Method for improving proportion of effective component A of avilamycin by enhancing component conversion

Abstract

The invention provides a method for improving the proportion of an effective component A of avilamycin by enhancing component conversion. Specifically, the invention provides a method for improving the proportion and / or yield of an effective component A of avilamycin, and the method comprises the following steps: providing an avilamycin production strain, enhancing the expression of AviO2, AviB1 and / or AviB2 proteins in the avilamycin production strain or increasing the copy number of aviO2, aviB1 and / or aviB2 genes in the avilamycin production strain, therefore, the proportion and / or the yield of the component A produced by fermentation of the avilamycin production strain are / is improved. According to the recombinant strain containing the multi-copy aviO2, aviB1 and / or aviB2 genes obtained by the method, the proportion of the effective component A of avilamycin can be increased from the source, so that the extraction process is simplified, the production cost is reduced, the genetic stability is good, and obvious social and economic benefits are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and more particularly relates to a method for improving the proportion of component A of avilamycin active ingredient by strengthening component conversion. BACKGROUND

[0002] Avilamycin is a multi-component oligosaccharide antibiotic produced by Streptomyces galbus, and its structure contains a orsellinic acid unit and a seven-sugar chain. Avilamycin is different from other currently clinically used antibiotics in terms of target site, and does not cause cross-resistance with known antibacterial drugs. It binds to the 50S subunit of bacterial ribosomes, which can block the entrance channel of aminoacyl-tRNA, block the synthesis of bacterial proteins, and ultimately achieve the purpose of inhibiting bacteria. Avilamycin has excellent application prospects in the domestic and foreign markets, and is currently mainly used as a feed additive to promote animal growth, or as a veterinary drug to treat bacterial infections.

[0003] The chemical structure of avilamycin is relatively complex, and its industrial production currently mainly relies on fermentation of Streptomyces galbus. The biosynthesis of avilamycin in the producing strain needs to go through a complex structural modification pathway, and the cascade regulation is not strict, resulting in the characteristic of component diversification of avilamycin. In addition to producing the effective component avilamycin A, Streptomyces galbus also produces the main impurity components avilamycin B, C and more than ten secondary impurity components.

[0004] Therefore, in order to further improve the industrial production of avilamycin active component A, it is urgent to develop a method for improving the proportion and / or yield of avilamycin A component from the source, and so far there has been no relevant report at home and abroad. SUMMARY

[0005] The purpose of the present application is to provide a method for improving the proportion of avilamycin active component A by strengthening component conversion.

[0006] Another purpose of the present application is to provide a recombinant strain for improving the proportion and / or yield of the main active component A of avilamycin from the source.

[0007] In the first aspect of the present application, a method for improving the proportion and / or yield of avilamycin active component A is provided, which comprises the steps of:

[0008] (a) providing an avilamycin-producing strain; and

[0009] (b) strengthening the expression of a target protein in the avilamycin-producing strain, or increasing the copy number of a target gene in the avilamycin-producing strain, so as to improve the proportion and / or yield of component A in the avilamycin-producing strain.

[0010] wherein the target protein is selected from the group consisting of AviO2, AviB1, AviB2, or a combination thereof;

[0011] The target gene is selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof.

[0012] In another preferred embodiment, the target protein is AviO2, or the target gene is aviO2.

[0013] In another preferred embodiment, the target protein is AviO2, AviB1 and AviB2, or the target gene is aviO2, aviB1 and aviB2.

[0014] In another preferred embodiment, the target protein comprises a wild-type protein and a mutant protein.

[0015] In another preferred embodiment, the AviO2 protein comprises a wild-type AviO2 protein and a mutant AviO2 protein.

[0016] In another preferred embodiment, the AviB1 protein comprises a wild-type AviB1 protein and a mutant AviB1 protein.

[0017] In another preferred embodiment, the AviB2 protein comprises a wild-type AviB2 protein and a mutant AviB2 protein.

[0018] In another preferred embodiment, the AviO2 protein has an amino acid sequence selected from the group consisting of:

[0019] (1) the amino acid sequence set forth in SEQ ID NO: 21; and / or

[0020] (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 21 and having dioxygenase (aKGD) activity.

[0021] In another preferred embodiment, the mutant AviO2 protein has an amino acid sequence selected from the group consisting of:

[0022] (i) one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid substitutions, deletions, alterations, insertions or additions to the sequence set forth in SEQ ID NO: 21 within the range of maintaining enzymatic activity; and / or

[0023] (ii) one or several (typically within 20, preferably within 10, more preferably within 5) amino acids are added at the C- and / or N-terminus of the sequence of SEQ ID NO: 21, resulting in an amino acid sequence.

[0024] In another preferred embodiment, the AviB1 protein has an amino acid sequence selected from the group consisting of:

[0025] (1) the amino acid sequence of SEQ ID NO: 23; and / or

[0026] (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 of SEQ ID NO: 23 and having pyruvate dehydrogenase activity.

[0027] In another preferred embodiment, the amino acid sequence of the mutant AviB1 protein is selected from the group consisting of:

[0028] (i) one or several (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids of the sequence of SEQ ID NO: 23 are replaced, deleted, altered, inserted or added within the range of maintaining the enzymatic activity, resulting in an amino acid sequence; and / or

[0029] (ii) one or several (typically within 20, preferably within 10, more preferably within 5) amino acids are added at the C- and / or N-terminus of the sequence of SEQ ID NO: 23, resulting in an amino acid sequence.

[0030] In another preferred embodiment, the AviB2 protein has an amino acid sequence selected from the group consisting of:

[0031] (1) the amino acid sequence of SEQ ID NO: 25; and / or

[0032] (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 of SEQ ID NO: 25 and having pyruvate dehydrogenase activity.

[0033] In another preferred embodiment, the amino acid sequence of the mutant AviB2 protein is selected from the group consisting of:

[0034] (i) one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid substitutions, deletions, alterations, insertions or additions to the sequence set forth in SEQ ID NO: 25, within the range of enzyme activity; and / or

[0035] (ii) one or more (usually 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acid additions to the C-terminus and / or N-terminus of the sequence set forth in SEQ ID NO: 25.

[0036] In another preferred embodiment, the target gene includes a wild-type gene and a mutant gene.

[0037] In another preferred embodiment, the aviO2 gene includes a wild-type aviO2 gene and a mutant aviO2 gene.

[0038] In another preferred embodiment, the aviBl gene includes a wild-type aviBl gene and a mutant aviBl gene.

[0039] In another preferred embodiment, the aviB2 gene includes a wild-type aviB2 gene and a mutant aviB2 gene.

[0040] In another preferred embodiment, the aviO2 gene has a nucleotide sequence selected from the group consisting of:

[0041] (1) the nucleotide sequence set forth in SEQ ID NO: 20;

[0042] (2) a nucleotide sequence having ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%, most preferably ≥ 99%) identity to the sequence set forth in SEQ ID NO: 20; and / or

[0043] (3) a nucleotide sequence obtained by truncating or adding 1-60 (preferably 1-30, more preferably 1-6) nucleotides to the 5' end and / or 3' end of the sequence set forth in SEQ ID NO: 20.

[0044] In another preferred embodiment, the aviBl gene has a nucleotide sequence selected from the group consisting of:

[0045] (1) the nucleotide sequence set forth in SEQ ID NO: 22;

[0046] (2) a nucleotide sequence having ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%, most preferably ≥ 99%) identity to the sequence set forth in SEQ ID NO: 22; and / or

[0047] (3) the nucleotide sequence obtained by truncating or adding 1-60 (preferably 1-30, more preferably 1-6) nucleotides to the 5'-end and / or the 3'-end of the sequence set forth in SEQ ID NO: 22.

[0048] In another preferred embodiment, the aviB2 gene has a nucleotide sequence selected from the group consisting of:

[0049] (1) the nucleotide sequence set forth in SEQ ID NO: 24;

[0050] (2) a nucleotide sequence having ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%, most preferably ≥ 99%) identity to the sequence set forth in SEQ ID NO: 24; and / or

[0051] (3) the nucleotide sequence obtained by truncating or adding 1-60 (preferably 1-30, more preferably 1-6) nucleotides to the 5'-end and / or the 3'-end of the sequence set forth in SEQ ID NO: 24.

[0052] In another preferred embodiment, the copy number of the target gene is increased to 2-fold, 3-fold, 4-fold, n-fold of the original.

[0053] In another preferred embodiment, the copy number of the target gene is increased to 2-fold of the original.

[0054] In another preferred embodiment, the proportion of the avermectin A component in the total avermectin components is increased in the range of 2%-30%, preferably 3%-20%, preferably 4%-15%, preferably 5%-10%.

[0055] In another preferred embodiment, the yield of the avermectin A component is increased in the range of 10%-70%, preferably 10%-30%, more preferably 30%-40%, most preferably 40%-70%.

[0056] In another preferred embodiment, the proportion of the avermectin A component in the total avermectin components is increased to more than 65%, preferably more than 66%, preferably more than 67%, preferably more than 68%, preferably more than 69%, preferably more than 70%, preferably more than 80%, preferably more than 90%.

[0057] In another preferred embodiment, the proportion of the avermectin B' component in the avermectin-producing strain is reduced to less than 10%, preferably less than 9%, preferably less than 8%, preferably less than 7%, preferably less than 6%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1%.

[0058] In another preferred embodiment, the chemical formula of the avermectin B' is C 59H 86 Cl2O 31 .

[0059] In another preferred embodiment, the avilamycin B" has a structure as shown in formula (I):

[0060]

[0061] wherein R1 is COCH(CH3)2 and R2 is H.

[0062] In another preferred embodiment, the avilamycin-producing strain comprises wild-type Streptomyces viridochromogenes and avilamycin industrial strain.

[0063] In another preferred embodiment, the avilamycin-producing strain is selected from the group consisting of LK171, Streptomyces viridochromogenes Tü57, or a combination thereof.

[0064] In another preferred embodiment, the avilamycin-producing strain is LK171.

[0065] In a second aspect of the present application, a method for constructing a recombinant strain with improved proportion and / or yield of avilamycin A component is provided, the method comprising the steps of:

[0066] (a) providing an avilamycin-producing strain; and

[0067] (b) increasing the copy number of a target gene in the avilamycin-producing strain, thereby obtaining the recombinant strain with improved proportion and / or yield of avilamycin A component;

[0068] wherein the target gene is selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof.

[0069] In another preferred embodiment, the copy number of the target gene is increased by 2-fold, 3-fold, 4-fold, n-fold.

[0070] In another preferred embodiment, the copy number of the target gene is increased by 2-fold.

[0071] In another preferred embodiment, the target gene comprises wild-type gene and mutant gene.

[0072] In another preferred embodiment, the aviO2 gene comprises wild-type aviO2 gene and mutant aviO2 gene.

[0073] In another preferred embodiment, the aviB1 gene comprises wild-type aviB1 gene and mutant aviB1 gene.

[0074] In another preferred embodiment, the aviB2 gene comprises a wild-type aviB2 gene and a mutant aviB2 gene.

[0075] In another preferred embodiment, the aviO2 gene has a nucleotide sequence selected from the group consisting of:

[0076] (1) a nucleotide sequence as set forth in SEQ ID NO: 20;

[0077] (2) a nucleotide sequence having ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%, most preferably ≥ 99%) identity to the sequence as set forth in SEQ ID NO: 20; and / or

[0078] (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 as set forth in SEQ ID NO: 20.

[0079] In another preferred embodiment, the aviB1 gene has a nucleotide sequence selected from the group consisting of:

[0080] (1) a nucleotide sequence as set forth in SEQ ID NO: 22;

[0081] (2) a nucleotide sequence having ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%, most preferably ≥ 99%) identity to the sequence as set forth in SEQ ID NO: 22; and / or

[0082] (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 as set forth in SEQ ID NO: 22.

[0083] In another preferred embodiment, the aviB2 gene has a nucleotide sequence selected from the group consisting of:

[0084] (1) a nucleotide sequence as set forth in SEQ ID NO: 24;

[0085] (2) a nucleotide sequence having ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%, most preferably ≥ 99%) identity to the sequence as set forth in SEQ ID NO: 24; and / or

[0086] (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 as set forth in SEQ ID NO: 24.

[0087] In another preferred embodiment, the recombinant strain is selected from the group consisting of: LK804EO2, LK804EO2B1B2, or a combination thereof.

[0088] In another preferred embodiment, the avilamycin-producing strain comprises a wild-type Streptomyces viridochromogenes and an avilamycin industrial strain.

[0089] In another preferred embodiment, the avilamycin-producing strain is selected from the group consisting of LK171, Streptomyces viridochromogenes Tü57, or a combination thereof.

[0090] In another preferred embodiment, the avilamycin-producing strain is LK171.

[0091] In another preferred embodiment, the avilamycin-producing strain comprises an attB site sequence recognized by the integrase in its genome, wherein the attB site sequence comprises an endogenous attB site sequence and / or an exogenous attB site sequence.

[0092] In another preferred embodiment, the integrase is selected from the group consisting of a VWB integrase, a ΦBT1 integrase, a ΦC31 integrase, a ΦTG1 integrase, or a combination thereof.

[0093] In another preferred embodiment, the avilamycin-producing strain comprises an attB site sequence as set forth in SEQ ID NO: 13 in its genome.

[0094] In another preferred embodiment, the proportion of the avilamycin A component in the total components of avilamycin in the recombinant strain is increased to a range of 2% to 30%, preferably 3% to 20%, preferably 4% to 15%, preferably 5% to 10%.

[0095] In another preferred embodiment, the yield of the avilamycin A component in the recombinant strain is increased to a range of 10% to 70%, preferably 10% to 30%, more preferably 30% to 40%, most preferably 40% to 70%.

[0096] In another preferred embodiment, the proportion of the avilamycin A component in the total components of avilamycin in the recombinant strain is increased to more than 65%, preferably more than 66%, preferably more than 67%, preferably more than 68%, preferably more than 69%, preferably more than 70%, preferably more than 80%, preferably more than 90%.

[0097] In another preferred embodiment, the proportion of the avilamycin B' component in the recombinant strain is decreased to less than 10%, preferably less than 9%, preferably less than 8%, preferably less than 7%, preferably less than 6%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1%.

[0098] In another preferred embodiment, the chemical formula of the avilamycin B' is C 59H 86 Cl2O 31 .

[0099] In another preferred embodiment, the avermectin B' has a structure as shown in formula (I):

[0100]

[0101] wherein R1 is COCH(CH3)2 and R2 is H.

[0102] In another preferred embodiment, the increasing the copy number of the target gene can be performed by a method selected from the group consisting of: protoplast transformation, electroporation, conjugative transfer, phage transduction, or a combination thereof.

[0103] In another preferred embodiment, the step (b) comprises:

[0104] (bi) constructing a recombinant expression vector containing single or multiple copies of the target gene; and

[0105] (bii) introducing the recombinant expression vector obtained in (a) into an avermectin-producing strain, thereby obtaining the recombinant strain with increased copy number of the target gene.

[0106] In another preferred embodiment, the method further comprises the step of:

[0107] (c) performing sequence verification and / or fermentation verification on the recombinant strain.

[0108] In another preferred embodiment, the conjugative transfer comprises the steps of:

[0109] (1) providing a Streptomyces utility plasmid comprising a complete integrase module, wherein the integrase module comprises: (al) a gene expressing integrase; and (a2) an attP site sequence recognized by the integrase;

[0110] (2) modifying the Streptomyces utility plasmid in (a) by: (bl) inserting the target gene; and (b2) optionally inserting a promoter; thereby constructing a plasmid with increased copy number of the target gene; and

[0111] (3) introducing the plasmid with increased copy number of the target gene obtained in (b) into an avermectin-producing strain by intergeneric conjugative transfer, thereby obtaining the recombinant strain with increased copy number of the target gene.

[0112] In another preferred embodiment, the conjugative transfer optionally comprises the step of:

[0113] (4) performing sequence verification and / or fermentation verification on the recombinant strain.

[0114] In another preferred embodiment, the integrase module is selected from the group consisting of: a VWB-attP integrase module, a ΦBT1-attP integrase module, a ΦC31-attP integrase module, a ΦTG1-attP integrase module, or a combination thereof.

[0115] In another preferred embodiment, the Streptomyces utility plasmid is selected from the group consisting of: pSOK804, pD-BT1, pSET152, or a combination thereof.

[0116] In another preferred embodiment, the Streptomyces utility plasmid is pSOK804.

[0117] In another preferred embodiment, the pSOK804 comprises a complete VWB-attP integrase module.

[0118] In another preferred embodiment, the promoter is selected from the group consisting of: P ermE* , P kasO* , P j23119 , or a combination thereof.

[0119] In another preferred embodiment, the promoter is P ermE* .

[0120] In another preferred embodiment, the plasmid for increasing the copy number of the target gene is selected from the group consisting of: pSOK804-P ermE* -aviO2, pSOK804-P ermE* -aviO2B1B2, or a combination thereof.

[0121] In another preferred embodiment, a construction primer is used in step (bii), and the sequence of the construction primer is selected from the group consisting of the nucleotide sequences as set forth in SEQ ID NOs: 14-17, or a combination thereof.

[0122] In another preferred embodiment, the method for sequence verification is PCR verification of the integration site attL fragment and / or the attR fragment.

[0123] In another preferred embodiment, a verification primer is used for sequence verification, and the sequence of the verification primer is selected from SEQ ID NO: 18 and / or SEQ ID NO: 19.

[0124] In another preferred embodiment, the method for fermentation verification is detecting and comparing the yields and / or proportions of avermectin A, B components in the recombinant strain and the avermectin-producing strain, respectively, through a three-stage fermentation of avermectin.

[0125] In a third aspect of the present application, a recombinant strain prepared by the method of the second aspect of the present application is provided, wherein the genome of the recombinant strain comprises an increased copy number of a target gene selected from the group consisting of aviO2, aviBl, aviB2, or a combination thereof.

[0126] In another preferred embodiment, the genome of the recombinant strain comprises 2-fold of the target gene, 3-fold of the target gene, 4-fold of the target gene, n-fold of the target gene.

[0127] In another preferred embodiment, the genome of the recombinant strain comprises 2-fold of the target gene.

[0128] In another preferred embodiment, the proportion of avilamycin A component in the total avilamycin components in the recombinant strain is increased by 2% to 30%, preferably 3% to 20%, preferably 4% to 15%, preferably 5% to 10% compared to the avilamycin-producing strain.

[0129] In another preferred embodiment, the yield of avilamycin A component in the recombinant strain is increased by 10% to 70%, preferably 10% to 30%, more preferably 30% to 40%, most preferably 40% to 70% compared to the avilamycin-producing strain.

[0130] In another preferred embodiment, the proportion of avilamycin A component in the total avilamycin components in the recombinant strain is increased to more than 65%, preferably more than 66%, preferably more than 67%, preferably more than 68%, preferably more than 69%, preferably more than 70%, preferably more than 80%, preferably more than 90%.

[0131] In another preferred embodiment, the proportion of avilamycin B component in the recombinant strain is decreased to less than 10%, preferably less than 9%, preferably less than 8%, preferably less than 7%, preferably less than 6%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1% compared to the avilamycin-producing strain.

[0132] In another preferred embodiment, the avilamycin-producing strain comprises a wild-type S. viridochromogenes and an avilamycin industrial strain.

[0133] In another preferred embodiment, the avilamycin-producing strain is selected from the group consisting of LK171, Streptomyces viridochromogenes Tü57, or a combination thereof.

[0134] In another preferred embodiment, the avilamycin-producing strain is LK171.

[0135] In another preferred embodiment, the recombinant strain is selected from the group consisting of: LK804EO2, LK804EO2B1B2, or a combination thereof.

[0136] In a fourth aspect of the present application, a polynucleotide having a sequence of the recombinant strain according to the third aspect of the present application or a derivative sequence thereof is provided.

[0137] In another preferred embodiment, the derivative sequence has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more homology with the sequence of the recombinant strain, preferably 98% or more homology, more preferably 99% or more homology.

[0138] In another preferred embodiment, the polynucleotide is RNA, DNA, or cDNA.

[0139] In a fifth aspect of the present application, an expression vector containing the polynucleotide according to the fourth aspect of the present application is provided.

[0140] In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof; preferably, the viral vector comprises lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0141] In another preferred embodiment, the expression vector comprises plasmid, cosmid.

[0142] In another preferred embodiment, the expression vector comprises a resistance selection marker and an element oriT required for intergeneric conjugative transfer.

[0143] In another preferred embodiment, the resistance selection marker is apramycin resistance gene.

[0144] In another preferred embodiment, the expression vector further comprises an element selected from the group consisting of: promoter, transcription enhancer element WPRE, long terminal repeat sequence LTR, and the like.

[0145] In another preferred embodiment, the promoter is selected from the group consisting of: P ermE* , P kasO* , P j23119 , or a combination thereof.

[0146] In another preferred embodiment, the promoter is P ermE * .

[0147] In a sixth aspect of the present application, a host cell containing the expression vector according to the fifth aspect of the present application or having integrated into its genome the polynucleotide according to the fourth aspect of the present application is provided.

[0148] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0149] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell.

[0150] In another preferred embodiment, the host cell is a Streptomyces cell.

[0151] In a seventh aspect of the present application, there is provided a use of an active ingredient selected from the group consisting of: a recombinant strain according to the third aspect of the present application, a polynucleotide according to the fourth aspect of the present application, an expression vector according to the fifth aspect of the present application, or a host cell according to the sixth aspect of the present application, or a combination thereof; for a use selected from the group consisting of:

[0152] (i) converting Avilamycin A, B' components into each other in vitro;

[0153] (ii) increasing the proportion and / or yield of Avilamycin A component;

[0154] (iii) preparing a recombinant strain producing high amounts of Avilamycin A component;

[0155] (iv) decreasing the proportion and / or content of Avilamycin B' component;

[0156] (v) preparing a recombinant strain with decreased Avilamycin B' component;

[0157] (vi) inhibiting the synthesis of bacterial proteins;

[0158] (vii) promoting the growth of animals; and / or

[0159] (viii) preparing a feed additive.

[0160] In another preferred embodiment, the transformed co-factor is selected from the group consisting of L-ascorbic acid, FeSO4, a-KG, TPP, pyruvic acid, or a combination thereof.

[0161] In an eighth aspect of the present application, there is provided a product comprising (a) a plasmid with increased copy number of a target gene selected from the group consisting of aviO2, aviBl, aviB2, or a combination thereof.

[0162] In another preferred embodiment, the product further comprises: (b) reagents required for intergeneric conjugative transfer.

[0163] In another preferred embodiment, the product further comprises: (c) a verification primer.

[0164] In another preferred embodiment, the sequence of the verification primer is selected from the group consisting of SEQ ID NO: 18 and / or SEQ ID NO: 19.

[0165] In another preferred embodiment, the plasmid with increased copy number of the target gene comprises a constitutive promoter. ermE * -aviO2, pSOK804-P ermE* -aviO2B1B2, or a combination thereof.

[0166] In another preferred embodiment, the plasmid with increased copy number of the target gene comprises a constitutive promoter.

[0167] In another preferred embodiment, the constitutive promoter is selected from the group consisting of P ermE* , P kasO* , P j23119 , or a combination thereof.

[0168] In another preferred embodiment, the product further comprises an instruction manual, wherein the instruction manual records a method for preparing a recombinant strain with improved proportion and / or yield of avilamycin A components using the plasmid with increased copy number of the target gene.

[0169] In a ninth aspect of the present application, a preparation combination is provided, which comprises:

[0170] (Z1) a Streptomyces application plasmid containing an integrase module; and

[0171] (Z2) a construction primer.

[0172] In another preferred embodiment, the Streptomyces application plasmid is selected from the group consisting of pSOK804, pD-BT1, pSET152, or a combination thereof.

[0173] In another preferred embodiment, the integrase module is selected from the group consisting of VWB-attP integrase module, ΦBT1-attP integrase module, ΦC31-attP integrase module, ΦTG1-attP integrase module, or a combination thereof.

[0174] In another preferred embodiment, the sequence of the construction primer is selected from the group consisting of nucleotide sequences as shown in SEQ ID NO: 14-17, or a combination thereof.

[0175] In another preferred embodiment, the preparation combination further comprises:

[0176] (Z3) reagents required for intergeneric conjugation transfer.

[0177] In another preferred embodiment, the preparation combination further comprises: (Z4) a verification primer.

[0178] In another preferred embodiment, the sequence of the verification primer is selected from the group consisting of SEQ ID NO: 18 and / or SEQ ID NO: 19.

[0179] In another preferred embodiment, the preparation combination is used for preparing a plasmid with increased copy number of a target gene selected from the group consisting of aviO2, aviBl, aviB2, or a combination thereof.

[0180] In another preferred embodiment, the preparation combination further comprises: (Z5) a plasmid with increased copy number of a target gene selected from the group consisting of aviO2, aviBl, aviB2, or a combination thereof, as a positive control.

[0181] In another preferred embodiment, the preparation combination further comprises an instruction manual, which describes a method for producing a recombinant strain with improved proportion and / or yield of Avilamycin A components using the preparation combination.

[0182] In the tenth aspect of the present application, there is provided a use of a target gene, or a target protein selected from the group consisting of AviO2, AviBl, AviB2, or a combination thereof; the target gene is selected from the group consisting of aviO2, aviBl, aviB2, or a combination thereof; for a use selected from the group consisting of:

[0183] (i) converting Avilamycin A, B' components into each other in vitro;

[0184] (ii) improving the proportion and / or yield of Avilamycin A component;

[0185] (iii) preparing a recombinant strain with high yield of Avilamycin A component;

[0186] (iv) reducing the proportion and / or content of Avilamycin B' component; and / or

[0187] (v) preparing a recombinant strain with reduced Avilamycin B' component.

[0188] In the eleventh aspect of the present application, there is provided an Avilamycin B' component, which has a chemical formula of C 59 H 86 Cl2O 31 .

[0189] In another preferred embodiment, the Avilamycin B' component has a structure as shown in formula (I):

[0190]

[0191] wherein R1 is COCH(CH3)2, and R2 is H.

[0192] In a twelfth aspect of the present application, there is provided use of the Avilamycin B' fraction as described in the eleventh aspect of the present application, as an impurity control for quality control of the Avilamycin production process.

[0193] In another preferred embodiment, the use comprises: as an impurity control, for quantitative analysis of impurities (Avilamycin B' fraction) in Avilamycin.

[0194] In another preferred embodiment, the Avilamycin is produced from a wild-type S. lividans or an Avilamycin industrial strain.

[0195] In another preferred embodiment, the Avilamycin is produced from a recombinant strain selected from the group consisting of: LK804EO2, LK804EO2B1B2, or a combination thereof.

[0196] In a thirteenth aspect of the present application, there is provided a method of preparing the Avilamycin B' fraction as described in the eleventh aspect of the present application, the method comprising the steps of:

[0197] (a) isolating the Avilamycin B' fraction from Avilamycin using HPLC detection conditions as follows:

[0198] The HPLC detection conditions are:

[0199] Mobile phase: Phase A: 95% water + 5% acetonitrile (10 mM ammonium acetate); Phase B: 5% water + 95% acetonitrile (10 mM ammonium acetate)

[0200] Injection volume: 20-30 μL Flow rate: 1 mL / min

[0201] DAD recording wavelength: 214 nm, no reference

[0202] Liquid gradient program (constant gradient):

[0203] 0-40 min: 38% Phase B.

[0204] In another preferred embodiment, the method further comprises the step of:

[0205] (b) further purifying the Avilamycin B' fraction.

[0206] In another preferred embodiment, the method further comprises the step of:

[0207] (c) structurally and / or functionally identifying the Avilamycin B' fraction.

[0208] It should be understood that, within the scope of the present application, all the technical features described above and the technical features described in detail hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0209] Figure 1 The plasmid map of pOJ260-ΔaviO2 is shown.

[0210] Figure 2 The plasmid map of pOJ260-ΔaviB1 is shown.

[0211] Figure 3 The plasmid map of pOJ260-ΔaviB2 is shown.

[0212] Figure 4 The fermentation results of mutant LK171ΔaviO2 are compared with those of the wild type (HPLC chart) are shown.

[0213] Figure 5 The fermentation results of mutant LK171ΔaviB1, LK171ΔaviB2 are compared with those of the wild type (HPLC chart) are shown.

[0214] Figure 6 The detection analysis of refined avilamycin B and B' (HPLC chart) is shown.

[0215] Figure 7 The structural formula of avilamycin B' component and its difference from avilamycin A component, B component and C component are shown.

[0216] Figure 8 The SDS-PAGE characterization map of AviO2, AviB1 and AviB2 proteins is shown.

[0217] Figure 9 AviO2, AviB1 and AviB2 can catalyze the generation of avilamycin A from avilamycin B' is shown.

[0218] Figure 10 The plasmid map of pSOK804-P ermE* -aviO2 is shown.

[0219] Figure 11 The plasmid map of pSOK804-P ermE* -aviO2B1B2 is shown.

[0220] Figure 12 The fermentation results of component-optimized recombinant strains LK804EO2, LK804EO2B1B2 and the starting strain LK171 (HPLC chart) are shown. DETAILED DESCRIPTION

[0221] The present inventors have found that knocking out aviO2, aviB1 and aviB2 in the avermectin biosynthesis gene cluster can greatly accumulate a new avermectin component B' and hardly produce the active ingredient avermectin A, and that avermectin B' is also accumulated as an impurity component in the fermentation product of the wild type of Streptomyces galbus, and that AviO2, AviB1 and AviB2 can catalyze the conversion of avermectin B' to avermectin A in vitro with the participation of oxygen, thiamine pyrophosphate (TPP) and the like, and thus, doubling the aviO2, aviB1 and / or aviB2 genes can increase the proportion and / or yield of avermectin A. Specifically, the present application uses the site-specific recombination mechanism to introduce a plasmid containing the target genes aviO2, aviB1 and / or aviB2 under the control of a strong constitutive promoter into the avermectin industrial strain by intergeneric conjugation to obtain the target recombinant strain. The recombinant strain of the present application can increase the proportion of the main component A of avermectin from the source, increase the yield of component A, reduce the production cost, and has obvious social and economic benefits. Moreover, the genetic stability of the recombinant strain of the present application is very good, and thus it is easy to achieve large-scale stable amplification, and the recombinant strain of the present application and the construction method thereof have high application value. The present application is completed on this basis.

[0222] TERMS

[0223] To enable a better understanding of the present disclosure, certain terms are defined first. As used in this application, unless specifically identified otherwise herein, each of the following terms shall have the meaning given below.

[0224] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0225] As used herein, the term "site-specific recombination" is a type of homologous recombination that depends on the association of small-range homologous sequences, which requires the participation of an integrase derived from a mycobacteriophage (such as ΦC31, VWB, ΦBT1) and specific sequences (such as attB / attP), and finally integrates the linearized plasmid into the corresponding site in the genome of the target strain.

[0226] As used herein, the term "introducing" or "transformation" refers to the transfer of a polynucleotide into a host cell (Streptomyces in the present application). Alternatively, the exogenous polynucleotide can be integrated into the host genome.

[0227] As used herein, the term "gene of interest" refers to a structural gene introduced into the genome of the original organism (Streptomyces in the present invention).

[0228] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window, which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein, and determining the number of positions for which the same residue occurs in both sequences. Typically, this is expressed as a percentage. Measurement of sequence identity of nucleotide sequences, amino acid sequences is well known to those skilled in the art.

[0229] aviO2, aviB1 / B2 genes and their expression in Streptomyces viridochromogenes

[0230] The aviO2 and aviB1 / B2 genes in the avermectin biosynthetic gene cluster are responsible for encoding a non-heme iron / alpha-ketoglutarate-dependent dioxygenase (aKGD) and a pyruvate dehydrogenase, respectively (Appl. Environ. Microbiol. 2005, 71(1): 400-406).

[0231] The nucleotide sequence of the aviO2 gene is 777 bp in length:

[0232] atgcctgacgaaaggagccgccgtatgggggccgtggaaactgtgatcgacgaggagcaccgcgcagccttccaggccgacggattcgcatcgctgccgcggctcgtggacgacacggagctggagtggctgcgcggtgtctacgatcgcctgttctccgagcacgccgacttcactacgggcgactacttcgacatcgccggccgccaggattccgacaggccggcccggctaccgcagatcgtgcgcccggagaaattcgtccccgagctggtcgagagtgcgcatttccagcggtgccgggcgatcgccgcgcagctgctggacatcccggaggacgagctcgacttctatgggcacgccattctgaagccgccgaactacggcgcggcaacgccctggcaccaggacgagggctacatggacccgcgctgccgccggcgcgggctcagcatatggaccccgctggacgaggccaccgtggacagcggctgcctgcactatgtgcccggcgcgcacctcggtcccgtgctgccgcaccggcacatcgacgacgacgaccgtatccgggggctggtgaccgacgcggtggacccggccgccggggtgcccgtccccctcgcctcgggtgaggccgtggtgcacgccctgcgcgctccgcattacgcgggccccaatctcaccgaccagacgcgccgggcctatgtgctggtcttcatgggggcggccgaagaggtcgccgaccccgagccgcgcccctggctggacaaggactga (SEQ ID NO:20)

[0233] The αKGD encoded by the aviO2 gene (i.e., the AviO2 protein) contains 258 amino acids:

[0234] MPDERSRRMGAVETVIDEEHRAAFQADGFASLPRLVDDTELEWLRGVYDRLFSEHADFTTGDYFDIAGRQDSDRPARLPQIVRPEKFVPELVESAHFQRCRAIAAQLLDIPEDELDFYGHAILKPPNYGAATPWHQDEGYMDPRCRRRGLSIWTPLDEATVDSGCLHYVPGAHLGPVLPHRHIDDDDRIRGLVTDAVDPAAGVPVPLASGEAVVHALRAPHYAGPNLTDQTRRAYVLVFMGAAEEVADPEPRPWLDKD (SEQ ID NO: 21)

[0235] The nucleotide sequence of the aviBl gene is 1005 bp long:

[0236]

[0237] The pyruvate dehydrogenase encoded by the aviBl gene contains 334 amino acids:

[0238] MTTEEGKPAMSAEPRAVRLYRTMRLIREFEERCLAMSLSGEIVGGIHPYIGQEAVAAGVCAALTDDDVVTSTHRGHGHVLAKGADPKRMLAELCGTTAGLNRGRGGSMHAADVSLGVYGANGIVGAGAPIAAGAAWSFQRRGDGRVAVAFFGDGALSQGVVLEAFNLAALWRLPVVFVCENNGYATSLPLDRALAGDPVERAAGFGLTARAVDGMDADAVADAAAEAVERCRRGGGPTFLDCRTYRFNGHHSFEEQVGLNYRDDAEVGEWRARDPLRTQSTRVDGATAGAIDAETSTLIDEAVEFARAGRAPDPAEALHHLYADGTTPRPGVMI (SEQ ID NO: 23)

[0239] The nucleotide sequence of the aviB2 gene is 963 bp long:

[0240] atggctgcacttgcgtacatcaccgcgctgaaccaggcgctgcacgagcagatggcgcgcgacgaccgcgtctgcgtcttcggcgaggacgtgcggatcggcctcacccaggtcgccaagggcctgcacgagcgcttcggcgacggacgcgtcgtcgacacccccctgtcggagcaggccttcaccagcctggccaccggcgcggcgatggcggggcagcgaccggtcgtggagtaccagatcccctcgctgctctatctggtgttcgagcagatcgccaaccaggcgcacaagttctcgctgatgaccggtgggcaggtggaggtcccggtcacctatctggtgccgggctccggttcgcgctcgggcatggccggccagcactccgaccatccctacagcctgttcgcacacgtcggcatcaagacggtcctgcccgccaccgcctcggacgcctacggcctgctgctctcggcggtccgtgacccggacccggtggccgtcttcgcgccgagcgcgctgatgggcacggtcgaggaggtctccggcgagctcggcccggtgccgctcgggtcggcccggatccaccgcacgggcgaggacgtcaccgtcgtcgccaccggccagtgcgtgcatgtcgccctggccgtcgcggaggccatggcggacgaggcgtcgatcgaggtggtggatccgcgcacgatctacccggtggactgggagacgatccgggcgtcggcggagaagaccggacggctggtcgtcatcgatgacgccaaccggatgtgcgggttcggcggcgaggtgctggccaccgcggcggagcagttcgatctgacggcccggccccggcgggtgacccggccggacggcgcggtcatcccctacgcgctcgtgctcgaccaggcgttgctgccggacgcggcccagctgaccgacgcgatccgtgccgtcctgaagtga (SEQ ID NO: 24)

[0241] The pyruvate dehydrogenase encoded by the aviB2 gene contains 320 amino acids:

[0242] MAALAYITALNQALHDEMARDDRVCVFGEDVRIGLTQVAKGLHERFGDGRVVDTPLSEQAFTSLATGAAMAGQRPVVEYQIPSLLYLVFEQIANQAHKFSLMTGGQVEVPVTYLVPGSGSRSGMAGQHSDHPYSLFAHVGIKTVLPATASDAYGLLLSAVRDPDPVAVFAPSALMGTVEEVSGELGPVPLGSARIHRTGEDVTVVATGQCVHVALAVAEAMADEASIEVVDPRTIYPVDWETIRASAEKTGRLVVIDDANRMCGFGGEVLATAAEQFDLTARPRRVTRPDGAVIPYALVLDQALLPDAAQLTDAIRAVLK (SEQ ID NO: 25)

[0243] The present application identifies a neglected major impurity component Avilamycin B' in the fermentation product of Streptomyces lividans, demonstrates that the aviO2, aviB1 and aviB2 genes are related to the conversion of Avilamycin A and B' components, and the ratio of Avilamycin A and B' components directly depends on the activities of αKGD AviO2 and pyruvate dehydrogenase AviB1 / B2, and the activity of αKGD AviO2 is the key to affect the ratio of Avilamycin A and B' components.

[0244] Genetic engineering of Streptomyces lividans can solve the problems of increasing the yield of Avilamycin A component and optimizing the components. For the introduction of the target DNA fragment, site-specific recombination can be used, which has high genetic stability and is not prone to reversion. There are multiple attB sequences recognized by actinophage ΦC31, VWB and ΦBT in the genome of Streptomyces lividans, and by intergeneric conjugation transfer, a plasmid containing the corresponding integrase and attP sequence and the target DNA fragment obtained in vitro can be introduced to realize the integration of the target gene fragment at a specific site of the chromosome.

[0245] The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can guide the correct binding of RNA polymerase to the target DNA template, activate the RNA polymerase to start gene transcription. Using different promoters can directly affect the transcription activity of the gene, thereby affecting the expression intensity of the protein. Preferably, the present application uses a constitutive strong promoter P ermE* .

[0246] The application utilizes the method for optimizing related genes by using multiplication components, can improve the copy number of target genes aviO2, aviB1 and / or aviB2, and uses the constitutive strong promoter PermE* to enhance the expression of target genes aviO2, aviB1 and / or aviB2, so that the recombinant strain for improving the proportion and / or yield of the main active ingredient A component of avilamycin is obtained from the source, and so far, the method has not been reported at home and abroad.

[0247] Integrase system

[0248] The integrase system is that the streptomycete phage uses its integrase (such as ΦC31, VWB, ФBT1, ФTG1 and the like) to mediate the specific recombination integration of attP site (phage attachment site) and attB site (bacterial attachment site on the host genome). The attB is composed of a sequence called BOB', and the attP is composed of POP'. O is a core sequence, which is common to attB and attP, and the sequences on both sides are B, B' and P, P', which are called arms. The phage DNA is circular, and is integrated into the host chromosome to become a linear sequence when recombining. After recombination, two new hybrid att sites are generated on both sides, the left side is called attL, which is composed of BOP', and the right side is attR, which is composed of POB'.

[0249] Three-stage fermentation

[0250] Generally, for the actinomycetes with relatively slow growth rate, two times of culture are needed in the seed culture process, first inoculated in the first-stage seed culture medium, then cultured by replacing the culture medium (second-stage seed culture medium) to obtain a large number of robust mycelia, and finally transferred to the fermentation medium for fermentation, which is the three-stage fermentation method.

[0251] Main advantages of the application

[0252] 1. By multiplying the target genes aviO2, aviB1 and / or aviB2, the recombinant strain for high-yield avilamycin A component can be obtained from the source, and the randomness of mutagenesis breeding is avoided.

[0253] 2. The proportion of avilamycin A component is improved, which can greatly simplify the extraction process of the target product, optimize the control of quality standards, and reduce the post-processing cost.

[0254] 3. The proportion of avilamycin A component is improved, and the yield of the main component A component is also improved, which indirectly reduces the production cost and produces obvious social and economic benefits.

[0255] 4. Since the gene multiplication method used is site-specific integration, the integration is irreversible, and after the introduction of the target gene, the target gene is distributed discretely, and homologous recombination is not easy to occur, so the genetic stability of the avilamycin engineering bacteria obtained by the method of the present application is very good.

[0256] The present application is further described below in connection with specific examples. It is understood that these examples are merely for illustrative purposes and do not limit the scope of the present application. In the following examples, the experimental methods not specified are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989); Genetic Manipulation of Streptomyces: A Laboratory Manual. 1985. DA Hopwood, The John Innes Foundation, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0257] General Materials and Methods

[0258] Unless otherwise specified, all plasmids and strains in the examples are conventional or commercially available.

[0259] Specifically, the strains involved in the examples include:

[0260] LK171, an industrial strain of avilamycin, provided by Luokang Biochemical Co., Ltd. LK171ΔaviO2, LK171ΔaviB1, LK171ΔaviB2, mutant strains obtained by knocking out the aviO2, aviB1, and aviB2 genes of the avilamycin biosynthetic gene cluster through homologous recombination double crossover. LK804EO2, LK804EO2B1B2, recombinant strains obtained by site-specific recombination, using LK171 as the starting strain, multiplying the target genes aviO2 or aviO2, aviB1, and aviB2, respectively.

[0261] The plasmids involved in the examples include:

[0262] The Streptomyces application plasmid pOJ260 is a conventional Streptomyces operation plasmid, which is preserved in the laboratory, and the sequence information is referred to GenBank: GU270843.1.

[0263] The Streptomyces application plasmid pSOK804 is a conventional Streptomyces operation plasmid, which is preserved in the laboratory, and the sequence information is referred to GenBank: LT545994.1.

[0264] Streptomyces pOJ260-ΔaviO2 is constructed by inserting the left and right homologous arms of the gene aviO2 into the Streptomyces conventional plasmid pOJ260 as a vector.

[0265] Streptomyces pOJ260-ΔaviB1 is constructed by inserting the left and right homologous arms of the gene aviB1 into the Streptomyces conventional plasmid pOJ260 as a vector.

[0266] Streptomyces pOJ260-ΔaviB2 is constructed by inserting the left and right homologous arms of the gene aviB2 into the Streptomyces conventional plasmid pOJ260 as a vector.

[0267] Streptomyces pSOK804-P ermE* -aviO2 is constructed by inserting the promoter P ermE* -aviO2 into the Streptomyces conventional plasmid pSOK804 as a vector.

[0268] Streptomyces pSOK804-P ermE* -aviO2B1B2 is constructed by inserting the promoter P ermE* -aviO2, aviB1, and aviB2 into the Streptomyces conventional plasmid pSOK804 as a vector.

[0269] The culture media involved in the examples include:

[0270] 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

[0271] 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

[0272] Seed medium (%): for primary fermentation of LK171 and its mutant strains, soluble starch 1.0, peptone 0.2, malt extract 1.0, yeast extract 0.6, glucose 0.4, CaCl2·2H2O 0.015, CaCO3 0.1, K2HPO3 0.05, tap water, pH 7.2 adjusted before sterilization

[0273] Fermentation medium (%): for fermentation of LK171 and its mutants, corn starch 8.0, soybean meal 2.0, NaNO30.2, (NH4)2SO40.02, glucose 1.6, NaCl 0.1, MgSO40.04, trace elements (MnCl2, ZnSO4, FeSO420 ppm, CoCl2, CuSO410 ppm), CaCl20.2, CaCO30.3, soybean oil 0.8, tap water, pH 7.2 adjusted before sterilization

[0274] Spore pre-germination medium (%): for germination of LK171 spores, yeast extract 0.5, casein amino acid 0.5, tap water, pH 7.2 adjusted before sterilization. 10 mM CaCl2was added after sterilization before use

[0275] Some experimental methods involved in the examples are as follows:

[0276] Method of conjugation transfer: Genetic Manipulation of Streptomyces: A Laboratory Manual. 1985. DA Hopwood, The John Innes Foundation

[0277] Method of fermentation of avermectins: strains grown on ISP-4 plates at 30°C for 5-7 days (cut together with agar blocks, about 1 cm in size 2 ) were inoculated into primary seed flasks and cultured at 28°C, 230 rpm for 48-72 hours to shake density. The primary seed (10% proportion) was inoculated into fermentation medium and cultured at 28°C, 230 rpm for 7-9 days.

[0278] Method of detection of avermectins: after fermentation, a certain amount of bacterial liquid was taken, an equal volume of methanol was added, ultrasonic treatment was performed for 10 minutes, and the liquid was left to stand for 1 hour. Centrifugation was performed at 12000 rpm for 10 minutes, and the supernatant was taken for HPLC and LC-MS analysis.

[0279] HPLC detection conditions 1 (general detection) of avermectins:

[0280] Mobile phase: A phase: 95% water + 5% acetonitrile (10 mM ammonium acetate); B phase: 5% water + 95% acetonitrile (10 mM ammonium acetate)

[0281] Injection volume: 20-30 μL Flow rate: 1 mL / min

[0282] DAD recording wavelength: 214 nm, no reference

[0283] Liquid gradient program (variable gradient):

[0284] 0 min: 20% Phase B

[0285] 0-10 min: linear increase to 45% Phase B

[0286] 10-27 min: linear increase to 62% Phase B

[0287] 27-28 min: linear decrease to 20% Phase B

[0288] 28-30 min: 20% Phase B

[0289] Avilamycin HPLC detection condition 2 (Avilamycin B and B' separation):

[0290] Mobile phase: A phase: 95% water + 5% acetonitrile (10 mM ammonium acetate); B phase: 5% water + 95% acetonitrile (10 mM ammonium acetate)

[0291] Injection volume: 20-30 μL Flow rate: 1 mL / min

[0292] DAD recording wavelength: 214 nm, no reference

[0293] Liquid gradient program (constant gradient):

[0294] 0-40 min: 38% Phase B

[0295] Example 1: Construction of knock-out plasmids pOJ260-ΔaviO2, pOJ260-ΔaviBl, pOJ260-ΔaviB2

[0296] The Streptomyces general use plasmid pOJ260 (GenBank: GU270843.1) contains an apramycin resistance gene and an element oriT required for conjugative transfer, and is a suicide-type knockout vector.

[0297] The vector was linearized by double digestion with Hindlll and EcoRI, and then the left and right homologous arm fragments required for knocking out the aviO2 gene were inserted at the enzyme digestion site to obtain the target plasmid pOJ260-ΔaviO2, the plasmid map of which is shown in Figure 1 .

[0298] The required primers are as follows:

[0299] pOJ260-ΔaviO2-Lf: acgacggccagtgccaagcttGCCCGCGGTGACGATCATCC (SEQ ID NO: 1)

[0300] pOJ260-ΔaviO2-Lr: TCGCCTGTTCCATTACGCGGGCCCCAATCT (SEQ ID NO: 2)

[0301] pOJ260-ΔaviB2-Rf: CCGCGTAATGGAACAGGCGATCGTAGACAC (SEQ ID NO: 3)

[0302] pOJ260-ΔaviB2-Rr: ctatgacatgattacgaattc GTCCGCGGATGCGTCTGACG (SEQ ID NO: 4)

[0303] The vector was linearized by double digestion with Hindlll and EcoRI, and then the left and right homologous arm fragments required for knocking out the aviBl gene were inserted at the cleavage sites to obtain the target plasmid pOJ260-ΔaviBl, the plasmid map of which is shown in Figure 2 .

[0304] The required primers are as follows:

[0305] pOJ260-ΔaviBl-Lf: cgacggccagtgccaagctt CCATCTGCGGGCGCTGAGCG (SEQ ID NO: 5)

[0306] pOJ260-ΔaviBl-Lr: CGTCGATGGCCGCCTCCTGGCCGATGTAGG (SEQ ID NO: 6)

[0307] pOJ260-ΔaviBl-Rf: CCAGGAGGCGGCCATCGACGCCGAGACCAG (SEQ ID NO: 7)

[0308] pOJ260-ΔaviBl-Rr: tatgacatgattacgaattc GCCCATGAGGTCCGCCACGAG (SEQ ID NO: 8)

[0309] The vector was linearized by double digestion with Hindlll and EcoRI, and then the left and right homologous arm fragments required for knocking out the aviB2 gene were inserted at the cleavage sites to obtain the target plasmid pOJ260-ΔaviB2, the plasmid map of which is shown in Figure 3 .

[0310] The required primers are as follows:

[0311] pOJ260-ΔaviB2-Lf: cgacggccagtgccaagctt GACGTGTTCCTGCACGCGAC (SEQ ID NO: 9)

[0312] pOJ260-ΔaviB2-Lr: GATCGAACTGGCGCTCGTGCAGGCCCTTGG (SEQ ID NO: 10)

[0313] pOJ260-ΔaviB2-Rf: GCACGAGCGCCAGTTCGATCTGACGGCCCG (SEQ ID NO: 11)

[0314] pOJ260-ΔaviB2-Rr: tatgacatgattacgaattcCGTGCCCACGACGGGAGTGC (SEQ ID NO: 12)

[0315] Example 2: Construction and fermentation verification of mutant strains LK171ΔaviO2, LK171ΔaviBl, LK171ΔaviB2

[0316] The plasmids pOJ260-ΔaviO2, pOJ260-ΔaviB1, and pOJ260-ΔaviB2 were introduced into the wild-type LK171 strain by intergeneric conjugation, and the conjugants were selected by streaking on AS-1 plates with both nalidixic acid and apramycin resistance. The grown conjugants were subcultured on antibiotic-free plates, and finally the successfully relaxed mutant strains were obtained. The knockout verification primers were used for PCR verification, and the correct mutant strains were used for subsequent three-stage fermentation.

[0317] The fermentation results of the mutant strain LK171ΔaviO2 compared with the wild type are shown in the HPLC chart as Figure 4 indicated.

[0318] The fermentation results of the mutant strains LK171ΔaviB1 and LK171ΔaviB2 compared with the wild type are shown in the HPLC chart as Figure 5 indicated.

[0319] From the above fermentation results, compared with the wild-type LK171, after knocking out the target genes aviO2, aviB1, or aviB2, the components of avilamycin A were no longer produced, and a new component of avilamycin different from component B was accumulated, which was named B`.

[0320] Example 3: Detection and analysis of avilamycin B' in fermentation product of wild type LK171 strain

[0321] The compound peaks between the components A and C of avilamycin of the wild-type LK171 strain were separated by semi-preparative HPLC using variable gradient elution conditions, and the collected fractions were subjected to HPLC detection analysis using constant gradient elution conditions with better separation.

[0322] The detection and analysis of refined avilamycin B and B` are shown in the HPLC chart as Figure 6 indicated.

[0323] By the above analysis with better resolution, it was found that the compound peak of Avilamycin B in the wild-type LK171 strain under the conventional HPLC detection conditions in the prior art was actually a mixture peak of Avilamycin B and B`. That is, Avilamycin B` is not only an intermediate produced by the mutant strain in Example 2, but also another major impurity component produced by fermentation of the wild type, which has been ignored due to its high similarity in molecular weight and polarity to the major impurity component Avilamycin B.

[0324] In addition, high-resolution mass spectrometry and NMR identification of the structure of Avilamycin B` were also performed, further confirming that Avilamycin B` is a new component different from Avilamycin B.

[0325] The structural formula of the Avilamycin B` component and its difference from the Avilamycin A component, B component, and C component are shown in Figure 7 .

[0326] Example 4: In vitro reconstitution of avilamycin A, B' components conversion

[0327] AviO2 belongs to non-heme iron / α-ketoglutarate-dependent dioxygenase (αKGD), which depends on the co-factors α-ketoglutarate (α-KG), Fe 2+ and L-ascorbic acid, and catalyzes the oxidation of hydroxyl to ketone in the presence of oxygen. AviB1 and AviB2 belong to pyruvate dehydrogenase, which can catalyze the decarboxylation of pyruvate in the presence of the co-factor thiamine pyrophosphate (TPP).

[0328] The soluble active AviO2, AviB1, and AviB2 proteins were obtained by expression and purification, and the SDS-PAGE characterization spectrum is shown in Figure 8 .

[0329] Using purified AviO2, AviB1, and AviB2, the activity was measured with Avilamycin B` as the substrate, L-ascorbic acid, FeSO4, α-KG, TPP, and pyruvate as the co-factors, the pH of the reaction system was 7.5, the activity measurement temperature was 30°C, and the reaction was overnight. The specific activity measurement conditions are as follows:

[0330]

[0331]

[0332] After the activity measurement was completed, an equal volume of methanol was added for quenching, and samples were taken for LC-MS analysis and detection, and the target molecule Avilamycin A was searched by extracting the ion stream (m / z = 1424-1426, +Na + ). The activity measurement results are shown in Figure 9 .

[0333] According to the results of the activity detection, the production of avilamycin A was detected from avilamycin B`, indicating that AviO2, AviB1 and AviB2 can catalyze the acetylation of avilamycin B` to generate avilamycin A.

[0334] Example 5: Multiplication of plasmid pSOK804-P ermE* - aviO2, pSOK804-P ermE* - Construction of aviO2B1B2

[0335] According to the analysis of the genome sequence of the avilamycin-producing strain Streptomyces viridochromogenes, it was found that there was a natural endogenous avi-VWB-attB site sequence (with a complete sequence of 53 bp), and the specific sequence was as follows:

[0336] CACCGCTCTCCTAAAGCGGGTGTCGCAGGTTCGAATCCTGCCGGGGG CACAAC (SEQ ID NO: 13)

[0337] The wild type LK171 contains the attB site required for the VWB integrase, which can be used to multiply the target gene aviO2 (and aviB1, aviB2), and the constitutive strong promoter P ermE * .

[0338] The Streptomyces conventional plasmid pSOK804 (GenBank: LT545994.1) contains a complete VWB integrase-attP module. Using pSOK804 as a vector, the target gene aviO2 (and aviB1, aviB2) is inserted, and the 5' end is connected with the promoter P ermE * . Finally, the plasmid pSOK804-P ermE * -aviO2 (inserting the target gene aviO2), pSOK804-P ermE* -aviO2B1B2 (inserting the target genes aviO2, aviB1 and aviB2), and the plasmid maps thereof are shown in Figure 10 、 Figure 11 .

[0339] The required primers are as follows:

[0340] 804-O2-F: tatgacatgattacgaattctacgaattcggtaccagccc (SEQ ID NO: 14)

[0341] 804-O2-R: aggtcgactctagaggatccTCAGTCCTTGTCCAGCCAGG (SEQ ID NO: 15)

[0342] 804-B1B2-f: tatgacatgattacgaattccagctatgacatgattacgaattcggtacc (SEQ ID NO: 16)

[0343] 804-B1B2-r: aggtcgactctagaggatcc TCACTTCAGGACGGCACGGA (SEQ ID NO: 17)

[0344] Example 6: Construction and fermentation verification of component-optimized recombinant strains LK804EO2, LK804EO2BlB2

[0345] The plasmid pSOK804-P ermE* -aviO2, pSOK804-P ermE * -aviO2B1B2 were introduced into wild type LK171 strain by intergeneric conjugation respectively. The conjugants were randomly picked and streaked on AS-1 plate with amphotericin and nalidixic acid double resistance, then the grown colonies were verified by PCR. The strain with pSOK804-P ermE * -aviO2 was named as LK804EO2, and the strain with pSOK804-P ermE * -aviO2B1B2 was named as LK804EO2B1B2.

[0346] The verification primers were as follows:

[0347] 804-CHECK-F: cggccggcgaatgctggtac (SEQ ID NO: 18)

[0348] 804-CHECK-R: gtccgcgaacggacgtacgc (SEQ ID NO: 19)

[0349] Three conjugants of each series were picked for fermentation to detect the yield and proportion of avermectin A, B components, and the starting strain LK171 was used as a control.

[0350] The fermentation results (HPLC chart) of the component-optimized recombinant strains and the starting strain LK171 are shown in Figure 12

[0351] ​According to the fermentation results, after the target gene aviO2 is multiplied by site-specific integration, compared with the starting strain LK171, the proportion of avermectin B component in the final fermentation product is significantly reduced from 10-15% to less than 5%, and the proportion of avermectin A component in the final fermentation product is significantly increased from 55-60% to more than 65%. Therefore, the proportion of avermectin A component can be increased by multiplying the aviO2, aviB1 and aviB2 genes.

[0352] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the application upon reading a further understanding of the above disclosure and these equivalents are also intended to fall within the scope of the application as defined by the appended claims.

Claims

1. A method for increasing the proportion and / or yield of the avermectin active ingredient Group A component, characterized in that, The method comprises the steps of: (a) providing an avermectin-producing strain; and (b) enhancing expression of a target protein in the avermectin-producing strain, or increasing copy number of a target gene in the avermectin-producing strain, thereby increasing the proportion and / or yield of Group A component in the avermectin-producing strain; wherein the target protein is selected from the group consisting of AviO2, AviB1, AviB2, or a combination thereof; the target gene is selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof.

2. A method of constructing a recombinant strain with improved proportions and / or yields of components of the avermectin A group, characterized in that, The method comprises the steps of: (a) providing an avermectin-producing strain; and (b) increasing copy number of a target gene in the avermectin-producing strain, thereby obtaining a recombinant strain with increased proportion and / or yield of Group A component of avermectin; wherein the target gene is selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof.

3. A recombinant bacterial strain prepared by the method of claim 2, wherein, The recombinant strain comprises a target gene with increased copy number in its genome, the target gene being selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof.

4. A polynucleotide, comprising: The polynucleotide has a sequence of the recombinant strain as claimed in claim 3, or a derivative sequence thereof.

5. An expression vector, characterized by, The expression vector contains the polynucleotide as claimed in claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector as claimed in claim 5, or has integrated into its genome the polynucleotide as claimed in claim 4.

7. Use of an active ingredient, characterized in that The active ingredient is selected from: the recombinant strain as claimed in claim 3, the polynucleotide as claimed in claim 4, the expression vector as claimed in claim 5, or the host cell as claimed in claim 6, or a combination thereof; for use in a method selected from the group consisting of: (i) converting Group A, B' components of avermectin into each other in vitro; (ii) increasing the proportion and / or yield of Group A component of avermectin active ingredient; (iii) preparing a recombinant strain with high yield of Group A component of avermectin; (iv) decreasing the proportion and / or content of Group B' component of avermectin; (v) preparing a recombinant strain with decreased Group B' component of avermectin; (vi) inhibiting synthesis of bacterial proteins; (vii) promoting animal growth; and / or (viii) preparing a feed additive.

8. A product characterized by, The product comprises (a) a plasmid with increased copy number of a target gene selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof.

9. A combination prepared by the process of claim 8, wherein the combination is prepared by the process of claim 8. The preparation kit comprises: (Z1) a Streptomyces application plasmid containing an integrase module; (Z2) a construction primer; (Z3) reagents required for intergeneric conjugation transfer; (Z4) a verification primer; and / or (Z5) a plasmid with increased copy number of a target gene selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof (as a positive control).

10. Use of a target gene, a target protein selected from the group consisting of AviO2, AviB1, AviB2, or a combination thereof; the target gene selected from the group consisting of aviO2, aviB1, aviB2, or a combination thereof; characterized in that, for use in a method selected from the group consisting of: (i) converting Group A, B' components of avermectin into each other in vitro; (ii) increasing the proportion and / or yield of Group A component of avermectin active ingredient; (iii) preparing a recombinant strain with high yield of Group A component of avermectin; (iv) decreasing the proportion and / or content of Group B' component of avermectin; and / or (v) preparing an avilamycin B' group component-reduced recombinant strain. (v) preparing an avilamycin B' group component-reduced recombinant strain.

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