus subtilis 168 (Molecular & Cellular Proteomics 13.4 (2014): 1008-1019) are calculated to screen out genes whose protein expression levels are higher than 10,000 under two treatment conditions (glucose as carbon source and amino acids as carbon source).

[0143] In addition, the ribosome binding site RBSymdA used in conjunction with PymdA and the ribosome binding site RBSsodA used in conjunction with PsodA in Bacillus subtilis 168 were also used as candidate ribosome binding sites. The nucleotide sequences of each ribosome binding site are shown in Table 5 below:

[0144] Table 5 List of nucleotide sequences of various ribosome binding sites

[0145] Using the recombinant plasmid pESC_PymdA as the starting plasmid, an RBS detection vector was constructed. Specifically, the ribosome binding sites except RBS0 in Table 5 were replaced with RBS0 in the expression cassette TserA-PymdA-tyrA-RBS0-sfGFP-TER-CmR in the recombinant plasmid pESC_PymdA. Taking the construction method of the RBStsf detection vector as an example, the following steps are included:

[0146] S2.1. Using the recombinant plasmid pESC_PymdA as a template, PCR amplification was performed using primers pESC_PymdA_For (nucleotide sequence shown in SEQ ID NO. 86) and pESC_PymdA_Rev (nucleotide sequence shown in SEQ ID NO. 87) to obtain a linearized fragment;

[0147] S2.2. Using the recombinant plasmid pESC_PymdA as a template, PCR amplification was performed using primers tyrA-RBS-all_For (nucleotide sequence shown in SEQ ID NO. 88) and tyrA-RBStsf_Rev (nucleotide sequence shown in SEQ ID NO. 89) to obtain the tyrA-RBStsf fragment;

[0148] S2.3. Use the Gibson reaction to connect the tyrA-RBStsf fragment with the linearized fragment in step S2.1 to obtain the RBStsf detection vector.

[0149] It is understood that the construction method of other RBS detection vectors can refer to steps S2.1 to S2.3, with the only difference being that the tyrA-RBStsf_Rev reverse primer in step S2.2 is replaced with the corresponding specific reverse primer. Those skilled in the art can infer other specific reverse primers based on the tyrA-RBStsf_Rev reverse primer, such as the tyrA-RBSsodA_Rev reverse primer for preparing the RBSsodA detection vector as shown in SEQ ID NO.90, and the tyrA-RBSfbaA_Rev reverse primer for preparing the RBSfbaA detection vector as shown in SEQ ID NO.91, and the tyrA-RBSaspB_Rev reverse primer for preparing the RBSaspB detection vector as shown in SEQ ID NO.92.

[0150] Each RBS detection vector was transformed into Bacillus subtilis (refer to the previous 1.3 for the specific method) to obtain transformants, and then the OD of the fermentation liquid of each transformant was detected according to the promoter strength detection method in 1.3. 600 The negative control is a fermentation broth sample of a transformant containing the RBS-free detection vector. The construction method of the RBS-free detection vector is carried out with reference to steps S2.1 to S2.3, with the only difference being that the tyrA-RBStsf_Rev reverse primer in step S2.2 is replaced with the corresponding specific reverse primer tyrA-RBS-free_Rev (nucleotide sequence as shown in SEQ ID NO.93). In addition, a fermentation broth sample of a transformant containing the recombinant plasmid pESC_PymdA is used as a positive control.

[0151] The test results are shown in Table 6 and Figure 3 below:

[0152] Table 6 Summary of fluorescence signal intensity of each sample

[0153] As shown in Table 6 and Figure 3 , compared with RBS0, the strengths of RBStsf, RBSsodA, RBSfbaA, RBSaspB, RBSthiC, RBSacpA, RBSpdhC, RBStufA, RBSgroEL, RBShag, RBSmdh and RBSaroA have obvious advantages, indicating that these RBSs are more compatible with the promoter PymdA.

[0154] Example 3

[0155] This embodiment provides an expression cassette, an expression vector, a recombinant microorganism, a method for heterologous expression of a tyrocryptidase synthesis gene cluster, and its use in synthesizing tyrocryptidase. The tyrocryptidase synthesis gene cluster comprises the tyrA to tyrE genes, and their NCBI accession numbers are C2W64_03774 to C2W64_03778, respectively.

[0156] The expression cassette comprises PymdA, tyrA gene, RBSsodA, tyrB gene, RBSfbaA, tyrC gene, RBSaspB, tyrD gene, RBSthiC, tyrE gene, terminator TrrnB, terminator TrpsI and terminator TtufA connected in series.

[0157] The expression vector replaces the "tyrA-RBS0-sfGFP" sequence in the recombinant plasmid pESC_PymdA with "tyrA-RBSsodA-tyrB-RBSfbaA-tyrC-RBSaspB-tyrD-RBSthiC-tyrE".

[0158] The expression vector is introduced into Bacillus subtilis ZL01 to obtain a first recombinant microorganism. The method for constructing the first recombinant microorganism is detailed in steps S3.1 to S3.5 below.

[0159] S3.1. Design primers for cloning and reconstruction of the Brevibacterium tyroin synthesis gene cluster (tyrA gene to tyrE gene). Detailed information on the primers is shown in Table 7 below:

[0160] Table 7 List of primers related to cloning and reconstruction of the Brevibacterium tyropexin synthesis gene cluster

[0161] S3.2. Using the genome of Brevibacillus laterosporus MG64 (NZ_QJJD00000000.1) as a template, PCR amplification was performed using primers tyrA_For and tyrA_RBSsodA_Rev to obtain the first fragment; PCR amplification was performed using primers tyrB_RBSsodA_For and tyrB_RBSfbaA_Rev to obtain the second fragment; PCR amplification was performed using primers tyrC1_RBSfbaA_For and tyrC1_Rev to obtain the third fragment; PCR amplification was performed using primers tyrC2_For and tyrC2_RBSaspB_Rev to obtain the fourth fragment; PCR amplification was performed using primers tyrD_RBSaspB_For and tyrD_RBSthiC_Rev to obtain the fifth fragment; PCR amplification was performed using primers tyrE_RBSthiC_For and tyrE_Rev to obtain the sixth fragment.

[0162] In step S3.2, the PCR reaction system for synthesizing each fragment is shown in Table 8 below:

[0163] Table 8 PCR reaction system list

[0164] In step S3.2, the PCR reaction procedure for synthesizing each fragment is: ① pre-denaturation at 95℃ for 3 minutes; ② denaturation at 95℃ for 15 seconds; ③ annealing at 55℃ for 15 seconds; ④ extension at 72℃ for 5 minutes; ⑤ repeat ② to ④ for 35 cycles; ⑥ 72℃ for 10 minutes.

[0165] S3.3. Using the recombinant plasmid pESC_PymdA as a template, perform PCR amplification using primers pESC_PymdA_For and pESC_PymdA_Rev to obtain the linearized vector backbone;

[0166] S3.4. The first to sixth fragments prepared in step S3.2 and the linearized vector backbone prepared in step S3.3 are jointly transformed into Saccharomyces cerevisiae BY4742. The first to sixth fragments and the linearized vector backbone undergo homologous recombination in Saccharomyces cerevisiae BY4742. Colony PCR (designed primers are used to perform PCR verification on the junctions between adjacent fragments) is used to identify transformants containing the correct expression vector, and the expression vector is extracted.

[0167] In step S3.4, the preparation method of the competent state of Saccharomyces cerevisiae BY4742 includes the following steps: picking Saccharomyces cerevisiae BY4742 and inoculating it into 5 mL of YPD liquid medium, and then placing it in a shaking culture at 30°C and 220 rpm for 12 hours, and then transferring the activated Saccharomyces cerevisiae to 50 mL of YPD liquid medium, and adjusting the starting OD 600Adjust to 0.2 and culture at 30°C and 220 r / min until OD 600 is between 0.7 and 1.0 (the culture time is about 5 hours) to obtain a culture solution; the culture solution is centrifuged at 3000 r / min for 5 minutes, 50 mL of pure water is added to the bacteria from which the supernatant is discarded, the solution is centrifuged at 3000 r / min for 5 minutes, the supernatant is discarded, and then 20 mL of 100 mmol / L lithium acetate is added. After mixing, the solution is centrifuged again at 3000 r / min for 5 minutes, the supernatant is discarded again, and finally 400 μL of 100 mmol / L lithium acetate is added to resuspend the cells to obtain a competent state of Saccharomyces cerevisiae BY4742.

[0168] In step S3.4, the method for transforming the first to sixth fragments and the linearized vector backbone into the competent state of Saccharomyces cerevisiae BY4742 comprises the following steps: taking 100 μL of the prepared competent state of Saccharomyces cerevisiae BY4742 into a 1.5 mL centrifuge tube, centrifuging at 13000 r / min for 30 s, removing the supernatant, adding 240 μL of PEG3350 aqueous solution (50 g of PEG3350 is added to pure water to a volume of 100 mL), 36 μL of lithium acetate with a concentration of 1 mol / L, 10 μL of single-stranded salmon sperm DNA with a concentration of 10 mg / mL (needed to be diluted before use), and the like. The transformation system was prepared by adding 74 μL of the first to sixth fragments (400 ng of each fragment was added and the total volume was 74 μL with water) and heat shocking the transformation system at 42°C for 40 min. The heat-shocked transformation solution was centrifuged at 5000 r / min for 1 min, the supernatant was discarded, 1 mL of water was added and mixed, 100 μL of the bacterial solution was evenly spread on an Sc-Leu plate, and the plate was cultured at 30°C for 48 h. After verification, transformants containing the correct expression vector were obtained.

[0169] S3.5. Transform the expression vector extracted in step S3.4 into Bacillus subtilis ZL01. The transformation method is similar to that in step 1.3 above. After verification, a first recombinant microorganism containing the expression vector is obtained.

[0170] The heterologous expression method of the tyroclast synthesis gene cluster of the present embodiment includes the steps of: selecting a first recombinant microbial monoclonal clone and placing it in 3 mL of LB liquid culture medium supplemented with 5 mg / L chloramphenicol, and culturing it at 30° C. and 220 rpm for 24 hours, so that the tyroclast synthesis gene cluster heterologously expresses and synthesizes tyroclasts. During the heterologous expression process, Bacillus subtilis ZL01 containing the recombinant plasmid pESC_PymdA (obtained by transforming the recombinant plasmid pESC_PymdA into Bacillus subtilis ZL01) was used as the first control sample, and Bacillus subtilis ZL01 containing the recombinant plasmid pESC_Orig1 (obtained by transforming the recombinant plasmid pESC_Orig1 into Bacillus subtilis ZL01) was used as the second control sample, wherein the recombinant plasmid pESC_Orig1 was obtained by replacing the PymdA-tyrA-RBS0-sfGFP sequence in the recombinant plasmid pESC_PymdA with the "Ptyr-tyrA-tyrB-tyrC-tyrD-tyrE" sequence, and the promoter Ptyr was derived from Brevibacillus laterosporus MG64.

[0171] After the fermentation culture was completed, 1 mL of the fermentation broth was lyophilized and then extracted with methanol. The extract was then subjected to LC-MS for qualitative and quantitative analysis of tyrocidine. Figure 4 shows the LC-MS peak spectrum of the polypeptide product obtained after the first recombinant microorganism expressed the exogenous tyrocidine synthesis gene cluster, demonstrating that tyrocidine was indeed expressed and synthesized at a high level. In addition, Figures 5 and 6 show the LC-MS spectra of the fermentation products of the first and second control samples, respectively. As shown in Figures 5 and 6, neither the first nor the second control sample was able to heterologously express the exogenous tyrocidine synthesis gene cluster to synthesize tyrocidine.

[0172] Example 4

[0173] This embodiment provides an expression cassette, an expression vector, a recombinant microorganism, a method for heterologous expression of a tauramamide synthesis gene cluster, and its use in synthesizing tauramamide. The tauramamide synthesis gene cluster includes the tauB gene (NCBI accession number BRLA_RS11475), the tauC gene (NCBI accession number BRLA_RS11470), and the tauA gene (NCBI accession number BRLA_RS11480).

[0174] The expression cassette comprises PymdA, tauB gene, RBStsf, tauC gene, RBSsodA, tauA gene, terminator TrrnB, terminator TrpsI and terminator TtufA connected in series.

[0175] The expression vector replaces the "tyrA-RBS0-sfGFP" sequence in the recombinant plasmid pESC_PymdA with the "tauB-RBStsf-tauC-RBSsodA-tauA" sequence.

[0176] The expression vector is introduced into Bacillus subtilis ZL01 to obtain a second recombinant microorganism. The method for constructing the second recombinant microorganism is detailed in steps S4.1 to S4.5 below.

[0177] S4.1. Design primers related to cloning and reconstruction of the tauramamide biosynthesis gene cluster (tauB gene, tauC gene, and tauA gene). Detailed information of the primers is shown in Table 9 below:

[0178] Table 9 List of primers for cloning and reconstruction of the tauramamide synthesis gene cluster

[0179] S4.2. Using the genome of Brevibacillus laterosporus LMG15441 (NZ_CP007806.1) as a template, PCR amplification was performed using primers tauB1_For and tauB1_Rev to obtain the first fragment; PCR amplification was performed using primers tauB2_For and tauB2_RBStsf_Rev to obtain the second fragment; PCR amplification was performed using primers tauC_RBStsf_For and tauC_RBSsodA_Rev to obtain the third fragment; PCR amplification was performed using primers tauA_RBSsodA_For and tauA_Rev to obtain the fourth fragment.

[0180] In step S4.2, the PCR reaction system for synthesizing each fragment is as shown in Table 7 of Example 3, and the PCR reaction procedure for synthesizing each fragment refers to the PCR reaction procedure in Example 3.

[0181] S4.3. Using the recombinant plasmid pESC_PymdA as a template, perform PCR amplification using primers pESC_PymdA_For and pESC_PymdA_Rev to obtain the linearized vector backbone;

[0182] S4.4. The first to fourth fragments prepared in step S4.2 and the linearized vector backbone prepared in step S4.3 are jointly transformed into the competent cell of Saccharomyces cerevisiae BY4742. The first to fourth fragments and the linearized vector backbone undergo homologous recombination in Saccharomyces cerevisiae BY4742. Colony PCR (designed primers are used to perform PCR verification on the junctions between adjacent fragments) is used to identify the transformants containing the correct expression vector, and the expression vector is extracted.

[0183] In step S4.4, the preparation method of competent cells of Saccharomyces cerevisiae BY4742 and the transformation method are both referred to Example 3.

[0184] S4.5. Transform the expression vector extracted in step S4.4 into Bacillus subtilis ZL01 using the same method as in step 1.3 above. After verification, obtain the second recombinant microorganism.

[0185] In this embodiment, the method for heterologous expression of the tauramamide synthetic gene cluster includes the following steps: picking a single clone of the second recombinant microorganism and placing it in 3 mL of LB liquid culture medium supplemented with 5 mg / L chloramphenicol, and culturing it at 30°C and 220 rpm for 24 hours to allow the tauramamide synthetic gene cluster to heterologously express and synthesize tauramamide. During the heterologous expression process, Bacillus subtilis ZL01 containing the recombinant plasmid pESC_PymdA (obtained by transforming the recombinant plasmid pESC_PymdA into Bacillus subtilis ZL01) was used as a third control sample, and Bacillus subtilis ZL01 containing the recombinant plasmid pESC_Orig2 (obtained by transforming the recombinant plasmid pESC_Orig2 into Bacillus subtilis ZL01) was used as a fourth control sample, wherein the recombinant plasmid pESC_Orig2 was obtained by replacing the "PymdA-tyrA-RBS0-sfGFP" sequence in the recombinant plasmid pESC_PymdA with the "Ptau-tauB-tauC-tauA" sequence, and the promoter Ptau was derived from Brevibacillus laterosporus LMG15441.

[0186] After the fermentation culture was completed, 1 mL of the fermentation broth was lyophilized and then extracted with methanol. The extract was then subjected to LC-MS for qualitative and quantitative analysis of tauramamide. Figure 7 shows the LC-MS peak spectrum of the polypeptide product obtained after Bacillus subtilis ZL01 expressed the exogenous tauramamide synthesis gene cluster, confirming that tauramamide was indeed expressed and synthesized at a high level. Figure 8 shows the LC-MS spectrum of the fermentation product of the third control sample. As shown in Figure 8, the third control sample was unable to heterologously express the tauramamide synthesis gene cluster to synthesize tauramamide. In addition, testing revealed that the fourth control sample was also unable to heterologously express the tauramamide synthesis gene cluster to synthesize tauramamide.

[0187] Example 5

[0188] This embodiment provides an expression cassette, an expression vector, a recombinant microorganism, a method for heterologous expression of a kurstakin synthetic gene cluster, and its use in synthesizing kurstakin. The kurstakin synthetic gene cluster comprises the kurA gene (NCBI accession number DJ93_5371), the kurB gene (NCBI accession number DJ93_5372), and the kurC gene (NCBI accession number DJ93_5373).

[0189] The expression cassette comprises PymdA, kurA gene, RBStsf, kurB gene, RBSsodA, kurC gene, terminator TrrnB, terminator TrpsI and terminator TtufA connected in series.

[0190] The expression vector replaces the "tyrA-RBS0-sfGFP" sequence in the recombinant plasmid pESC_PymdA with the "kurA-RBStsf-kurB-RBSsodA-kurC" sequence.

[0191] The expression vector is introduced into Bacillus subtilis ZL01 to obtain a third recombinant microorganism. The construction method of the third recombinant microorganism is detailed in steps S5.1 to S5.5 below.

[0192] S5.1. Design primers related to cloning and reconstruction of the kurstakin synthetic gene cluster (kurA gene, kurB gene, and kurC gene). Detailed information of the primers is shown in Table 10 below:

[0193] Table 10 List of primers related to cloning and reconstruction of the kurstakin synthetic gene cluster

[0194] S5.2. Using the genome of Bacillus mycoides ATCC 21929 (NZ_JMQC00000000.1) as a template, PCR amplification was performed using primers kurA_For and kurA_RBStsf_Rev to obtain the first fragment; PCR amplification was performed using primers kurB_RBStsf_For and kurB_RBSsodA_Rev to obtain the second fragment; PCR amplification was performed using primers kurC1_RBSsodA_For and KurC1_Rev to obtain the third fragment; PCR amplification was performed using primers KurC2_For and KurC2_Rev to obtain the fourth fragment.

[0195] In step S5.2, the PCR reaction system for synthesizing each fragment is as shown in Table 7 of Example 3, and the PCR reaction procedure for synthesizing each fragment refers to the PCR reaction procedure in Example 3.

[0196] S5.3. Using the recombinant plasmid pESC_PymdA as a template, perform PCR amplification using primers pESC_PymdA_For and pESC_PymdA_Rev to obtain a linearized vector backbone.

[0197] S5.4. The first to fourth fragments prepared in step S5.2 and the linearized vector backbone prepared in step S5.3 are jointly transformed into the competent cell of Saccharomyces cerevisiae BY4742. The first to fourth fragments and the linearized vector backbone undergo homologous recombination in Saccharomyces cerevisiae BY4742. Colony PCR (designed primers are used to perform PCR verification on the junctions between adjacent fragments) is used to identify the transformants containing the correct expression vector, and the expression vector is extracted.

[0198] In step S5.4, the preparation method of competent cells of Saccharomyces cerevisiae BY4742 and the transformation method are both referred to Example 3.

[0199] S5.5. The expression vector extracted in step S5.4 is transformed into Bacillus subtilis ZL01. The transformation method is similar to that in step 1.3 above. After verification, the third recombinant microorganism is obtained.

[0200] The heterologous expression method of the kurstakin synthetic gene cluster in this embodiment includes the following steps: picking a single clone of the third recombinant microorganism and placing it in 3 mL of LB culture medium supplemented with 5 mg / L chloramphenicol, and culturing it at 30° C. and 220 rpm for 24 hours to allow the kurstakin synthetic gene cluster to heterologously express and synthesize kurstakin. During the heterologous expression process, Bacillus subtilis ZL01 containing the recombinant plasmid pESC_PymdA (obtained by transforming the recombinant plasmid pESC_PymdA into Bacillus subtilis ZL01) was used as the fifth control sample, and Bacillus subtilis ZL01 containing the recombinant plasmid pESC_Orig3 (obtained by transforming the recombinant plasmid pESC_Orig3 into Bacillus subtilis ZL01) was used as the sixth control sample, wherein the recombinant plasmid pESC_Orig3 is the recombinant plasmid pESC_PymdA in which the "PymdA-tyrA-RBS0-sfGFP" sequence is replaced with the "Pkur-kurA-kurB-kurC" sequence, and the promoter Pkur is derived from Bacillus mycoides ATCC 21929.

[0201] After the fermentation, 1 mL of the fermentation broth was lyophilized and extracted with methanol. The extract was then analyzed for kurstakin qualitatively and quantitatively by LC-MS. Figure 9 shows the LC-MS peaks of the products obtained after Bacillus subtilis ZL01 expressed the exogenous kurstakin synthesis gene cluster, demonstrating that kurstakin was indeed expressed and synthesized at a high level. Testing revealed that neither the fifth nor the sixth control samples heterologously expressed the kurstakin synthesis gene cluster and produced kurstakin.

[0202] The above is a detailed introduction to the heterologous expression method and application of an expression cassette, expression vector, recombinant microorganism, and non-ribosomal peptide synthesis gene cluster provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the technical solutions and core ideas of the present application. Those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An expression cassette, characterized in that The expression cassette comprises a promoter, a non-ribosomal peptide synthesis gene cluster and a terminator connected in series, wherein the non-ribosomal peptide synthesis gene cluster comprises N synthetic genes connected in series, N is a positive integer greater than or equal to 2, and at least one ribosome binding site is provided between two adjacent synthetic genes; the promoter, the terminator and the ribosome binding site are derived from Bacillus subtilis.

2. The expression cassette according to claim 1, characterized in that A ribosome binding site is located between two adjacent synthetic genes; and / or When the expression cassette comprises two or more ribosome binding sites, the two or more ribosome binding sites are different from each other; and / or The non-ribosomal peptide synthesis gene cluster is selected from the group consisting of a tyroclast synthesis gene cluster, a tauramamide synthesis gene cluster, a kurstakin synthesis gene cluster, a polymyxin synthesis gene cluster, a daptomycin synthesis gene cluster, a bleomycin synthesis gene cluster, auriporcine synthesis gene cluster, a tridecaptin synthesis gene cluster, a bacitracin synthesis gene cluster, a vancomycin synthesis gene cluster, a cyclosporin synthesis gene cluster, a nicotinoic acid synthesis gene cluster, a bouvardin synthesis gene cluster or an epothilone synthesis gene cluster.

3. The expression cassette according to claim 1, characterized in that The promoter is selected from one or more of PymdA, PahpC, PsodA, Phbs, PrpsO, PrplM, PrpsU, Pveg, PrplGB, Ppta, Pcdd, PyukE, PserA, Picd, PrpoA, PrpsL, PrplS and PtufA; and / or The terminator is selected from one or more of TrrnB, TrpsI and TtufA; and / or The ribosome binding site is selected from one or more of RBStsf, RBSsodA, RBSfbaA, RBSaspB, RBSthiC, RBSacpA, RBSpdhC, RBStufA, RBSgroEL, RBShag, RBSmdh, RBSaroA, RBSicd, RBSrplL, RBSahpC, RBS0, RBSfusA, RBSpdhD, RBSrpsG, RBSrpsE, RBSpdhB, RBSatpD, RBSrpsP, RBSymdA, RBSmtnA, RBSrpmC, RBSrplk, RBSglyA, RBShbs, RBSgndA, RBSypfD and RBSpgk.

4. The expression cassette according to claim 1, characterized in that The expression cassette further comprises a resistance gene, and the resistance gene is located at the 3' end of the terminator; optionally, the resistance gene is a chloramphenicol resistance gene.

5. The expression cassette according to any one of claims 1 to 4, characterized in that The non-ribosomal peptide synthesis gene cluster is used for synthesizing tyroclast peptides, and the N synthesis genes include tyrA gene, tyrB gene, tyrC gene, tyrD gene and tyrE gene.

6. The expression cassette according to claim 5, characterized in that The expression cassette comprises PymdA, tyrA gene, ribosome binding site RBSsodA, tyrB gene, ribosome binding site RBSfbaA, tyrC gene, ribosome binding site RBSaspB, tyrD gene, ribosome binding site RBSthiC, tyrE gene, terminator TrrnB, terminator TrpsI and terminator TtufA connected in series.

7. The expression cassette according to any one of claims 1 to 4, characterized in that The non-ribosomal peptide synthesis gene cluster is used to synthesize tauramamide, and the N synthesis genes include tauB gene, tauC gene and tauA gene.

8. The expression cassette according to claim 7, characterized in that The expression cassette comprises a promoter PymdA, a tauB gene, a ribosome binding site RBStsf, a tauC gene, a ribosome binding site RBSsodA, a tauA gene, a terminator TrrnB, a terminator TrpsI and a terminator TtufA connected in series.

9. The expression cassette according to claim 1, characterized in that The non-ribosomal peptide synthesis gene cluster is used to synthesize kurstakin, and the N synthesis genes include kurA gene, kurB gene and kurC gene.

10. The expression cassette according to claim 9, characterized in that The expression cassette comprises a promoter PymdA, a kurA gene, a ribosome binding site RBStsf, a kurB gene, a ribosome binding site RBSsodA, a kurC gene, a terminator TrrnB, a terminator TrpsI and a terminator TtufA connected in series.

11. An expression vector, characterized in that: Comprising an expression cassette as claimed in any one of claims 1 to 10.

12. The expression vector according to claim 11, characterized in that The expression vector is a recombinant plasmid obtained by replacing a fragment with the 2235th site as the starting point and the 4422nd site as the end point in the yeast plasmid pESC-Leu with the amyE gene derived from Bacillus subtilis, and replacing a fragment with the 904th site as the starting point and the 1048th site as the end point within the amyE gene with the expression cassette.

13. A recombinant microorganism, characterized in that The method comprises a host, and the expression cassette according to any one of claims 1 to 10 or the expression vector according to claim 11 or 12 located in the body of the host.

14. The recombinant microorganism according to claim 13, characterized in that The host is yeast or Bacillus subtilis.

15. A method for heterologous expression of a non-ribosomal peptide synthesis gene cluster, characterized in that: The steps include: Providing a first recombinant microorganism, wherein the first recombinant microorganism comprises yeast, and the expression cassette according to any one of claims 1 to 10 or the expression vector according to claim 11 or 12 located in the body of the yeast; extracting the expression cassette or the expression vector, and introducing the expression cassette or the expression vector into Bacillus subtilis to obtain a second recombinant microorganism; and The second recombinant microorganism is fermented and cultured to express the non-ribosomal peptide synthesis gene cluster.

16. The heterologous expression method according to claim 15, characterized in that: The yeast is Saccharomyces cerevisiae BY4742; and / or The Bacillus subtilis is Bacillus subtilis ZL01.

17. Use of the expression cassette according to any one of claims 1 to 10, or the expression vector according to claim 11 or 12, or the recombinant microorganism according to claim 13 or 14, or the heterologous expression method according to claim 15 or 16 in synthesizing non-ribosomal peptides.

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