A method for increasing the proportion of component A of avilamycin active ingredient

Abstract

The present application provides a method for increasing the proportion of component A of avilamycin active ingredient. Specifically, the present application provides a method for increasing the proportion and / or yield of component A of avilamycin active ingredient, which comprises the steps of: providing an avilamycin-producing strain, overexpressing AviZ1 protein in the avilamycin-producing strain or doubling the aviZ1 gene in the avilamycin-producing strain, thereby increasing the proportion and / or yield of component A in the avilamycin-producing strain. The recombinant strain containing multiple copies of aviZ1 gene obtained by the method of the present application can increase the proportion of component A, the main component of avilamycin, from the source, thereby simplifying the extraction process, reducing the production cost, and having good genetic stability, obvious social and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to obtaining a target gene that multiplies on the chromosome of Streptomyces using gene recombination methods. aviZ1 Recombinant strains and their applications. Background Technology

[0002] Avilamycin is a multi-component oligosaccharide antibiotic produced by *Streptomyces viride*, with a structure comprising a meta-dichloroformate unit and a hepta-saccharide chain. Unlike most antibiotics, avilamycin targets the uL16 and CTC proteins of the 50S ribosomal subunit, blocking tRNA binding at the A site and thus inhibiting bacterial protein synthesis. In addition to the active component avilamycin A, *Streptomyces viride* also produces avilamycin C and other minor impurity components. Heilman W. et al. determined the minimum inhibitory concentrations (MICs) of avilamycin A and C against different bacterial strains, showing that avilamycin A exhibited higher antibacterial activity than avilamycin C, with some strains showing a 10-fold difference in activity.

[0003] Avilamycin C, as the main byproduct, differs from A only in the structure of the two carbon units on the terminal glycosyl group: an acetyl group in component A and a hydroxyethyl group in component C. Therefore, conventional methods such as recrystallization, macroporous resin adsorption, and chemical oxidizing agents are insufficient for the efficient separation or conversion of the structurally similar effective component A and the byproduct C.

[0004] Currently, methods to increase the yield of the active component of avilamycin mainly include optimizing culture medium composition and fermentation conditions, mutagenesis breeding, and genome shuffling technology breeding. These methods are inconsistent in their effectiveness, time-consuming, and suffer from poor strain genetic stability. Furthermore, the industrial production and large-scale use of this strain in China is still in its infancy, and the fermentation industry system is not yet fully developed. Existing industrial production strains generally suffer from genetic instability, low fermentation titers, and poor component ratios, resulting in low domestic avilamycin self-sufficiency rates. Therefore, rationally designing genetic modification of strains to alter relevant metabolic pathways through genetic engineering to increase the proportion and yield of avilamycin component A has considerable industrial value and social benefits. To date, there have been no reports on improving the fermentation performance of strains by modifying the biosynthetic pathway of avilamycin. There is an urgent need in this field to develop a method that can increase the proportion of component A, the active ingredient of avilamycin, from the source. Summary of the Invention

[0005] The purpose of this invention is to provide a method for increasing the proportion and / or yield of the active ingredient A component of avilamycin.

[0006] Another objective of this invention is to provide a method for constructing recombinant strains with increased proportion and / or yield of avilamycin A component, so as to obtain optimized strains with higher antibacterial activity and simplify the process flow, which has significant social and economic benefits.

[0007] In a first aspect of the invention, a method is provided to increase the proportion and / or yield of component A, the active ingredient of avilamycin, the method comprising the steps of: providing an avilamycin-producing strain, and overexpressing the AviZ1 protein in the avilamycin-producing strain or overexpressing the AviZ1 protein in the avilamycin-producing strain. aviZ1 Gene multiplication increases the proportion and / or yield of component A in the avilamycin-producing strain.

[0008] In another preferred embodiment, the AviZ1 protein includes wild-type AviZ1 protein and mutant AviZ1 protein.

[0009] In another preferred embodiment, the AviZ1 protein has an amino acid sequence selected from the group consisting of: (1) The amino acid sequence as shown in SEQ ID NO: 22; and / or (2) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 22 and having aldehyde reductase activity.

[0010] In another preferred embodiment, the amino acid sequence of the mutant AviZ1 protein is selected from the group consisting of: (i) The amino acid sequence obtained by substituting, deleting, altering, inserting or adding one or more (usually 1-50, preferably 1-30, more preferably 1-20, best 1-10) amino acids in the sequence shown in SEQ ID NO: 22 within the range of maintaining enzyme activity.

[0011] (ii) Adding one or more (usually up to 20, preferably up to 10, and more preferably up to 5) amino acids to the C-terminus and / or N-terminus of the sequence shown in SEQ ID NO: 22 to obtain the amino acid sequence.

[0012] In another preferred embodiment, the aviZ1 Genes include wild type aviZ1 Genes and mutants aviZ1 Gene.

[0013] In another preferred embodiment, the aviZ1 The gene has a nucleotide sequence selected from the following group: (1) The nucleotide sequence shown in SEQ ID NO: 21; (2) A nucleotide sequence having ≥90% (preferably ≥95%, more preferably ≥98%) identity with the sequence shown in SEQ ID NO: 21; and / or (3) The nucleotide sequence obtained by truncating or adding 1-60 (preferably 1-30, more preferably 1-6) nucleotides at the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 21; Furthermore, the nucleotide sequences in (2) and (3) have the activity of positively regulating the synthesis of avilamycin A component.

[0014] In another preferred embodiment, the proportion of avilamycin A component in the total avilamycin components is increased in the range of 10%-20%, more preferably 10%-12%, more preferably 13%-16%, and most preferably 17%-20%.

[0015] In another preferred embodiment, the yield of the avilamycin A component is increased by 10%-70%, more preferably 10%-30%, even more preferably 30%-40%, and most preferably 40%-70%.

[0016] In another preferred embodiment, the aviZ1 Genes multiply to 2, 3, 4, or n times their original size.

[0017] In another preferred embodiment, the aviZ1 The number of genes doubled.

[0018] In another preferred embodiment, the avilamycin-producing strain includes wild-type green chromogenic Streptomyces and avilamycin industrial strains.

[0019] In another preferred embodiment, the avilamycin-producing strain is selected from: LK171, Streptomyces viridochromogenes Tü57, or a combination thereof.

[0020] In another preferred embodiment, the avilamycin-producing strain is LK171.

[0021] In a second aspect of the invention, a method is provided for constructing a recombinant strain with an increased proportion and / or yield of avilamycin A component, the method comprising the steps of: providing an avilamycin-producing strain, and doubling the proportion and / or yield of avilamycin A component in the avilamycin-producing strain. aviZ1 Genes, thereby obtaining aviZ1 The recombinant strain with gene duplication.

[0022] In another preferred embodiment, the recombinant strain contains aviZ1 Genes multiply to 2, 3, 4, or n times their original size.

[0023] In another preferred embodiment, the recombinant strain contains aviZ1 The number of genes doubled.

[0024] In another preferred embodiment, the aviZ1 Genes include wild type aviZ1 Genes and mutants aviZ1 Gene.

[0025] In another preferred embodiment, the aviZ1 The gene has a nucleotide sequence selected from the following group: (1) The nucleotide sequence shown in SEQ ID NO: 21; (2) A nucleotide sequence having ≥90% (preferably ≥95%, more preferably ≥98%) identity with the sequence shown in SEQ ID NO: 21; and / or (3) The nucleotide sequence obtained by truncating or adding 1-60 (preferably 1-30, more preferably 1-6) nucleotides at the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 21; Furthermore, the nucleotide sequences in (2) and (3) have the activity of positively regulating the synthesis of avilamycin A component.

[0026] In another preferred embodiment, the aviZ1 The gene sequence is shown in SEQ ID NO: 21.

[0027] In another preferred embodiment, the recombinant strain is selected from the LK804EZ series, LK804KZ series, LKBTEZ series, LKBTKZ series, or a combination thereof.

[0028] In another preferred embodiment, the avilamycin-producing strain includes wild-type green chromogenic Streptomyces and avilamycin industrial strains.

[0029] In another preferred embodiment, the avilamycin-producing strain is selected from: LK171, Streptomyces viridochromogenes Tü57, or a combination thereof.

[0030] In another preferred embodiment, the avilamycin-producing strain is LK171.

[0031] In another preferred embodiment, the genome of the avilamycin-producing strain contains an attB site sequence recognized by the integrase, wherein the attB site sequence includes endogenous attB site sequences and / or exogenous attB site sequences.

[0032] In another preferred embodiment, the integrase is selected from the group consisting of VWB integrase, ФBT1 integrase, ФC31 integrase, ФTG1 integrase, or combinations thereof.

[0033] In another preferred embodiment, the genome of the avilamycin-producing strain contains the attB site sequence as shown in SEQ ID NO: 5 and / or the attB site sequence as shown in SEQ ID NO: 6.

[0034] In another preferred embodiment, the proportion of avilamycin A component in the recombinant strain is increased by 10%-20% compared to the avilamycin producing strain, more preferably by 10%-12%, more preferably by 13%-16%, and most preferably by 17%-20%.

[0035] In another preferred embodiment, the yield of avilamycin A component in the recombinant strain is increased by 10%-70% compared to the avilamycin-producing strain, more preferably by 10%-30%, even more preferably by 30%-40%, and most preferably by 40%-70%.

[0036] In another preferred embodiment, the multiplication can be performed by methods selected from the group consisting of: protoplast transformation, electroporation, conjugation transfer, phage transduction, or combinations thereof.

[0037] In another preferred embodiment, the doubling step includes: (a) Constructing a system containing single or multiple copies aviZ1 Recombinant gene expression vectors; and (b) The recombinant expression vector obtained in (a) was introduced into an avilamycin-producing strain to obtain aviZ1 The recombinant strain with gene duplication.

[0038] In another preferred embodiment, the method further includes the step of: (c) Perform sequence verification and / or fermentation verification on the recombinant strain.

[0039] In another preferred embodiment, the bonding transfer step includes: (1) A Streptomyces application plasmid is provided, the Streptomyces application plasmid containing a complete integrase module, wherein the integrase module contains: (a1) a gene capable of expressing an integrase; and (a2) an attP site sequence recognized by the integrase; (2) The Streptomyces described in (a) were modified using plasmids as follows: (b1) Insertion of the target gene aviZ1 ; and (b2) optionally insert promoters; thereby constructing to obtain aviZ1 Gene duplication plasmids; and (3) Transferring the data obtained in (b) through indirect combination of subspecies. aviZ1 Gene duplication plasmids were introduced into an avilamycin-producing strain to obtain... aviZ1 The recombinant strain with gene duplication.

[0040] In another preferred embodiment, the engagement transfer optionally includes the step of: (4) Perform sequence verification and / or fermentation verification on the recombinant strain.

[0041] In another preferred embodiment, the integrase module is selected from the group consisting of: VWB-attP integrase module, ФBT1-attP integrase module, ФC31-attP integrase module, ФTG1-attP integrase module, or a combination thereof.

[0042] In another preferred embodiment, the integrase module is a VWB-attP integrase module and / or a ФBT1-attP integrase module.

[0043] In another preferred embodiment, the VWB-attP integrase module comprises a gene capable of expressing VWB integrase and an attP site sequence recognized by VWB integrase, wherein the attP site sequence is the sequence shown in SEQ ID NO: 19.

[0044] In another preferred embodiment, the ФBT1-attP integrase module comprises a gene capable of expressing ФBT1 integrase and an attP site sequence recognized by ФBT1 integrase, wherein the attP site sequence is the sequence shown in SEQ ID NO: 20.

[0045] In another preferred embodiment, the Streptomyces plasmid used is selected from: pSOK804, pD-BT1, pSET152, or a combination thereof.

[0046] In another preferred embodiment, the Streptomyces plasmid used is pSOK804, pD-BT1, or a combination thereof.

[0047] In another preferred embodiment, the pSOK804 comprises a complete VWB-attP integrase module.

[0048] In another preferred embodiment, the pD-BT1 comprises a complete ФBT1-attP integrase module.

[0049] In another preferred embodiment, the promoter is selected from: PermE 、PkasO Pj23119, or a combination thereof.

[0050] In another preferred embodiment, the promoter is PermE. 、PkasO , or a combination thereof.

[0051] In another preferred embodiment, the aviZ1 The gene duplication plasmid was selected from the following group: pSOK804-PermE - aviZ1 pSOK804-PkasO - aviZ1 pD-BT1-PermE - aviZ1 pD-BT1-PkasO - aviZ1 , or a combination thereof.

[0052] In another preferred embodiment, the pSOK804-PermE - aviZ1 Includes VWB-attP integrase module, target gene aviZ1 and promoter P ermE .

[0053] In another preferred embodiment, the pSOK804-PkasO - aviZ1 Includes VWB-attP integrase module, target gene aviZ1 and promoter P kasO .

[0054] In another preferred embodiment, the pD-BT1-PermE - aviZ1 Includes the ФBT1-attP integrase module and target gene. aviZ1 and promoter P ermE .

[0055] In another preferred embodiment, the pD-BT1-PkasO - aviZ1 Includes the ФBT1-attP integrase module and target gene. aviZ1 and promoter P kasO .

[0056] In another preferred embodiment, step (b) uses construction primers whose sequences are selected from nucleotide sequences as shown in SEQ ID NO: 7-14, or combinations thereof.

[0057] In another preferred embodiment, the sequence verification method is to perform PCR verification on the integration site attL fragment and / or attR fragment.

[0058] In another preferred embodiment, the sequence verification uses verification primers whose sequences are selected from nucleotide sequences shown in SEQ ID NO: 15-18, or combinations thereof.

[0059] In another preferred embodiment, the fermentation verification method is to detect and compare the yield and / or proportion of avilamycin A and C components in the recombinant strain and the avilamycin producing strain through avilamycin three-stage fermentation.

[0060] In a third aspect of the invention, a recombinant strain prepared by the method described in the second aspect of the invention is provided, the genome of which includes a multiplied... aviZ1 Gene.

[0061] In another preferred embodiment, the genome of the recombinant strain includes 2 times the amount of aviZ1 Genes, 3 times aviZ1 Genes, 4 times aviZ1 Genes, n times aviZ1 Gene.

[0062] In another preferred embodiment, the genome of the recombinant strain includes 2 times the amount of aviZ1 Gene.

[0063] In another preferred embodiment, the proportion of avilamycin A component in the recombinant strain is increased by 10%-20% compared to the avilamycin producing strain, more preferably by 10%-12%, more preferably by 13%-16%, and most preferably by 17%-20%.

[0064] In another preferred embodiment, the yield of avilamycin A component in the recombinant strain is increased by 10%-70% compared to the avilamycin-producing strain, more preferably by 10%-30%, even more preferably by 30%-40%, and most preferably by 40%-70%.

[0065] In another preferred embodiment, the recombinant strain is selected from the LK804EZ series, LK804KZ series, LKBTEZ series, LKBTKZ series, or a combination thereof.

[0066] In another preferred embodiment, the amino acid sequence of the recombinant strain is shown as protein_id=AAK83185.1.

[0067] In a fourth aspect of the invention, an AviZ1 protein is provided, said AviZ1 protein being expressed by a recombinant strain as described in the third aspect of the invention.

[0068] In another preferred embodiment, the AviZ1 protein has an amino acid sequence selected from the group consisting of: (1) The amino acid sequence as shown in SEQ ID NO: 22; and / or (2) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 22 and having aldehyde reductase activity.

[0069] In another preferred embodiment, the AviZ1 protein may be purified.

[0070] In another preferred embodiment, the AviZ1 protein may have an optional tag sequence for assisting expression and / or purification.

[0071] In another preferred embodiment, the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.

[0072] In a fifth aspect of the invention, a polynucleotide is provided that encodes the AviZ1 protein as described in the fourth aspect of the invention, or has a sequence of a recombinant strain as described in the third aspect of the invention or a derivative thereof.

[0073] In another preferred embodiment, the derived sequence has more than 95% homology with the recombinant strain sequence, more preferably more than 98% homology, and even more preferably more than 99% homology.

[0074] In another preferred embodiment, the recombinant strain sequence includes multiple folds of... aviZ1 Gene.

[0075] In another preferred embodiment, the polynucleotide may be RNA, DNA, or cDNA.

[0076] In a sixth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the fifth aspect of the invention.

[0077] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the viral vector includes lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0078] In another preferred embodiment, the expression vector includes plasmids and viscous particles.

[0079] In another preferred embodiment, the expression vector contains an resistance selection marker and the element oriT necessary for intergeneric synergistic transfer.

[0080] In another preferred embodiment, the resistance selection marker is an apramycin resistance gene.

[0081] In another preferred embodiment, the expression vector further includes a selection from the group consisting of promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0082] In another preferred embodiment, the promoter is selected from: PermE 、PkasO Pj23119, or a combination thereof.

[0083] In a seventh aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the sixth aspect of the invention, or having a genome containing polynucleotides as described in the fifth aspect of the invention.

[0084] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0085] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0086] In another preferred embodiment, the host cell is a Streptomyces cell.

[0087] In an eighth aspect of the invention, there is provided an use of an active ingredient selected from: recombinant strains as described in the third aspect of the invention, AviZ1 protein as described in the fourth aspect of the invention, polynucleotides as described in the fifth aspect of the invention, expression vectors as described in the sixth aspect of the invention, or host cells as described in the seventh aspect of the invention, or combinations thereof, wherein the active ingredient can be used for purposes selected from the group consisting of: (i) Interconversion of avilamycin A and C components in vitro; (ii) Increase the proportion and / or yield of avilamycin active ingredient A component; (iii) Preparation of recombinant strains producing high yields of avilamycin A fraction; and / or (iv) Inhibit bacterial protein synthesis.

[0088] In another preferred embodiment, the transformation includes an oxidation reaction or a reduction reaction.

[0089] In another preferred embodiment, the oxidation reaction partially or completely converts component C into component A.

[0090] In another preferred embodiment, the reduction reaction partially or completely converts component A into component C.

[0091] In another preferred embodiment, the cofactor for the oxidation reaction is NADPH.

[0092] In another preferred embodiment, the cofactor for the reduction reaction is NADP. + .

[0093] In another preferred embodiment, the oxidation reaction is performed at a pH of 8.5.

[0094] In another preferred embodiment, the reduction reaction is performed at a pH of 7.5.

[0095] In a ninth aspect of the invention, a product is provided, the product comprising (a) aviZ1 Gene duplication plasmid.

[0096] In another preferred embodiment, the product may further include: (b) reagents required for interspecific fusion transfer.

[0097] In another preferred embodiment, the product may further include: (c) a verification primer.

[0098] In another preferred embodiment, the sequence of the verification primer is shown in SEQ ID NO: 15-18.

[0099] In another preferred embodiment, the aviZ1 The gene duplication plasmid was selected from the following group: pSOK804-PermE - aviZ1 pSOK804-PkasO - aviZ1 pD-BT1-PermE - aviZ1 pD-BT1-PkasO - aviZ1 , or a combination thereof.

[0100] In another preferred embodiment, the aviZ1 Gene duplication plasmids contain constitutive promoters.

[0101] In another preferred embodiment, the constitutive promoter is selected from: promoter PermE 、PkasO Pj23119, or a combination thereof.

[0102] In another preferred embodiment, the product may further include an instruction manual describing how to use it. aviZ1 A method for preparing recombinant strains with increased proportion and / or yield of avilamycin A component using gene-multiplying plasmids.

[0103] In a tenth aspect of the invention, a preparation combination is provided, the preparation combination comprising: (Z1) Streptomyces containing an integrase module applied to plasmids; and (Z2) Construct primers; In another preferred embodiment, the Streptomyces plasmid used is selected from: pSOK804, pD-BT1, pSET152, or a combination thereof.

[0104] In another preferred embodiment, the integrase module is selected from the group consisting of: VWB-attP integrase module, ФBT1-attP integrase module, ФC31-attP integrase module, ФTG1-attP integrase module, or a combination thereof.

[0105] In another preferred embodiment, the sequence of the constructing primer is shown in SEQ ID NO: 7-14.

[0106] In another preferred embodiment, the preparation combination may further include: (Z3) is a reagent required for indirect combination transfer.

[0107] In another preferred embodiment, the preparation combination may further include: (Z4) verification primers.

[0108] In another preferred embodiment, the sequence of the verification primer is shown in SEQ ID NO: 15-18.

[0109] In another preferred embodiment, the product may further include a specification describing a method for using a recombinant strain to produce an increased proportion and / or yield of avilamycin A component.

[0110] In the eleventh aspect of the present invention, it is provided that aviZ1 The uses of the gene and AviZ1 protein, selected from the following groups: (i) Interconversion of avilamycin A and C components in vitro; (ii) Increase the proportion and / or yield of avilamycin active ingredient A component; and / or (iii) Prepare a recombinant strain that produces high levels of avilamycin A component.

[0111] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0112] Figure 1 pOJ260-Δ was displayed aviZ1 Plasmid map.

[0113] Figure 2 The mutant Δ was shown aviZ1 The fermentation results are compared with those of the wild type by HPLC.

[0114] Figure 3 pSOK804-P was displayed ermE - aviZ1 Plasmid map.

[0115] Figure 4 pSOK804-P was displayed kasO - aviZ1 Plasmid map.

[0116] Figure 5 pD-BT1-P was displayed. ermE - aviZ1 Plasmid map.

[0117] Figure 6 pD-BT1-P was displayed. kasO - aviZ1 Plasmid map.

[0118] Figure 7 The HPLC chromatograms of the fermentation results of the optimal strain LK804KZ-2 and the starting strain LK171 are shown. Figure 8 This shows an SDS-PAGE protein characterization gel image of the AviZ1 protein. Figure 9 The results show the activity assays performed using purified AviZ1, with avilamycin A and C fractions as substrates, NADPH or NADP+ as cofactors, and the pH values ​​for the reduction and oxidation reactions being 7.5 and 8.5, respectively. Detailed Implementation

[0119] Through extensive and in-depth research, the inventors of this invention have made the first discovery of genes. aviZ1 A positive regulatory relationship between avilamycin A component and the gene was discovered, and a site-specific integration mechanism was found that allows the gene in the native bacteria to... aviZ1 A method to double the proportion and / or yield of avilamycin A component.

[0120] Specifically, this invention utilizes a site-specific integration mechanism to integrate the target gene containing a strong promoter. aviZ1The plasmid is used to rapidly recombine the target gene requiring enhanced expression into the corresponding attB locus on the Streptomyces chromosome via intermetagenesal fusion transfer, thereby introducing it into the avilamycin-producing strain to enhance the expression of the component-optimized gene. Using a three-stage fermentation method for avilamycin, the modified recombinant strain is fermented. The resulting recombinant strain can increase the proportion of the main component A from the source, thus simplifying the extraction process, increasing the yield of component A, and reducing production costs, resulting in significant social and economic benefits. Furthermore, because the avilamycin-engineered strain of this invention has excellent genetic stability, its large-scale stable amplification is easily achieved. Therefore, the recombinant multiplicative strain and its construction method of this invention have high practicality.

[0121] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0122] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0123] As used in this article, the term "site-specific recombination" refers to a type of homologous recombination that relies on the pairing of small homologous sequences. It requires the participation of integrase (such as VWB, ФBT1) and specific recombination sites (such as attB / attP) to ultimately integrate the foreign plasmid into the genome of the target strain.

[0124] As used herein, the terms “introduction” or “transformation” refer to the transfer of exogenous polynucleotides into host cells (Streptomyces in this invention). Optionally, the exogenous polynucleotides may be integrated into the host genome.

[0125] As used herein, the term "target gene" refers to a foreign gene introduced into the genome of a native organism (Streptomyces in this invention).

[0126] As used herein, the terms "recombinant strain", "recombinant multiplying strain", "recombinant multiplying strain of the present invention", "avilamycin engineered strain", "avilamycin recombinant strain", "recombinant strain of avilamycin ..." are used interchangeably with "recombinant strain of avilamycin". aviZ1 "Gene-multiplying recombinant strains", aviZ1 Terms such as "gene-multiplying strain" are used interchangeably and all refer to strains constructed based on the principle of gene recombination using the method of increasing the proportion of avilamycin A component of this invention. aviZ1 Genetically engineered recombinant strains of avilamycin can achieve doubled expression of the gene.

[0127] As used herein, the terms “Avilamycin A component ratio” and “Avilamycin A component ratio” are used interchangeably, both referring to the proportion of avilamycin A component in the total avilamycin components. The total avilamycin components refer to avilamycin produced by industrial strains of avilamycin or wild-type green chromogenic streptomyces, which includes avilamycin A component, avilamycin C component, and other minor impurity components.

[0128] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or protein, etc.) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of sequence identity of nucleotide sequences and amino acid sequences is a method well known to those skilled in the art.

[0129] aviZ1 Genes and AviZ1 protein Genes in the avilamycin biosynthesis gene cluster (approximately 50 kb in length) aviZ1 It is responsible for encoding aldehyde-ketone reductase, and the nucleotide sequence of this gene is 993 bp in length. aviZ1 The nucleotide sequence of the gene can be found in GenBank: AF333038.2, 29508..30500, i.e. SEQ ID NO: 21), encoding 330 amino acids (the amino acid sequence of the AviZ1 protein can be found in GenBank: AAK83185.1, i.e. SEQ ID NO: 22).

[0130] This invention is the first to demonstrate that this gene is associated with the transformation of avilamycin A and C components, and it was found that this gene can positively regulate the synthesis of avilamycin A component. Knocking out the gene... aviZ1 Subsequently, avilamycin A fraction ceases to be produced, while avilamycin C fraction accumulates. Therefore, the proportion of avilamycin A fraction directly depends on the activity of aldehyde-ketone reductase AviZ1, which in turn is related to gene dosage, gene promoter strength, and the fermentation environment. Therefore, targeting... aviZ1 Genetic engineering can effectively increase the proportion and / or content of component A.

[0131] In addition, the AviZ1 protein can also interconvert avilamycin A and C components in vitro using oxidation or reduction reactions.

[0132] It should be understood that the present invention described aviZ1 Genes include wild type aviZ1 Genes and mutants aviZ1A gene having a nucleotide sequence selected from the group consisting of: (1) the nucleotide sequence shown in SEQ ID NO: 21; (2) a nucleotide sequence having ≥90% (preferably ≥95%, more preferably ≥98%) identity with the sequence shown in SEQ ID NO: 21; and / or (3) a nucleotide sequence obtained by truncating or adding 1-60 (preferably 1-30, more preferably 1-6) nucleotides at the 5' end and / or 3' end of the sequence shown in SEQ ID NO: 21; and the nucleotide sequences in (2) and (3) having the activity of positively regulating the synthesis of avilamycin A component.

[0133] It should be understood that the AviZ1 protein of the present invention includes wild-type AviZ1 protein and mutant AviZ1 protein, having an amino acid sequence selected from the group consisting of: (1) the amino acid sequence shown in SEQ ID NO: 22; and / or (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 22 and having aldehyde reductase activity. In the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Adding one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. Therefore, the amino acid sequence of the AviZ1 protein of the present invention also includes amino acid sequences obtained by substitution, deletion, alteration, insertion, or addition of amino acids, and by adding one or more amino acids to the C-terminus and / or N-terminus.

[0134] In this invention, preferably, it also includes "conserved variants of the AviZ1 protein," referring to polypeptides formed by replacing up to 10, more preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the AviZ1 protein of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0135] Table 1 Genetic engineering of Streptomyces With the advancement of biotechnology, genetic engineering of Streptomyces can solve problems such as increasing yield and optimizing components in the fermentation process of industrial strains for production. The introduction of exogenous DNA fragments can be achieved through homologous recombination or site-specific integration techniques. Site-specific integration mediated by bacteriophage integrase has become a routine method for introducing exogenous DNA fragments into Streptomyces due to its high strain stability and low susceptibility to reversion mutations. Based on the attB site sequence recognized by which integrase exists in the Streptomyces genome, the corresponding attP integrase module can be constructed in vitro using plasmids. Finally, the recombinant plasmid is introduced into the target Streptomyces host, achieving specific integration of the target gene onto the chromosome. Recombinant strains obtained through this method exhibit good stability.

[0136] promoter A promoter is a DNA sequence located upstream of the 5' end of a structural gene. It guides RNA polymerase to bind correctly to the target DNA template, activating RNA polymerase and thus initiating gene transcription. The region where RNA polymerase binds to the promoter is called the promoter region. Generally, in prokaryotes, this is the -10 region and the -35 region. Differences in promoter structure, such as variations in the nucleotide sequence between the -10 and -35 regions, can affect promoter activity. Therefore, using different promoters can directly influence gene transcription activity, thereby affecting gene expression. This invention uses the strongly constitutive promoter P derived from erythromycin-producing bacteria. ermE And the strong constitutive promoter P from Streptomyces azureense kasO Ultimately, this enhances the target gene. aviZ1 This enhances the expression of avilamycin by increasing the proportion of its main active ingredient, component A, at the source.

[0137] VWB, ФBT1 integrase system The VWB-attP integrase system is a system in which streptococci utilize their VWB integrase to mediate specific recombination and integration of attP and attB sites. The conserved sequences of the corresponding attB-attP sites are as follows: attPsite:ACGTTCTCTCCTAAAGCGGGTGTCGCAGGTTCGAATCCTGCCGGGGGCACAACCTGCA(SEQ ID NO: 19) attBsite:CACCGCTCTCCTAAAGCGGGTGTCGCAGGTTCGAATCCTGCCGGGGGCACAAG (SEQID NO: 5) The ФBT1-attP integrase system is a system in which streptococci utilize their ФBT1 integrase to mediate specific recombination and integration of attP with the attB site. The conserved sequences of the corresponding attB-attP sites are as follows: attPsite:TGCTGGGTTGTTGTCTCTGGACAGTGATCCATGGGAAACTACTCAGCA (SEQ ID NO:20) attBsite:CTGCCGTCCTTGACCAGGTTTTTGACGAAAGTGATCCAGATGATCCAGCTCCACACCCCGAA (SEQ ID NO: 6) attB consists of a sequence called BOB', while attP consists of POP'. O is the core sequence, common to both attB and attP, while the flanking sequences are B, B' and P, P', collectively called arms. Phage DNA is circular and integrates into the host chromosome during recombination, becoming a linear sequence. After recombination, two new hybridization att sites are generated on either side: the left one, called attL, consists of BOP', and the right one, called attR, consists of POB'.

[0138] aviZ1 Gene multiplication plasmid The present invention aviZ1 In gene duplication plasmids aviZ1 Genes can be multiplied to 2, 3, 4, or n times their original size using the method described in this invention.

[0139] Preferably, aviZ1 The gene-multiplying plasmid contains gene-selected from PermE 、PkasO The constitutive promoters obtained from Pj23119, or combinations thereof, are Pj23119, or combinations thereof. aviZ1 The gene duplication plasmid was selected from the following group: pSOK804-PermE - aviZ1 pSOK804-PkasO - aviZ1 pD-BT1-PermE - aviZ1 pD-BT1-PkasO - aviZ1 or combinations thereof. Typically, the present invention aviZ1 Gene multiplication plasmids have the following properties: Figure 3-6 The plasmid map shown.

[0140] aviZ1 Recombinant strains with gene duplication The present invention aviZ1The proportion of avilamycin A component in the gene-multiplying recombinant strain is increased by 10%-20%, preferably 10%-12%, more preferably 13%-16%, and most preferably 17%-20%; the yield of avilamycin A component is increased by 10%-70%, preferably 10%-30%, more preferably 30%-40%, and most preferably 40%-70%. The recombinant strains obtained by the method of this invention are selected from: LK804EZ series, LK804KZ series, LKBTEZ series, LKBTKZ series, or combinations thereof; wherein the LK804EZ series guides the insertion plasmid pSOK804-P. ermE - aviZ1 The strain, LK804KZ series, guides the insertion of plasmid pSOK804-P kasO - aviZ1 The strain, LKBTEZ series, guide plasmid pD-BT1-P ermE - aviZ1 The strain, LKBTKZ series, guide plasmid pD-BT1-P kasO - aviZ1 strains.

[0141] It should be understood that aviZ1 Gene multiplication can occur multiple times, such as 2 times, 3 times, 4 times... n times.

[0142] Third-stage fermentation Generally, for actinomycetes with relatively slow growth rates, two stages of culture are required in the seed culture process. First, the bacteria are inoculated into a primary seed culture medium, and then cultured in a secondary seed culture medium to obtain a large number of robust cells. Finally, the bacteria are transferred to a fermentation medium for fermentation. This is the three-stage fermentation method.

[0143] The main advantages of this invention include: (1) The recombinant multiplying strain of the present invention multiplies the target gene. aviZ1 This can increase the proportion of avilamycin A component from the source, avoiding the randomness of mutation breeding.

[0144] (2) The method of the present invention for increasing the proportion and / or yield of avilamycin A component can greatly simplify the extraction process of the target product, shorten the process cycle, optimize the control of quality standards, and reduce post-processing costs.

[0145] (3) The method of increasing the proportion and / or yield of avilamycin A component of the present invention increases the yield and fermentation potency of the main component A component, indirectly reducing production costs. The increase of avilamycin A component can obtain better antibacterial activity and generate significant social and economic benefits.

[0146] (4) Since the gene duplication method used is site-specific integration, the integration is irreversible. At the same time, the target gene is discretely distributed after introduction and is not easy to undergo homologous recombination. Therefore, the avilamycin recombinant engineered bacteria obtained by the method of the present invention have good genetic stability and can be stably amplified on a large scale.

[0147] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0148] Sequence information SEQ ID NO: 1 pOJ260-Δ aviZ1 -Lf primers acgacggccagtgccaagcttACCGGGGGTGCCGGCTTC SEQ ID NO: 2 pOJ260-Δ aviZ1 -Lr primers CGGGAAGCTGGTTGGCGCGCAGGAAACCGGCGTCGCTGTT SEQ ID NO: 3 pOJ260-Δ aviZ1 -Rf primers TGCGCGCCAACCAGCTTCCCGAGCACCTGGAGGACAGCGTC SEQ ID NO: 4 pOJ260-Δ aviZ1 -Rr primers ctatgacatgattacgaattcGTCGAGCAGATGGGCGTAGG SEQ ID NO: 5 Endogenous avi-VWB-attB site sequence CACCGCTCTCCTAAAGCGGGTGTCGCAGGTTCGAATCCTGCCGGGGGCACAAG SEQ ID NO: 6 Endogenous avi-ФBT1-attB site sequence CTGCCGTCCTTGACCAGGTTTTTGACGAAAGTGATCCAGATGATCCAGCTCCACACCCCGAA SEQ ID NO: 7 804-PermE - aviZ1 upstream primer tatgacatgattacgaattcGGGGACGGGGCATCTCCGTGG SEQ ID NO: 8804-PermE - aviZ1 Downstream primer tatgacatgattacgaattcGGGGACGGGGCATCTCCGTGG SEQ ID NO: 9804-PkasO - aviZ1 upstream primer tatgacatgattacgaattcGGGGACGGGGCATCTCCGTGG SEQ ID NO: 10 804-PkasO - aviZ1 Downstream primer tcatggagggcgcgaagctttgttcacattcgaacggtct SEQ ID NO: 11 BT1-PermE - aviZ1 upstream primer agctcggtacccggggatccGGTACCCGGGTCGCACGACG SEQ ID NO: 12 BT1-PermE - aviZ1 Downstream primer gttagttaggctaaaagcttGGGGACGGGGCATCTCCGTGG SEQ ID NO: 13 BT1-PkasO - aviZ1 upstream primer tcgcgcgcggccgcggatccGGGGACGGGCATCTCCGTGG SEQ ID NO: 14 BT1-PkasO - aviZ1 Downstream primer ctggggggagttatggggatccGGTACCCGGGTCGCACGACG SEQ ID NO: 15 VWBattL upstream primer AGACGTAGTCGATGGCCAGC SEQ ID NO: 16 VWBattL downstream primer GCCCTCATCTGTTACGCCGG SEQ ID NO: 17 BTattL upstream primer GGGTCGTACGACTTCACCCG SEQ ID NO: 18 BTattL downstream primer CCGCCTTCAGGTCACTGTCC SEQ ID NO: 19 Conserved sequence of attP site recognized by VWB integrase ACGTTCTCTCCTAAAGCGGGTGTCGCAGGTTCGAATCCTGCCGGGGGCACAACCTGCA SEQ ID NO: 20 Conserved sequence of attP site recognized by ФBT1 integrase TGCTGGGTTGTTGTCTCTGGACAGTGATCCATGGGAAACTACTCAGCA SEQ ID NO: 21 aviZ1 nucleotide sequence of a gene gtgaggtaccgcacgctgggacggcaaggccccgaggtctcctccgtctgcctgggcacctgggcgctgagcgggttctggggcagccggaccgagccggccgtcgaggcggtccggcgcgcgttcgacctgggcgtcaacttcttcgacaccgcgcacgcctacggcgcgggcatggccgaggcgggactggcccgcggcctgggcgacctgctgcgcacccgtcgctccgacatcgtcatctcgacgaagggcggcctggagctgcggggcgacggtgtggtccgcaacagcgacgccggtttcctgcgcgccaacctgaccgacagcctgcgctcgctggggaccgactacgtggacgtgttcctggtccactggcccgacccgagggtcccgccggccgagacggcgggtgcgctggcggggttcgtggaggaagggctcgcccggtacgtgggcgtctccaacttcaccgtcgaggaaatggccgaattctcctccgtcgtcaccccgcaggtcgcccaggtgccgttcaacatgctcgaccgcggcatcgagaagcaggtgctgccgcactgcgccgctgccgggatcggcgtcatggggtggtcggcgctcgcgcacggggtgctggcgggagcgctccgtcccggccaggtgttcccgcccgacgactggcgcgcctactccccgaccttccagggcgaacgcttcgccaagctgctcgcggccgtggaccggctgaaggagttcgccgccgaacgcgggcacagtgtcgcccagctcgcgcttgcctgggtgctggcccacccctcgggagtcatcccggtcatcggcgcccagcttcccgagcacctggaggacagcgtccgggccgtggacctcgacctcgacgaggccgagctgcgcgccctggacgaactgctggccgacgcgcccgagctggacggcagcggggaccaaccgcccgcgcgcgaagagaggtcggtctga SEQ ID NO: 22 Amino acid sequence of AviZ1 protein mryrtlgrqgpevssvclgtwalsgfwgsrtepaveavrrafdlgvnffdtahaygagmaeaglarglgdllrtrrsdivistkgglelrgdgvvrnsdagflranltdslrslgtdyvdvflvhwpdprvppaetagalagfveeglaryvgvsnftveemaef ssvvtpqvaqvpfnmldrgiekqvlphcaaagigvmgwsalahgvlagalrpgqvfppddwraysptfqgerfakllaavdrlkefaaerghsvaqlalawvlahpsgvipvigaqlpehledsvravdldldeaelraldelladapeldgsgdqppareersv General Materials and Methods general material Unless otherwise specified, all plasmids and strains in the examples are conventional or commercially available.

[0149] strain LK171 is an industrial strain of avilamycin, provided by Greencon Biochemical Co., Ltd.

[0150] LK171Δ aviZ1 To knock out the avilamycin biosynthesis gene cluster through homologous recombination double crossover aviZ1 Mutant strains obtained from genes.

[0151] LK804MEZ1, LK804KZ1, LKBTMEZ1, and LKBTKZ1 are site-specific integration strains that use LK171 as the starting strain to multiply the target gene. aviZ1 The recombinant strain obtained.

[0152] plasmid The Streptomyces plasmid pSOK804 is a routine operation plasmid for Streptomyces, which is stored in our laboratory. The sequence information is referenced from GenBank: LT545994.1.

[0153] The Streptomyces application plasmid pD-BT1 was constructed in our laboratory using pSOK804 as the starting vector and the VWB integrase module was replaced with the ФBT1 integrase module.

[0154] Streptomyces application of plasmid pOJ260-Δ aviZ1 To construct the gene knockout required for this application, the Streptomyces plasmid pOJ260 (a standard operation plasmid for Streptomyces) was used as a vector to insert and knock out the gene. aviZ1 The left and right homologous arms.

[0155] Streptomyces application of plasmid pSOK804-P ermE - aviZ1 For the construction of this application, the Streptomyces plasmid pSOK804 (a standard operating plasmid for Streptomyces) was used as a vector, and the promoter P was inserted. ermE With genes aviZ1 .

[0156] Streptomyces application of plasmid pSOK804-P kasO - aviZ1 To construct the vector for this application, the Streptomyces plasmid pSOK804 (a standard operating plasmid for Streptomyces) was used, and the promoter P was inserted. kasO With genes aviZ1 .

[0157] Streptomyces application of plasmid pD-BT1-P ermE - aviZ1 To construct the vector for this application, the Streptomyces plasmid pD-BT1 (a standard operating plasmid for Streptomyces) was used, and the promoter P was inserted. ermE With genes aviZ1 .

[0158] Streptomyces application of plasmid pD-BT1-P kasO - aviZ1 To construct the vector for this application, the Streptomyces plasmid pD-BT1 (a standard operating plasmid for Streptomyces) was used, and the promoter P was inserted. kasO With genes aviZ1 .

[0159] culture medium ISP-4 medium (%): soluble starch 1.0, K2HPO4 0.1, MgSO4 0.1, NaCl 0.1, (NH4)2SO4 0.2, CaCO3 0.2, FeSO4 0.0001, MnCl2·6H2O 0.0001, ZnSO4 0.0001, agar 2.0, pH 7.2.

[0160] AS-1 medium (%): yeast extract 0.1, soluble starch 0.5, NaCl 0.25, Na2SO4 1.0, L-alanine 0.02, L-arginine 0.05, agar 2.0, pH 7.2.

[0161] Avilamycin primary seed culture medium (%): soluble starch 1.0, yeast extract 0.6, malt extract 1.0, tryptone 0.2, glucose 0.4, dipotassium hydrogen phosphate 0.05, calcium carbonate 0.1, calcium chloride dihydrate 0.015, pH 7.2.

[0162] Avilamycin secondary seed culture medium (%): corn starch 2.0, soybean meal 1.5, glucose 0.5, yeast extract 0.2, calcium chloride dihydrate 0.2, calcium carbonate 0.3, sodium chloride 0.1, magnesium sulfate 0.02, ammonium sulfate 0.02, pH 7.2.

[0163] Avilamycin fermentation medium (%): corn starch 8.0, soybean meal 2.0, glucose 1.6, sodium nitrate 0.2, ammonium sulfate 0.02, magnesium sulfate 0.04, sodium chloride 0.1, trace elements (ferrous sulfate, manganese chloride, zinc sulfate 20 ppm, copper sulfate, cobalt chloride 10 ppm), calcium chloride 0.2, calcium carbonate 0.3, soybean oil 0.8, pH 7.2.

[0164] General Method Validation of the attB integration site on the chromosome of Streptomyces Site-specific recombination integrase can accurately recognize the bacterial attachment site attB and the attP site in the plasmid, catalyzing the integration between attB and attP to form attL and attR. Since attL and attR do not exhibit significant duplication and cannot serve as substrates for integrase recombination, the reaction is unidirectional and irreversible, and can be stably passaged with the host. Therefore, by designing primers flanking the attB site and the attP site in the plasmid, combined PCR verification was performed based on the recombined attL and attR, followed by further sequencing confirmation.

[0165] Methods of joint transfer The procedures were performed using the standard methods described in the Genetic Manipulation of Streptomyces (A Laboratory Manual. 1985. DA Hopwood The John Innes Foundation).

[0166] Fermentation method of avilamycin The strain grown on ISP-4 plates at 30°C for 5-7 days (cut off along with the agar block, approximately 1 cm in size) 2 The primary seed culture was inoculated into a shake flask containing the secondary seed culture and cultured at 28°C and 230 rpm for 48-72 hours until concentrated. The primary seed culture (10% inoculum) was then inoculated into a shake flask containing the secondary seed culture and cultured at 28°C and 230 rpm for 24 hours. The secondary seed culture (10% inoculum) was then inoculated into the fermentation medium and cultured at 28°C and 230 rpm for 7-9 days. Samples were then taken from the flasks for avilamycin analysis.

[0167] Methods for determining the potency of avilamycin After fermentation, a certain amount of bacterial culture was taken, an equal volume of methanol was added, the mixture was sonicated for 10 minutes, allowed to stand for 1 hour, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken for HPLC analysis. The remaining sample was stored in a -20℃ refrigerator.

[0168] HPLC detection conditions for avilamycin Mobile phases: Phase A: 95% water, 5% acetonitrile (10 mM ammonium acetate); Phase B: 5% water, 95% acetonitrile (10 mM ammonium acetate) Injection volume: 10-20 μL; Flow rate: 1 mL / min DAD recording wavelengths: 214 nm, 254 nm, 280 nm, without reference. Liquid phase gradient program: 0 min: 20% B phase 0-10 min: Linear increase to 45% B phase 10-27 min: Linear increase to 62% B phase 27-28 min: linear decrease to 20% in phase B 28-30 min: 20% B phase Example 1. Knockout of plasmid pOJ260-Δ aviZ1 Construction The Streptomyces plasmid pOJ260 (GenBank: GU270843.1), used routinely, contains the apramycin resistance gene and the oriT gene required for conjugation transfer; it is a suicide knockout vector. The vector was linearized using HindIII and EcoRI double digestion, followed by insertion of a knockout gene at the digestion sites. aviZ1 The required left and right homologous arm fragments of the gene were used to obtain the target plasmid pOJ260-Δ. aviZ1 .

[0169] pOJ260-Δ aviZ1 plasmid maps as follows Figure 1 As shown, the required primers are as shown in SEQ ID NO: 1-4.

[0170] Example 2. Mutant strain Δ aviZ1 Construction and fermentation verification Plasmid pOJ260-Δ aviZ1 Introduced into the wild-type LK171 strain via indirect zygotic transfer. Conjugates were selected and streaked onto AS-1 plates with nadolpicolone and apramycin resistance. The resulting conjugates were then validated by single-crossover PCR. Afterward, the conjugates were relaxed to obtain either successfully relaxed mutants or reverted wild-type strains. PCR validation was performed using knockout validation primers. Correctly validated mutants underwent subsequent tertiary fermentation to verify the gene. aviZ1 Whether it is related to the conversion of avilamycin A and C components.

[0171] like Figure 2 The HPLC chromatogram shown indicates that the mutant strain Δ aviZ1 The fermentation results were compared with those of the wild type. From Figure 2 The fermentation results shown indicate that, compared to the wild type, knocking out the target gene resulted in the elimination of avilamycin A fraction, while avilamycin C fraction accumulated, suggesting that the gene... aviZ1 It is highly correlated with the expression of component A. Therefore, it can be increased by doubling. aviZ1 Genes to increase the proportion of avilamycin A component.

[0172] Example 3. Construction of multiplication plasmids Analysis of the genome sequence of *Streptomyces viride*, the avilamycin-producing bacterium, revealed one natural endogenous avi-VWB-attB site sequence (with a complete 53 bp sequence) and one natural endogenous avi-ФBT1-attB site sequence (with a complete 62 bp sequence), as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0173] Because wild-type LK171 contains the attB site required for the VWB and ФBT1 integrase systems, these two sites can be used to multiply the target gene. aviZ1 At the same time, strong constitutive promoters are used respectively. P ermE Or a stronger constitutive promoter P kasO .

[0174] The Streptomyces plasmid pSOK804 (GenBank: LT545994.1), used routinely, contains a complete VWB-attP integrase module. The Streptomyces plasmid pD-BT1 (constructed by the inventors) contains a complete ФBT1-attP integrase module. The target gene was inserted using either pSOK804 or pD-BT1 as a vector. aviZ1At the same time, a promoter is attached to the 5' end of the gene. P ermE or promoter P kasO The final plasmid pSOK804-P was constructed. ermE - aviZ1 pSOK804-P kasO - aviZ1 pD-BT1-P ermE - aviZ1 and pD-BT1-P kasO - aviZ1 .

[0175] The plasmid maps of the above four plasmids are as follows: Figures 3-6 As shown, the primers required for construction are shown in SEQ ID NO: 7-14.

[0176] Example 4: Construction and fermentation validation of component-optimized recombinant strains plasmid pSOK804-P ermE - aviZ1 pSOK804-P kasO - aviZ1 pD-BT1-P ermE - aviZ1 and pD-BT1-P kasO - aviZ1 The conjugates were introduced into the wild-type LK171 strain via indirect genus-specific transfer. Approximately 10 conjugates were randomly selected and streaked onto AS-1 plates with both apramycin and nalidixic acid resistance. The resulting colonies were then validated by PCR using the attL fragment at the integration site.

[0177] The validation PCR product was sequenced, and the sequencing results showed that it was consistent with the expected attL sequence.

[0178] Therefore, the plasmid pSOK804-P was introduced. ermE - aviZ1 The strain was named the LK804EZ series and the plasmid pSOK804-P was introduced. kasO - aviZ1 The strain was named the LK804KZ series and the plasmid pD-BT1-P was introduced. ermE - aviZ1 The strain was named the LKBTEZ series and the plasmid pD-BT1-P was introduced. kasO - aviZ1 The strain was named the LKBTKZ series.

[0179] The designed attL sequence verification primers are shown in SEQ ID NO: 15-18.

[0180] Three zygotes from each series were selected and subjected to three-stage fermentation in 500 mL Erlenmeyer flasks to detect the yield and ratio of avilamycin A and C components, while the starting strain LK171 was used as a control.

[0181] Based on the fermentation results, the target gene was multiplied through site-specific integration. aviZ1 Subsequently, compared with the starting strain LK171, the yield and proportion of avilamycin A component were significantly improved. Among them, strain LK804KZ-2 had the highest yield of A component and the lowest C / A ratio.

[0182] The HPLC chromatograms of the fermentation results of the optimal strain LK804KZ-2 and the starting strain LK171 are shown below. Figure 7 As shown, the yield of component A increased from 0.30 g / L (starting strain LK171) to 1.19 g / L, and the C / A ratio increased from 0.77 (starting strain LK171) to 0.19.

[0183] Example 5: In vitro reconstitution of avilamycin A and C components AviZ1 belongs to the AKR (aldehyde-ketone reductase) superfamily. These enzymes typically contain NAD(P)H or NAD(P) compounds. + As a cofactor, it catalyzes redox reactions, reducing carbonyl groups to alcohols, or the reverse reaction.

[0184] The soluble, active protein AviZ1 was expressed and purified. The SDS-PAGE protein characterization gel image of this protein is shown below. Figure 8 As shown, the AviZ1 protein size is 36.4 kDa, consistent with the predicted value, indicating that the expressed and purified AviZ1 protein was correctly identified by SDS-PAGE. Furthermore, the expression bands are uniform, indicating that the AviZ1 protein expressed and purified in this invention achieved high purity.

[0185] The activity of purified AviZ1 was measured, using avilamycin A and C fractions as substrates, with NADPH or NADP as the chosen substrate. + As cofactors, the pH value for the reduction reaction was chosen to be 7.5, and the pH value for the oxidation reaction was chosen to be 8.5. The activity assay reaction temperature was 30℃, and the reaction was carried out overnight. The specific activity assay system is shown in Table 2: Table 2 After the activity assay was completed, an equal volume of methanol was added for quenching. Samples were then taken for low-resolution LC-MS analysis. The extracted ion current (m / z = 1420-1426, +NH4+) was measured. + Search for target molecules and measure activity results as follows: Figure 9 As shown.

[0186] according to Figure 9 The activity test results showed that for the reduction reaction, component A was almost completely converted into component C, while for the oxidation reaction, most of component C was converted into component A, leaving only a small amount of substrate. Therefore, AviZ1 can interconvert components A and C of avilamycin.

[0187] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for increasing the proportion and / or yield of avilamycin's active ingredient, component A, characterized in that, The method includes the steps of: providing an avilamycin-producing strain, and overexpressing the AviZ1 protein in the avilamycin-producing strain or overexpressing the AviZ1 protein in the avilamycin-producing strain. aviZ1 Gene duplication, thereby increasing the proportion and / or yield of component A in the avilamycin-producing strain; The amino acid sequence of the AviZ1 protein is shown in SEQ ID NO:

22. aviZ1 The nucleotide sequence of the gene is shown in SEQ ID NO:

21.

2. The method as described in claim 1, characterized in that, The aviZ1 The gene multiplies to 2, 3, or 4 times its original size.

3. The method as described in claim 1, characterized in that, The avilamycin-producing strains include wild-type green chromogenic Streptomyces and avilamycin industrial strains.

4. The method as described in claim 1, characterized in that, The avilamycin-producing strain was selected from: Streptomyces viridochromogenes Tü57.

5. A method for constructing a recombinant strain with an increased proportion and / or yield of avilamycin A component, characterized in that, The method includes the steps of: providing an avilamycin-producing strain, and multiplying the avilamycin-producing strain... aviZ1 Genes, thereby obtaining aviZ1 The recombinant strain with gene duplication, wherein the proportion and / or yield of the avilamycin A component in the recombinant strain is increased; The aviZ1 The nucleotide sequence of the gene is shown in SEQ ID NO:

21.

6. The method as described in claim 5, characterized in that, The recombinant strain aviZ1 The gene multiplies to 2, 3, or 4 times its original size.

7. The method as described in claim 5, characterized in that, The recombinant strains are selected from the LK804EZ series, LK804KZ series, LKBTEZ series, LKBTKZ series, or combinations thereof.

8. The method as described in claim 5, characterized in that, The genome of the avilamycin-producing strain contains an attB site sequence recognized by integrase, wherein the attB site sequence includes endogenous attB site sequences and / or exogenous attB site sequences.

9. The method as described in claim 8, characterized in that, The integrase is selected from the group consisting of VWB integrase, ФBT1 integrase, ФC31 integrase, ФTG1 integrase, or a combination thereof.

10. The method as described in claim 8, characterized in that, The genome of the avilamycin-producing strain contains the attB site sequence as shown in SEQ ID NO: 5 and / or the attB site sequence as shown in SEQ ID NO:

6.

11. The method as described in claim 5, characterized in that, The multiplication can be achieved by methods selected from the group consisting of: protoplast transformation, electroporation, conjugation transfer, phage transduction, or combinations thereof.

12. The method as described in claim 5, characterized in that, The doubling steps include: (a) Constructing a system containing single or multiple copies aviZ1 Recombinant gene expression vectors; and (b) The recombinant expression vector obtained in (a) was introduced into an avilamycin-producing strain to obtain aviZ1 The recombinant strain with gene duplication.

13. The method as described in claim 5, characterized in that, The method further includes the steps of: performing sequence verification and / or fermentation verification on the recombinant strain.

14. The method as described in claim 11, characterized in that, The bonding transfer step includes: (1) A Streptomyces application plasmid is provided, the Streptomyces application plasmid containing a complete integrase module, wherein the integrase module contains: (a1) a gene capable of expressing an integrase; and (a2) an attP site sequence recognized by the integrase; (2) The Streptomyces described in (1) were modified using plasmids as follows: (b1) Insertion of the target gene aviZ1 ; and (b2) insert promoters; thereby constructing and obtaining aviZ1 Gene duplication plasmids; and (3) Transfer the data obtained in (2) through indirect combination of subspecies. aviZ1 Gene duplication plasmids were introduced into an avilamycin-producing strain to obtain... aviZ1 The recombinant strain with gene duplication.

15. The method as described in claim 14, characterized in that, The joining transfer also includes the step of: (4) Perform sequence verification and / or fermentation verification on the recombinant strain.

16. The method as described in claim 14, characterized in that, The integrase module is selected from the group consisting of: VWB-attP integrase module, ФBT1-attP integrase module, ФC31-attP integrase module, ФTG1-attP integrase module, or a combination thereof.

17. The method as described in claim 14, characterized in that, The integrase module is a VWB-attP integrase module and / or a ФBT1-attP integrase module.

18. The method as described in claim 17, characterized in that, The VWB-attP integrase module comprises a gene expressing VWB integrase and an attP site sequence recognized by VWB integrase, wherein the attP site sequence is as shown in SEQ ID NO:

19.

19. The method as described in claim 17, characterized in that, The ФBT1-attP integrase module comprises a gene expressing ФBT1 integrase and an attP site sequence recognized by ФBT1 integrase, wherein the attP site sequence is as shown in SEQ ID NO:

20.

20. The method as described in claim 14, characterized in that, The Streptomyces plasmid used is selected from: pSOK804, pD-BT1, pSET152, or a combination thereof.

21. The method as described in claim 14, characterized in that, The Streptomyces used plasmids are pSOK804, pD-BT1, or a combination thereof.

22. The method as described in claim 21, characterized in that, The pSOK804 contains a complete VWB-attP integrase module.

23. The method as described in claim 21, characterized in that, The pD-BT1 contains a complete ФBT1-attP integrase module.

24. The method as described in claim 14, characterized in that, The promoter is selected from: PermE 、PkasO Pj23119, or a combination thereof.

25. The method as described in claim 14, characterized in that, The promoter is PermE. 、PkasO , or a combination thereof.

26. The method as described in claim 14, characterized in that, The aviZ1 The gene duplication plasmid was selected from the following group: pSOK804-PermE - aviZ1 pSOK804-PkasO - aviZ1 pD-BT1-PermE - aviZ1 pD-BT1-PkasO - aviZ1 or a combination thereof; pSOK804-PermE - aviZ1 Includes VWB-attP integrase module, target gene aviZ1 and promoter P ermE ; pSOK804-PkasO - aviZ1 Includes VWB-attP integrase module, target gene aviZ1 and promoter P kasO ; The pD-BT1-PermE - aviZ1 Includes the ФBT1-attP integrase module and target gene. aviZ1 and promoter P ermE ; pD-BT1-PkasO - aviZ1 Includes the ФBT1-attP integrase module and target gene. aviZ1 and promoter P kasO .

27. The method as described in claim 14, characterized in that, In step (2), a construction primer is used, the sequence of which is selected from the nucleotide sequences shown in SEQ ID NO: 7-14, or a combination thereof.

28. The method as described in claim 13 or 15, characterized in that, The sequence verification method is to perform PCR verification on the attL fragment and / or attR fragment at the integration site.

29. The method as described in claim 13 or 15, characterized in that, The sequence verification used verification primers, the sequences of which were selected from nucleotide sequences as shown in SEQ ID NO: 15-18, or combinations thereof.

30. The method as described in claim 13 or 15, characterized in that, The fermentation verification method involves three-stage fermentation with avilamycin, during which the yield and / or proportion of avilamycin A and C components in the recombinant strain and the avilamycin-producing strain are detected and compared.

31. The method as described in claim 5, characterized in that, The method includes the step of: using (a) aviZ1 Product preparation of gene-multiplying plasmids: recombinant strains with increased proportion and / or yield of avilamycin A component.

32. The method as described in claim 31, characterized in that, The product also includes: (b) reagents required for indirect fusion transfer.

33. The method as described in claim 32, characterized in that, The product also includes: (c) verification primers.

34. The method as described in claim 5, characterized in that, The method includes the step of: using a recombinant strain that produces an increased proportion and / or yield of avilamycin A component in a preparation combination, said preparation combination comprising: (Z1) Streptomyces containing an integrase module applied to plasmids; and (Z2) Construct primers.

35. The method as described in claim 34, characterized in that, The preparation combination also includes: (Z3) reagents required for indirect combination and transfer.

36. The method as described in claim 35, characterized in that, The preparation combination also includes: (Z4) verification primer.

37. A kind aviZ1 The uses of the gene and AviZ1 protein, characterized in that, For use in the group selected below: (i) Interconversion of avilamycin A and C components in vitro; (ii) Increase the proportion and / or yield of avilamycin active ingredient A component; and / or (iii) Prepare a recombinant strain that produces high levels of avilamycin A component.

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