CN110563783B

CN110563783B - High-efficiency low-toxicity tetramycin B derivative and directed high-yield metabolic engineering method thereof

Info

Publication number: CN110563783B
Application number: CN201910761912.3A
Authority: CN
Inventors: 康前进; 盛勇; 白林泉; 欧一新
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2020-11-17
Anticipated expiration: 2039-08-16
Also published as: CN110563783A

Abstract

The invention discloses a high-efficiency low-toxicity tetramycin B derivative and a directed high-yield metabolic engineering method thereof; the tetramycin B derivative has a molecular formula of C₃₅H₅₅NO₁₂The chemical structural formula is as follows:

the invention adopts a metabolic engineering strategy, obtains the mutant strain SY05 with high yield of the tetramycin B derivative by enhancing the supply of the precursor malonyl coenzyme A of the polyketide chain extension unit and modifying genes tetrK and tetrF after overexpression of the tetramycin biosynthesis gene cluster, improves the yield of the tetramycin B derivative in the strain by 40 percent compared with that in the original strain SY04, and lays a foundation for the industrial production and application of the high-efficiency low-toxicity tetramycin B derivative.

Description

High-efficiency low-toxicity tetramycin B derivative and directed high-yield metabolic engineering method thereof

Technical Field

The invention belongs to the field of bioengineering and technology, and relates to a high-efficiency low-toxicity tetramycin B derivative and a directed high-yield metabolic engineering method thereof; in particular to a method for improving the yield of a tetramycin B derivative in a mutant strain SY05 by 40 percent compared with the yield of a starting strain SY04 by enhancing the supply of malonyl coenzyme A serving as a polyketide chain extension unit precursor and the expression of tetrK and tetrF serving as a post-modification gene cluster of a tetramycin biosynthesis gene in Streptomyces hygroscopicus Beijing variant SY04 by means of metabolic engineering and genetic engineering.

Background

The common polyene macrolide antibiotics have good prevention and treatment effects on pathogenic fungal infection. But the antifungal activity of the compound is lower than that of other antifungal medicines, and the compound is easy to be combined with cholesterol on the cell membrane of a mammal, so that the hemolytic toxicity is higher. The disadvantage of this pharmacological property of polyene antibiotics severely limits their widespread use. The research reports that cytochrome P450 monooxygenase responsible for carboxylation in genes modified after biosynthetic genes such as polyene antibiotics (amphotericin, nystatin and pimaricin) are inactivated by using a genetic engineering means, and a decarboxylation precursor compound with lower hemolytic toxicity and better antifungal activity can be generated, so that the clinical application of the polyene antibiotics has good application prospect. However, compared with the wild strain, the yield of decarboxylation precursor compounds of the cytochrome P450 monooxygenase gene deletion mutant strain is obviously reduced, and the phenomenon seriously limits the industrial directional high yield of polyene derivatives.

Decarboxylated tetramycin B derivatives

Compared with the tetramycin B, the derivative has lower hemolytic toxicity and better antifungal activity, but the yield of the tetramycin B derivative in the cytochrome P450 monooxygenase tetrG gene deletion mutant SY04 which is responsible for carboxylation is reduced by 59 percent compared with the yield of the tetramycin B in the original strain SY 02. This phenomenon severely limits the industrial application of the tetramycin B derivative, and thus high production of the tetramycin B derivative by means of metabolic engineering and genetic engineering is imminent.

Disclosure of Invention

The invention aims to provide a high-efficiency low-toxicity tetramycin B derivative and a directed high-yield metabolic engineering method thereof; the yield of the high-efficiency low-toxicity tetramycin B derivative is improved by means of metabolic engineering and genetic engineering in the streptomyces spinosa water-absorption Beijing variety SY04, and a foundation is laid for the industrial oriented production of the derivative.

The purpose of the invention is realized by the following technical scheme.

In a first aspect, the invention relates to a high-efficiency low-toxicity tetramycin B derivative with a molecular formula of C₃₅H₅₅N0₁₂The chemical structure formula is as follows:

in a second aspect, the present invention relates to a metabolic engineering method for high yield of the high-efficiency low-toxicity tetramycin B derivative, which comprises the following steps:

s1, performing in-frame deletion on polyketone synthase gene nysI in a nystatin biosynthetic gene cluster in a strain streptomyces spinosa Beijing mutant CGMCC4.1123 to obtain a mutant strain with the gene nysI deleted in the same frame;

s2, inactivating a P450 monooxygenase gene tetrG in a tetramycin biosynthesis gene cluster in the same-frame deletion mutant strain to obtain a gene tetrG inactivated mutant strain;

s3, obtaining a mutant strain with high yield of the tetramycin B derivative by enhancing the supply of malonyl coenzyme A as a precursor of a polyketide chain extension unit and the expression of tetrK and tetrF as post-modification genes of a tetramycin biosynthesis gene cluster in the mutant strain with the inactivated tetrG gene;

s4, fermenting and culturing the mutant strain of the high-yield tetramycin B derivative to obtain the high-efficiency low-toxicity tetramycin B derivative.

As one embodiment of the invention, the mutant strain with the gene nysI deletion in frame is a genetically engineered strain Streptomyces hygroscopicus Beijing variant (Streptomyces hygrospinococcus var. beijingensis) SY 02.

As one embodiment of the invention, the gene tetrG inactivated mutant strain is genetically engineered strain Streptomyces hygroscopicus Beijing variant (Streptomyces hygrospinococcus var. beijingensis) SY 04.

As an embodiment of the invention, the mutant strain of the high-yield tetramycin B derivative is a genetically engineered strain Streptomyces hygroscopicus Beijing variant (Streptomyces hygrospinococcus var. beijingensis) SY 05.

In the present invention, step S1 specifically includes the following steps:

s11, constructing a double-exchange plasmid pJQK503 for the in-frame deletion of the polyketide synthase gene nysI in the nystatin biosynthetic gene cluster;

s12, transferring the plasmid pJQK503 into receptor bacterium streptomyces spinosus Beijing variant CGMCC4.1123 through conjugation between escherichia coli and streptomyces;

s13, carrying out PCR verification on the genomic DNA of the mutant strain obtained in the step S12 to obtain the mutant strain with the same frame deletion of the polyketide synthase gene nysI in the nystatin biosynthetic gene cluster.

The construction of the plasmid pJQK503 is that a left and a right homologous arm fragments delta Nys-L and delta Nys-R which are correctly sequenced are respectively inserted between XbaI/HindIII double enzyme cutting sites of the plasmid pJTU 1278; and the homologous arm fragment delta Nys-L is inserted between the XbaI/BspTI enzyme cutting sites, and the homologous arm fragment delta Nys-R is inserted between the BspTI/HindIII enzyme cutting sites.

The sequence of the homologous arm fragment delta Nys-L is shown in SEQ ID NO. 5; the sequence of the homologous arm fragment delta Nys-R is shown in SEQ ID NO. 6.

In the present invention, step S2 specifically includes the following steps:

s21, constructing a P450 monooxygenase gene tetrG inactivation plasmid pJQK513 used in the tetramycin biosynthesis gene cluster;

s22, inactivating the P450 monooxygenase gene tetrG in the gene nysI in-frame deletion mutant strain by the intergeneric conjugative transfer of the streptomycete and escherichia coli on the plasmid pJQK 513;

s23, carrying out PCR verification on the genomic DNA extracted from the mutant strain obtained in the step S22 to obtain the mutant strain with the inactivated P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster.

In step S21, the inactivation of the P450 monooxygenase gene tetrG in the tetramycin biosynthetic gene cluster is achieved by mutating the active site amino acid Cys340 of the gene tetrG to Ala.

The sequence of the P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster is shown as SEQ ID NO. 15.

The construction of the plasmid pJQK503 is that right and left homologous arm fragments C340A-tetrG-L and C340A-tetrG-R which are correctly sequenced are respectively inserted between EcoRI/XbaI double enzyme cutting sites of the plasmid pJTU 1278; and the homologous arm fragment C340A-tetrG-L is inserted between EcoRI/HindIII enzyme cutting sites, and the homologous arm fragment C340A-tetrG-R is inserted between HindIII/XbaI enzyme cutting sites.

The sequence of the homologous arm fragment C340A-tetrG-L is shown in SEQ ID NO. 10; the sequence of the homologous arm fragment C340A-tetrG-R is shown in SEQ ID NO. 11.

In the present invention, step S3 includes the following steps:

s31, constructing modified genes tetrK and tetrF expression integration type plasmids pJQK512 used for heterogeneously expressing acc genes and strengthening tetramycin biosynthesis gene clusters;

s32, integrating the plasmid pJQK512 to the chromosome of the gene tetrG inactivated mutant strain through the conjugation and transfer between streptomycete and escherichia coli to obtain the mutant strain with high yield of the tetramycin B derivative.

In step S31, the acc gene is derived from Streptomyces coelicolor M152 and consists of three genes of accA2, accB and accE, wherein the accB and accE genes share one promoter in the Streptomyces coelicolor M152; wherein, the sequence of the accA2 gene is shown in SEQ ID NO. 40; the accB gene sequence is shown in SEQ ID NO. 41; the accE gene sequence is shown in SEQ ID NO. 42.

accA2 gene sequence, SEQ ID No. 40:

accB gene sequence, SEQ ID No. 41:

accE gene sequence, SEQ ID NO. 42:

in the present invention, heterologous expression of the acc gene is achieved by placing the accA2 and accBE genes under a strong promoter PermE, respectively.

In step S31, the post-modifier tetrK gene sequence is shown in SEQ ID NO. 43.

tetrK gene sequence, SEQ ID No. 43:

in step S31, the post-modifier tetrF gene sequence is shown in SEQ ID NO. 44.

tetrF gene sequence, SEQ ID No. 44:

in step S31, the genes tetrK and tetrF were modified after the amplification of the tetramycin biosynthetic gene cluster by placing both tetrK and tetrF genes under the strong promoter KasOp.

In step S31, the plasmid pJQK512 is constructed by subjecting the plasmid pJQK508 to double restriction with MfeI/KpnI, recovering a 3822bp enzyme-cleaved DNA fragment, and ligating the DNA fragment to the plasmid pJQK511 subjected to double restriction with MfeI/KpnI by T4 ligase to obtain the plasmid pJQK 512.

Plasmid pJQK508 was constructed by digesting plasmid SK (+) -ermEp-accA 2-ermEp-accBE with XbaI alone and recovering 3838-bp size target fragment ermEp-accA 2-ermEp-accBE/XbaI, and ligating this fragment with XbaI alone digested vector pSET152 to obtain plasmid pJQK 508.

Wherein, the construction of the plasmid SK (+) -ermEp-accA 2-ermEp-accBE comprises the following steps:

XbaI, KpnI, BglII and NcoI enzyme cutting sites are respectively introduced at two ends of a PCR fragment ermEp-accA 2, and BglII, MfeI, XbaI and NcoI enzyme cutting sites are respectively introduced at two ends of the PCR fragment ermEp-accBE; connecting to pBluescript SK (+) vector treated by EcoRV, performing BglII/NcoI double digestion, recovering fragments SK (+) -ermEp-accA 2/(BglII/NcoI) and ermEp-accBE/(BglII/NcoI);

the DNA fragment ermEp-accBE/(BglII/NcoI) recovered by enzyme digestion is connected to the SK (+) -ermEp-accA 2/(BglII/NcoI) plasmid fragment by T4DNA ligase to obtain the plasmid SK (+) -ermEp-accA 2-ermEp-accBE.

The plasmid pJQK511 was constructed by ligating the BspTI/BamHI double-digested DNA target fragment KasOp-tetrF to the BspTI/BamHI double-digested plasmid pJQK510 by T4DNA ligase to obtain a plasmid pJQK 511.

The construction of the plasmid pJQK510 comprises the following steps:

using the total DNA of the Beijing variant SY02 genome of Streptomyces hygroscopicus as a template, respectively using KasOp-tetrK-1-F/R, KasOp-tetrK-2-F/R, KasOp-tetrF-1-F/R and KasOp-tetrF-2-F/R as primers, amplifying by using high-fidelity DNA polymerase to obtain PCR fragments for enhancing the expression of genes tetrK and tetrF, wherein the PCR fragments are marked as KasOp-tetrK and KasOp-tetrF, wherein XbaI, KpnI, MunI, BspTI and BamHI enzyme cutting sites are respectively introduced at two ends of the PCR fragment KasOp-tetrF, and BspTI and BamHI enzyme cutting sites are respectively introduced at two ends of the PCR fragment;

connecting the PCR product after gel recovery to a pBluescript SK (+) vector treated by EcoRV, carrying out blue-white screening and sequencing on the PCR product to be correct, carrying out double enzyme digestion on the PCR product respectively through XbaI/BamHI and BspTI/BamHI, and recovering a target fragment KasOp-tetrK; the recovered DNA fragment KasOp-tetrK digested with XbaI/BamHI was ligated to XbaI/BamHI-digested vector pJQK509 to obtain plasmid pJQK 510.

In step S2, the fermentation culture is to inoculate the spore suspension of the mutant strain with high yield of the tetramycin B derivative into a TBSY seed culture medium, culture the mutant strain at 220rpm and 30 ℃ for 24h, transfer the mutant strain to the fermentation culture medium in a proportion of 5%, and then culture the mutant strain at 220rpm and 30 ℃ for 120h to obtain a fermentation culture solution.

The TSBY seed culture medium is prepared from 30g of Oxoid tryptone bean soup powder, 103g of sucrose and 5g of Difco yeast extract, and distilled water is added to the mixture until the volume is 1L and the pH value is 7.2.

The fermentation medium comprises corn flour 10g, soluble starch 20g, soybean cake powder 10g, KH₂PO₄ 0.2g、 NaCl 3g、NH₄Cl 3g、CaCO₃4g, adding distilled water to a constant volume of 1L, and adjusting the pH value to 7.0 by using 1M sodium hydroxide solution.

In a third aspect, the invention relates to a production strain of the high-efficiency low-toxicity tetramycin B derivative, wherein the strain is Streptomyces hygroscopicus var beijing (beijinginnsis) SY05 with the preservation number of CGMCC NO. 18370.

The mutant strain SY02 is Streptomyces hygroscopicus Beijing variant (Beijing. Beijing) which has been preserved in the general microbiological center of China Committee for culture Collection of microorganisms (Beijing city, Chaoyang district, Beicheng West Lu No.1, institute of microbiology, China academy of sciences) in 2019 at 17 th month, and the preservation number is CGMCC NO. 18241.

The mutant strain SY04 is Streptomyces hygroscopicus Beijing variant (Beijing. beijingningnsis) which has been preserved in the general microorganism center of China Committee for culture Collection of microorganisms (Beijing city rising district West Lu No.1 Beijing institute, China academy of sciences microorganism research institute) 8.6 days in 2019, and the preservation number is CGMCC NO. 18369.

The mutant strain SY05 is Streptomyces hygroscopicus Beijing variant (Beijing. beijingningnsis) which has been preserved in the general microorganism center of China Committee for culture Collection of microorganisms (Beijing city rising district West Lu No.1 Beijing institute, China academy of sciences microorganism research institute) 8.6 days in 2019, and the preservation number is CGMCC NO. 18370.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts a metabolic engineering strategy, and enhances the supply of the polyketide chain extension unit precursor malonyl coenzyme A and the expression of the tetramycin biosynthesis gene cluster post-modification genes tetrK and tetrF, so that the yield of the tetramycin B derivative in the mutant strain SY05 is increased by 40 percent compared with that of the original strain SY04, and a foundation is laid for the industrial application and production of the high-efficiency low-toxicity tetramycin B derivative.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram of construction of plasmid pJQK503 for the in-frame deletion group of nySi gene;

FIG. 2 is a same frame deletion mutant strain SY02PCR verification agarose gel electrophoresis picture;

FIG. 3 is a schematic diagram of the construction of plasmid pJQK513 for inactivating P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster;

FIG. 4 is a PCR-verified agarose gel electrophoresis image of a tetrG inactivated mutant strain SY 04;

FIG. 5 is a schematic diagram of the construction of an integrative plasmid pJQK512 for heterologous expression of ACC genes and for enhancing expression of the post-modification genes tetrK and tetrF of the tetramycin biosynthetic gene cluster;

FIG. 6 is HPLC analysis chart of fermentation liquor of original strain SY04 and mutant strain SY 05;

FIG. 7 is a high performance liquid chromatography HPLC UV absorption chart of a tetramycin B derivative;

FIG. 8 is a mass spectrometric detection of a tetramycin B derivative;

FIG. 9 is a bar graph of fermentation yields of tetramycin A, B derivatives in strains SY04 and SY 05.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of protection of the present invention. The following examples are examples of experimental methods not specified under specific conditions, according to conventional conditions or manufacturer's recommended conditions.

Example 1 construction of Streptomyces spinosus Beijing variant SY02

Step 1: constructing a double-exchange plasmid pJQK503 for deleting the nysI of the polyketide synthase gene in the nystatin biosynthetic gene cluster in the same frame;

as shown in figure 1, the total DNA of the Beijing variant CGMCC4.1123 (purchased from China general microbiological culture Collection center) genome of Streptomyces erythraea is used as a template, and delta Nys-L-F/R and delta Nys-R-F/R are used as primers respectively, and a left homologous arm fragment and a right homologous arm fragment for gene deletion are obtained by using high-fidelity DNA polymerase amplification and are marked as delta Nys-L and delta Nys-R (sequences are shown as SEQ ID NO.5 and SEQ ID NO. 6), wherein XbaI and BspTI enzyme cutting sites are respectively introduced into two ends of the delta Nys-L homologous arm fragment, and BspTI and HindIII enzyme cutting sites are respectively introduced into two ends of the delta Nys-R homologous arm, so as to facilitate the construction of a plasmid pJQK 503. The PCR products of the left and right homologous arms after gel recovery are connected to a pBluescript SK (+) vector treated by EcoRV, and after blue-white screening and sequencing are correct, the target fragments are recovered by XbaI/BspTI and BspT1/HindIII double enzyme digestion respectively. The recovered left and right homologous arm fragments were simultaneously ligated to vector pJTU1278 digested with XbaI and HindIII to obtain plasmid pJQK 503. In order to verify the correctness of the construction of the plasmid pJQK503, the plasmid is digested by XbaI/HindIII to obtain a digested fragment with the size of 3.2kbp, which indicates that the construction of the plasmid pJQK503 is correct.

The primer sequences used in step 1 are shown in table 1 below:

TABLE 1

Primer name	Base sequence
		ΔNys-L-F	5′-TCTAGAGGGTGGTGGCGAGGGAGT-3' (XbaI cleavage site) SEQ IDNO.1
ΔNys-L-R	5′-CTTAAGGCGGCGGGTCAGCGAGCTG-3' (BspTI cleavage site) SEQ ID NO.2
		ΔNys-R-F	5′-CTTAAGGTCGGCGTATTGGTTTTC-3' (BspTI cleavage site) SEQ ID NO.3
ΔNys-R-R	5′-AAGCTTACCTGTTGCACGTCGTTC-3' (HindIII cleavage site) SEQ ID NO.4

Step 2: transferring the plasmid pJQK503 constructed in the first step into receptor strain Streptomyces spinosus Beijing variant CGMCC4.1123 through the intergeneric conjugation of escherichia coli and streptomyces;

the plasmid pJQK503 which has been constructed in step 1 is introduced into the host ET12567/pUZ8002 by the calcium transformation method. The transformant was taken out and cultured overnight at 37 ℃ in LB medium containing three antibiotics chloramphenicol (50. mu.g/mL), kanamycin (50. mu.g/mL) and ampicillin (100. mu.g/mL), and the overnight culture was transferred to LB medium containing three antibiotics chloramphenicol (50. mu.g/mL), kanamycin (50. mu.g/mL) and ampicillin (100. mu.g/mL) at a ratio of 10% for 3 hours, and then the cells were washed 2 times with fresh antibiotic-free LB medium to sufficiently remove the antibiotics in the culture. Simultaneously preparing about 10 fresh spores of Streptomyces spinosus Beijing variant⁸Rinsing with TES solution for 2-3 times, suspending spores with 0.5mL TES solution, thermally shocking in 50 deg.C water bath for 10min, rapidly cooling to room temperature, adding isovolume 2 × spore pre-germination culture solution (formula: 1% Difco yeast extract, 1% Difco casein amino acid, CaCl)₂0.1M (5M stock solution is required to be prepared and added into yeast extract/casein amino acid solution after being separately sterilized at high temperature) and 0.5uM calcium chloride solution,the spores were then cultured at 37 ℃ for 2.5 hours for a pregermination procedure. Centrifugally collecting spores, rinsing the spores with LB culture solution without antibiotics for 2 times, then suspending the spores in an appropriate amount of LB culture solution, fully shaking and scattering the spores on a mixer, and performing a method according to the ratio of 10⁷∶10⁸The donor-acceptor ratio of (1) and Escherichia coli cells are mixed and then uniformly coated on an MS plate without antibiotics, the plate is transferred to an incubator at 30 ℃ after being dried and cultured for 17 hours, then the plate is taken out, 1.5mL of sterile water containing thiostrepton (0.5mg) and nalidixic acid (1mg) is covered on the MS plate, and the plate is dried and then transferred to the incubator at 30 ℃ for culture. Single colony binders can be seen on the plate after 4-5 days. After 2-3 rounds of antibiotic-free relaxation, spores were collected on MS plates without any antibiotic at 10%^-4、 10^-5、10^-6And (3) performing gradient dilution and plate coating, and finally verifying the correctness of the binding molecules by adopting V-delta Nys-F/R as a PCR primer to obtain a mutant strain SY 02.

FIG. 2 is a same frame deletion mutant strain SY02PCR verification agarose gel electrophoresis picture, and as can be seen from FIG. 2, when PCR verification is performed by taking wild type strain genome DNA as a template, the band is 664 bp; and when PCR verification is carried out by taking genomic DNA of the mutant strain with the nysI gene in-frame deletion as a template, the band is 403 bp. The result of this experiment proves the success of the construction of the mutant strain SY 02.

The sequences of the primers used in step 2 are shown in table 2:

TABLE 2

Primer name	Base sequence
		V-ΔNys-F	5′-CGTTCGAACGCCTCCCAG-3′ SEQ ID NO.7
V-ΔNys-R	5′-CGATTTCCCCGCCCTCAT-3′ SEQ ID NO.8

Example 2 construction of Streptomyces spinosus Beijing variant SY04

Step 1: construction of P450 monooxygenase gene tetrG inactivation plasmid pJQK513 in tetramycin biosynthesis gene cluster

As shown in FIG. 3, the total DNA of Beijing variant SY02 genome of Streptomyces hygroscopicus is used as a template, C340A-tetrG-L-F/R and C340A-tetrG-R-F/R are respectively used as primers, left and right homologous arm fragments for gene mutation are obtained by high-fidelity DNA polymerase amplification and are marked as C340A-tetrG-L and C340A-tetrG-R (the sequences are shown as SEQ ID NO.9 and 10), wherein EcoRI and HindIII cleavage sites are respectively introduced at the two ends of the homologous arm fragment C340A-tetrG-L, HindIII and XbaI cleavage sites are respectively introduced at the two ends of the homologous arm C340A-tetrG-R, and the 3 'end of the homologous arm C340A-tetrG-L and the 5' end of the homologous arm C340-tetrG-R can mutate the cysteine 340 of the tetrG at the site in the tetrG as alanine (Ala 340 trG) of the homologous arm C340 TraI (SEQ ID NO. 3) in the homologous arm C340, restriction enzyme HindIII restriction sites introduced into the mutation sites in tetrG facilitate later verification of the mutants. The left and right homologous arm PCR products after gel recovery are connected to a pBluescript SK (+) vector treated by EcoRV, and after blue-white screening and sequencing are correct, the target fragments are recovered by EcoRI/HindIII and HindIII/XbaI double enzyme digestion respectively. The recovered left and right homologous arm fragments were ligated to vector pJTU1278 digested with EcoRI and XbaI at the same time to obtain plasmid pJQK 513. In order to verify the correctness of the construction of the plasmid pJQK513, the EcoRI/XbaI double-enzyme digestion plasmid is adopted to obtain an enzyme digestion fragment with the size of 3.0-kbp, which indicates that the construction of the plasmid pJQK513 is correct.

The primer sequences used in step 1 are shown in table 3 below:

TABLE 3

Primer name	Base sequence
		C340A-tetrG-L-F	5′-GAATTCACACCACGACCAGTTCGCAG-3' (EcoRI cleavage site) SEQ ID NO.11
C340A-tetrG-L-R	5′-AAGCTTTGGGGCAGAACCTGGTGCGG-3' (HindIII cleavage site) SEQ ID NO.12
		C340A-tetrG-R-F	5′-AAGCTTGGTGGATCCCGTAGCCGAAC-3' (HindIII cleavage site) SEQ ID NO.13
C340A-tetrG-R-R	5′-TCTAGAAGAAGTTGGCGTACAACTAC-3' (XbaI cleavage site) SEQ ID NO.14

The sequence of the P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster is shown as SEQ ID NO. 15:

step 2: the plasmid pJQK513 constructed in the step 1 is transferred and introduced into receptor bacterium streptomyces spinosus Beijing variant SY02 through conjugation between escherichia coli and streptomyces.

The plasmid pJQK513 constructed in the first step was introduced into the host ET12567/pUZ8002 by the calcium transformation method. The transformant was taken out and cultured overnight at 37 ℃ in LB medium containing three antibiotics chloramphenicol (50. mu.g/mL), kanamycin (50. mu.g/mL) and ampicillin (100. mu.g/mL), and the overnight culture was transferred to LB medium containing three antibiotics chloramphenicol (50. mu.g/mL), kanamycin (50. mu.g/mL) and ampicillin (100. mu.g/mL) at a ratio of 10% for 3 hours, and then the cells were washed 2 times with fresh antibiotic-free LB medium to sufficiently remove the antibiotics from the culture. Simultaneously preparing about 10 fresh spores of Streptomyces spinosus Beijing variant⁸Rinsing with TES solution for 2-3 times, suspending spores with 0.5ml of TES solution, thermally shocking in 50 deg.C water bath for 10min, rapidly cooling to room temperature, adding equal volume of 2 × spore pre-germination culture solution (formula: 1% Difco yeast extract, 1% Difco casein amino acid, CaCl)₂0.1M (5M stock solution is required to be prepared and added into yeast extract/casein amino acid solution after being separately sterilized at high temperature) and 0.5uM calcium chloride solution, and then the pre-germination process of the spores is carried out after 2.5 hours of culture at 37 ℃. Collecting spores by centrifugation, rinsing spores with LB culture solution without antibiotic for 2 times, then suspending the spores in an appropriate amount of LB culture solution, shaking thoroughly on a mixer and scattering the spores, according to 10⁷∶10⁸The donor-acceptor ratio of (1) and Escherichia coli cells are mixed and then uniformly coated on an MS flat plate without antibiotics, the flat plate is transferred to an incubator at 30 ℃ after being dried and cultured for 17 hours, then the flat plate is taken out, 1.5mL of sterile water containing thiostrepton (0.5mg) and nalidixic acid (1mg) is used for covering the MS flat plate, and the flat plate is dried and then transferred to the incubator at 30 ℃ for culture. Generally, after 4-5 days, a single colony binder grows out on the plate. After 2-3 rounds of non-antibiotic relaxation, spores were collected on MS plates without any antibiotic at 10^-4、10^-5、 10^-6Gradient dilution plating is carried out, and finally V-C340A-tetrG-F/R is used as a PCR primer to verify the correctness of the binding molecules.

FIG. 4 is a PCR-verified agarose gel electrophoresis diagram of a tetrG inactivated mutant strain SY04, which can be obtained from FIG. 4, wherein PCR can obtain a DNA fragment with the size of 1425bp by using genomic DNAs of strains SY02 and SY04 as templates, and then enzyme digestion verification is performed on the PCR products by using HindIII restriction endonuclease, and the experimental result proves that the construction of the mutant strain SY04 is successful, wherein the size of the PCR products obtained by using SY02 as the template is unchanged after enzyme digestion by HindIII, and the sizes of the DNA fragments respectively of 1091bp and 334bp are obtained by enzyme digestion of the PCR products obtained by using SY04 as the template.

The primer sequences used in step 2 are shown in table 4 below:

TABLE 4

Primer name	Base sequence
		V-C340A-tetrG-F	5′-GACCAGGACGAACGGAAGGT-3′ SEQ ID NO.16
V-C340A-tetrG-R	5′-GTGCTGATGCTGCTGCTCAT-3′ SEQ ID NO.17

Example 3 fermentation of Tetramycin B derivatives and purification of the preparation thereof

Step 1: construction of integrative plasmid pJQK512 for heterologous expression of acc gene and reinforcement of expression of tetrK and tetrF after-modification genes of tetramycin biosynthesis gene cluster

As shown in FIG. 5, PCR fragments for enhancing the expression of genes tetrK and tetrF were obtained by high fidelity DNA polymerase amplification using the total DNA of Streptomyces hygroscopicus Beijing variant SY02 genome as a template and KasOp-tetrK-1-F/R and KasOp-tetrF-2-F/R as primers, respectively, and were labeled as KasOp-tetrK and KasOp-tetrF (sequences are shown in SEQ ID NO.18 and 19), wherein XbaI, KpnI, MunI, BspTI and BamHI cleavage sites were introduced at both ends of the PCR fragment KasOp-tetrK, and BspTI and BamHI cleavage sites were introduced at both ends of the PCR fragment KasOp-tetrF, respectively. The PCR product after gel recovery is connected to a pBluescript SK (+) vector treated by EcoRV, and after blue-white screening and sequencing are correct, the PCR product is subjected to double enzyme digestion by XbaI/BamHI and BspTI/BamHI respectively, and a target fragment is recovered. The DNA fragment recovered by XbaI/BamHI digestion, KasOp-tetrK, was ligated to the XbaI and BamHI double digested vector pJQK509 (plasmid pJQK509 was a laboratory preliminary construction, specifically obtained by ligating the DNA fragment carrying KasOp-tetrK (BglII/NotI) to the BglII/NotI treated pSET152 plasmid by T4 ligase), to obtain plasmid pJQK 510. In order to verify the correctness of the construction of the plasmid pJQK510, the plasmid is digested by XbaI/BamHI to obtain a digested fragment with the size of 1318bp, which indicates that the construction of the plasmid pJQK510 is correct.

The BspTI/BamHI double-digested DNA target fragment KasOp-tetrF was ligated to BspTI/BamHI double-digested plasmid pJQK510 by T4DNA ligase to obtain plasmid pJQK 511. In order to verify the correctness of the construction of the plasmid pJQK511, the BspTI/BamHI plasmid is used for double digestion to obtain a digestion fragment with the size of 301bp, which indicates that the construction of the plasmid pJQK511 is correct.

By taking the total DNA of Streptomyces coelicolor M152 genome as a template, respectively taking ermEp-accA 2-1-F/R, ermEp-accA 2-2-F/R, ermEp-accBE-1-F/R and ermEp-acBE-2-F/R as primers, amplifying by using high-fidelity DNA polymerase to obtain PCR fragments for heterologously expressed genes accA2, accB and accE, and marking the PCR fragments as ermEp-accA 2 and ermEp-accBE (sequences are shown as SEQ ID NO.20 and 21), wherein XbaI, KpnI, BglII and NcoI enzyme digestion sites are respectively introduced at two ends of the PCR fragment ermEp-accA 2, and BglII, XbaI enzyme digestion sites are respectively introduced at two ends of the PCR fragment ermEpcBE. The PCR product after gel recovery is connected to a pBluescript SK (+) vector treated by EcoRV, and after blue white spot screening and correct sequencing, the PCR product is subjected to BglII/NcoI double enzyme digestion to recover target fragments SK (+) -ermEp-accA 2/(BglII/NcoI) and ermEp-accBE/(BglII/NcoI). The DNA fragment ermEp-accBE/(BglII/NcoI) recovered by enzyme digestion is connected to the SK (+) -ermEp-accA 2/(BglII/NcoI) plasmid fragment by T4DNA ligase to obtain the plasmid SK (+) -ermEp-accA 2-ermEp-accBE. In order to verify the construction correctness of the plasmid SK (+) -ermEp-accA 2-ermEp-accBE, the plasmid XbaI is adopted to carry out single enzyme digestion to obtain an enzyme digestion fragment with the size of 3838-bp, which indicates that the plasmid SK (+) -ermEp-accA 2-ermEp-accBE is constructed correctly.

And (3) performing single digestion treatment on the plasmid SK (+) -ermEp-accA 2-ermEp-accBE with correct digestion verification through XbaI, recovering a target fragment ermEp-accA 2-ermEp-accBE/XbaI with the size of 3838-bp, and connecting the fragment with the vector pSET152 subjected to single digestion treatment through XbaI through blue white spot screening to obtain the plasmid pJQK 508. In order to verify the correctness of the construction of the plasmid pJQK508, the MfeI/KpnI double-enzyme digestion plasmid is adopted, and an enzyme section segment with the size of 3822bp can be obtained, so that the construction of the plasmid pJQK508 is correct.

After the plasmid pJQK508 which is verified to be correct by enzyme digestion is subjected to double enzyme digestion by MfeI/KpnI, an enzyme digestion DNA fragment with the size of 3822bp is recovered, and the plasmid pJQK512 (with two copies of tetrK, one copy of tetrF and one copy of ACC gene expression Cassette) is obtained by connecting the plasmid pJQK511 subjected to double enzyme digestion by MfeI/KpnI through T4 ligase. In order to verify the correctness of the construction of the plasmid pJQK512, the MfeI/KpnI double enzyme digestion plasmid is adopted, and the enzyme digestion fragment with the size of 3822bp can be obtained, so that the construction of the plasmid pJQK512 is correct.

The sequences of the primers used in step 1 are shown in table 5:

TABLE 5

Step 2:

the plasmid pJQK512 constructed in the step 1 is transferred and introduced into receptor bacterium streptomyces spinosus Beijing variant SY04 through conjugation between escherichia coli and streptomyces.

The plasmid pJQK512 constructed in the step 1 is introduced into E.coli ET12567/pUZ8002 by a chemical transformation method. The transformant was taken out and cultured overnight at 37 ℃ in LB medium containing three antibiotics chloramphenicol (50. mu.g/mL), kanamycin (50. mu.g/mL) and apramycin (50. mu.g/mL), the overnight culture was transferred to LB medium containing three antibiotics chloramphenicol (50. mu.g/mL), kanamycin (50. mu.g/mL) and ampicillin (100. mu.g/mL) at a ratio of 10% for 3 hours, and then the culture was washed 2 times with fresh antibiotic-free LB medium to sufficiently remove the antibiotics from the culture. Simultaneously preparing about 10 fresh spores of Streptomyces spinosus Beijing variant SY04⁸Rinsing with TES solution for 2-3 times, suspending spores with 0.5mL TES solution, thermally shocking in 50 deg.C water bath for 10min, cooling with circulating water to room temperature, adding equal volumes of 2 × spore pre-germination culture solution (formula: Difco yeast extract 1%, Difco casein amino acid 1%, CaCl 20.1M (5M stock solution needs to be prepared, separately sterilizing at high temperature, adding into yeast extract/casein amino acid solution), and 0.5uM calcium chloride solution, culturing at 37 deg.C for 2.5 hr, performing pre-germination process, centrifuging to collect spores, rinsing spores with LB culture solution containing no antibiotic for 2 times, re-suspending the spores in appropriate amount of LB culture solution, shaking and scattering the spores on a mixer, and culturing according to 10mL⁷∶10⁸The donor-acceptor ratio of (1) and the cells of the Escherichia coli are mixed and then uniformly coated on an MS plate without antibiotics, the plate is transferred to an incubator at 30 ℃ after being dried and cultured for 17 hours, then the plate is taken out, 1.5mL of sterile water containing apramycin (0.5mg) and nalidixic acid (1mg) is covered on the MS plate, and the plate is dried and then transferred to the incubator at 30 ℃ for culture. And (3) generally, after 4-5 days, a single colony of a binder grows out on the plate, and finally, the correctness of the binder is verified by adopting V-ermEp-accA 2-F/R as a PCR primer, so that a mutant strain SY05 is obtained.

The primer sequences used in step 2 are shown in table 6:

TABLE 6

Example 4 fermentation culture of the original Strain Streptomyces spinosus SY04 and the mutant Strain SY05

The formula of a seed culture medium (TSBY) is 30g of Oxoid tryptone bean soup powder, 103g of sucrose and 5g of Difco yeast extract, and distilled water is added to the mixture to reach a constant volume of 1L.

The fermentation medium comprises corn flour 10g, soluble starch 20g, soybean cake powder 10g, KH₂PO4 0.2g， NaCl 3g，NH4Cl 3g，CaCO₃4g, adding distilled water to a constant volume of 1L, and adjusting the pH value to 7.0 by using 1M sodium hydroxide solution.

The seed culture medium and the fermentation culture medium prepared according to the formula are respectively filled in 500mL triangular flasks with springs, 100mL culture medium is respectively filled in each flask, and the mixture is sterilized at 121 ℃ for 20min under high pressure. Transferring a certain amount of original strain Streptomyces spinosus SY04 and mutant strain SY05 spore suspension into a seed culture medium, culturing for 24h in a shaker at 30 ℃ at a rotating speed of 220rpm to obtain a fermented seed liquid, finally transferring the fermented seed liquid into the fermented culture medium according to an inoculation ratio of 5%, culturing for 5 days in the shaker at 30 ℃ at the rotating speed of 220rpm, collecting the fermented culture liquid, and detecting and analyzing according to the following method.

After obtaining a fermentation culture product, firstly adding methanol solution with twice volume (210mL) into the fermentation culture product, uniformly mixing and carrying out ultrasonic treatment for 15min to obtain a fermentation extract. And (3) centrifuging 1mL of fermentation extract in a 1.5mL centrifuge tube at 12000rpm for 5-8 min, and filtering the obtained supernatant through a 0.22-micron organic filter membrane to obtain a fermentation product sample for HPLC and LC-MS analysis.

The apparatus for detecting tetramycin and derivatives thereof is American Agilent 1260 series high performance liquid chromatography, and the used analysis liquid chromatographic column is an Agilent Eclipse TC-C18 chromatographic column with specification of 4.6mm × 250mm and particle size of 5 μm. The mobile phase is 67% of water containing 0.1% of formic acid and 33% of acetonitrile, the flow rate is 0.4mL/min, the detection wavelength is 304nm, and the column temperature is 30 ℃.

FIG. 7 is a high performance liquid chromatography HPLC ultraviolet absorption chart of the tetramycin B derivative, as can be seen from FIG. 7, the maximum ultraviolet absorption wavelength of the tetramycin B derivative is 304 nm;

FIG. 8 is a spectrum of substance derived from tetramycin B, as shown in FIG. 8, by MS₂O+H]⁺、 [M+H]⁺And [ M + Na]⁺The molecular weight of the ion peak is completely matched with that of the corresponding ion peak of the tetramycin B derivative;

FIG. 6 is HPLC analysis diagram of fermentation liquid of original strain SY04 and mutant strain SY05, and FIG. 9 is bar chart of fermentation yield of tetramycin A, B derivative in original strain SY04 and SY 05. The strain SY05 obtained from the figure 6 and the figure 9 integrates the plasmid pJQK512, so that the tetramycin A derivative is converted into the tetramycin B derivative more, and the yield of the tetramycin B derivative is greatly improved;

table 7 shows the concrete fermentation data of the starting strain SY04 and the mutant strain SY 05;

TABLE 7

From table 7, it can be seen: the yield of the tetramycin B derivative in the mutant strain SY05 is improved by 40 percent compared with that of the original strain, and the mutant strain SY04 with an empty vector pSET 152: : the yield of the tetramycin B derivative in pSET152 was not significantly changed compared to strain SY04, further indicating that the improved yield of the tetramycin B derivative in strain SY05 is due to the enhanced supply of the polyketide chain extension unit precursor malonyl-CoA and the expression of the post-tetramycin biosynthesis gene cluster modification genes tetrK and tetrF.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> high-efficiency low-toxicity tetramycin B derivative and directed high-yield metabolic engineering method thereof

<130> DAG41389

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial sequence Δ Nys-L-F (Artificial sequence)

<400> 1

tctagagggt ggtggcgagg gagt 24

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence Δ Nys-L-R (Artificial sequence)

<400> 2

cttaaggcgg cgggtcagcg agctg 25

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence Δ Nys-R-F (Artificial sequence)

<400> 3

cttaaggtcg gcgtattggt tttc 24

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence Δ Nys-R (Artificial sequence)

<400> 4

aagcttacct gttgcacgtc gttc 24

<210> 5

<211> 1511

<212> DNA

<213> Artificial sequence Δ Nys-L (Artificial sequence)

<400> 5

tctagagggt ggtggcgagg gagtgggcga cgtcgagcag gtcggcgtgc ggggcgtcgt 60

cgagggcggc gcgcagggcc gcggcctggc cgcgcagggc gtcgggggtg cggccggaga 120

gcaggaccgg gacggtgccc ggcgtcacgg gggcggtggt cggcgtggtg tcggtgccgg 180

ccggggcgtg gggcgtcccg ggctcctcgg gcggtgcctc gggggcctgc tcgatgatgg 240

tgtgggcgtt ggtgccgctg atgccgaagg aggagacggc ggcgcggcgc ggggcgccgg 300

tggcgggcca gtcggtgggg tcggtgagca ggcgcaccgc gccctgcgac cagtcgacgt 360

gccgggtggg ttcctcggcg tgcagggtgc ggggcagcac gccgtgccgc atcgccagca 420

ccatcttgat gacgccggcg acgccggcgg cggcctgggt gtggctgagg ttggacttga 480

ccgagccgag cagcagcggc cggtccgcgg ggcggttctg gccgtaggtg gcgagcagtg 540

cctgggcctc gacggggtcg cccagggtgg tgccggtgcc gtgtgcctcg acgacgtcga 600

cgtccccggc ggtcagtcgg gcgttgacca gggcctgttg gatgacctgc tgctgggagg 660

ggccgttggg ggcggtcagg ccgttggagg cgccgtcctg gttgacggcg gtgccgcgga 720

cgacggcgag gacctcgtgg ccgttgcgga cggcgtcgga gaggcgttcc aggacgagga 780

tgccgacgcc ctcggcccag ccggtgccgt cggcggtgtc gccgaaggag cggcagcgac 840

cgtccgcgga cagtccgccc tggcggccca tctcgatgaa ggtgccgggg ccggacatga 900

tggtgacgcc gccggccagg gcgagggtgc actcgccggc ccgcagtgcc tgggtggcca 960

ggtggacggc gacgagcgag gaggagcagg cggtgtcgac ggtgacggcg gggccgaccg 1020

tgccgaaggt gtaggagatg cggccggaca gcacgctgcc ggtgttgccg gtgagttgga 1080

agccctcggc gccgtcggcg gggccgaccc ggtagtcctg cgccatggcg ccgatgaaga 1140

cgccggtgcg gctgcccttc acggacgtcg ggtcgatgcc ggcgcgttcg aacgcctccc 1200

aggcggattc caggacgacg cgctgctgcg ggtccatggc gaccgcctcg cgcggcgaga 1260

tgccgaagaa ctccgcgtcg aagtcggggg cgtcgtgcag gaatccgccg gctgcggtcg 1320

gtgcggtggc gagtgcctcc aggtcccagc cgcggtcggt ggggaacggg ctgacgccgt 1380

gttcgccggc cgcgaccagt cgccagaggt cttcggggct gcggacgcct ccggggtagc 1440

ggcaggtcat gccgatgatg gccaccggtt cctgggccgc cgattccagc tcgctgaccc 1500

gccgccttaa g 1511

<210> 6

<211> 1698

<212> DNA

<213> Artificial sequence Nys-R (Artificial sequence)

<400> 6

cttaaggtcg gcgtattggt tttcgcggga aacctgcgcc aggtcggaat cgaccatcat 60

gcgcatgagg gcggggaaat cgacgtccgg tttccagccg aggcggtcgc gggccttggc 120

gctgtcggcg cacagcacct cgacctcggc gggccgcacc aggtcggggt cgatgacgac 180

gtggtcctgc cagttcaggc cgacgtgttc gaaggcgagc cggaccgcgt cgcgcaccga 240

gtgcatctgg ccggtgccga tgacgtagtc gtcgccggcg tcctgctgga gcatcaggtg 300

catggcgcgg acgtagtcgc cggcgtagcc ccagtcgcgg accgcgtcga ggttgcccag 360

ggagagcttg tcctggaggc cgtgcttgat gcgggcgacc gcgagggaga tcttgcgggt 420

gacgaactcc tggccgcggc gcggggactc gtggttgaag agcatcccgg agaccgcgta 480

catgccgaag gactcgcggt agttgcgggt gatgtagtgg ccgtacgcct tggccgcgcc 540

gtacgggctg cgcggatgga agagcgtggt ctcgcgctgc ggggtctccg ccgccttgcc 600

gaacatctcc gaggaggacg cctggtagaa gcggatctga ccgcgcgggt tggcggtgcg 660

ggaggtggac aggccgctga ccatgcggat ggcctccagc atccgcagca cgcccatgcc 720

gttgacctcg gtgacgagtt ccgcctgctg ccaggacatc gggacgaacg agatcgcgcc 780

gaggttgtag acctcgtcgg gctgcacgcc gtcgaccgcg gagaccaggc tcccctggtc 840

catcaggtcg ccgtcgatga agttcagttc ggtggcgagc cggctgacgc gggacttgcg 900

ggggttcgcc tggccgcgga tcagacccca cacctggtag ccccgcgaca gcaggtgctc 960

cgcgagatag gagccgtcct ggccggtgat tccggtgatc agcgctcgtt tggacatggc 1020

caacccttct cgatcacgaa agagaccgtc caaaccctgg agctcggcca cgacctggcg 1080

tcgaatacgc agtccggacg atagataccg gcacgctcgc aagccgcgga agacgctgtc 1140

aacgcatccc ggagttttta gggaattccc gagttccgga ccgtcaggag tgcgccgtcg 1200

tcgccaacac tagtgagatt tcggtgcgga atgcggtgca atgacgtggg tcggcggaac 1260

ggccgaccgc gtccaccacc gagcggcccg gcgccgccgt caaccggggt gttcctccgt 1320

gctgctgaga ctcctgcggg cgcagttgcg cccctacgcg ggggccacct ccgccctcgt 1380

cgccctccaa ctcgtccaga tcctcggcac gttgctgctg ccgacgctgg gtgccgcgct 1440

catcgaccag ggcgtggtgc gcgccgaccg cgatcgggtg gccgagctgg gggcggtgat 1500

ggtggcggtg gccgtggtgc agatcgcggc ggcgctcggg gcggcggcgt tggcggcgcg 1560

gacctcgacg gcgatgggcc gcgacctgcg gtccgcggtg ttccgccgca tcctggactt 1620

ctccgcccgc gaggtcgggc ggttcggcac gccgtcgctg ctcacccgtg ccgtgaacga 1680

cgtgcaacag gtaagctt 1698

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence V-. DELTA. Nys-F (Artificial sequence)

<400> 7

cgttcgaacg cctcccag 18

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence V-. DELTA. Nys-R (Artificial sequence)

<400> 8

cgatttcccc gccctcat 18

<210> 9

<211> 1526

<212> DNA

<213> Artificial sequence C340A-tetrG-L (Artificial sequence)

<400> 9

gaattcacac cacgaccagt tcgcaggcga gcgccgcccg cacccgctcg gcgagcgcgg 60

gcgcgaccgc ggcgcccgcc gtccgcacct ggtggggggc gaaccccgcc gacccctccg 120

acgactccaa ccggcccatg acgtcccaca gttgcgtggg gacggcgaag acgacgcgcg 180

ggtcgtgctc ccgggccagg gcgaggaaac ggtccgcgtc ccagtcggtg agcacgacct 240

ggcggcaggc ggtggacagc gccgcgtgca cggcctggag gccgaacaga tgggtcatcg 300

ggcaggccgt gaggaccgtg cccgcgaagg cgtcggcggc ctcggcggtc acggccgcgg 360

tgttcgacag caggccgtcg tggctgtgca gacacagcct gggccgcggt gaggccgtcc 420

cggacgacgg gaccaggacg aacggaaggt cgggggtgac ctccaccggc cggggcccgc 480

tgcccgccca ggccgccagc agcccgtcca acgaaccgat ccccggctcc gcaccgccgg 540

tcgccggccc gccggccagc aacaccccgc gcagcgacgg cacgccgtcc aacagcgcac 600

gggccgtcgc cggatcctcg ccctcggcgc ccgacaggac gaggaggacg ggctccgcgc 660

ggtccagcag tgcgcgcacc tcgcccaccg cggtgccctg gtgcaggggc agcagcaccg 720

ccccgacggc cgcgaccgcc aggtgcaggg tctgcaactc ccagctgttc ggcagccgca 780

ccgccaccac gtcgccgggc gcgacgtgcg actcctggag gccgcgggcc agcgcgtcga 840

cgtccgcccg ccactcggcc cacgtccacg agcggtcccc gtcgacgagg gccaccgcat 900

cgggcgcgga cgccacggcg gacgcgaaca cctcggggag cgtgcggccc cccggtgtgc 960

cggtgccgtc ggacggcggg tcgatcacat agtcatcggt gtggacgggc accatcactc 1020

ccggatgtcg agcgcttgcg tcggttgggg ggatccggca ggtggatcgc ccggtgcggg 1080

cgtcagccct cgtgtgcggt gatcgccccg gaggggcaca gcgccgccgc gatgcggacc 1140

ttggccgcct cgtcggggcc gggcgcggcg agcagggtga cgatcccgtc ctcgtcctgg 1200

tcgaagacct gtggcgcgga cagcacgcac tggccggcgc ccacgcagcg ctccgaatcg 1260

atggtgatac gcatgccgat gtctctcctg caaggggatc ggggtccacc ggcggctact 1320

ggcgcaccgg gcggacggga ctggccttcc gctggcggga cggtgcctac cagcggacgg 1380

gaagctgctc caggccgtag agcacgccgt cgtacttgat gctcaaaccc tcatcgggaa 1440

cggcgagttg gatgtcggga atgcgctcga agagcttgcg gtaggcgatc tccatctcga 1500

cccgcaccag gttctgcccc aaagct 1526

<210> 10

<211> 1477

<212> DNA

<213> Artificial sequence C340A-tetrG-R (Artificial sequence)

<400> 10

aagcttggtg gatcccgtag ccgaacgcca cgtgatgacg ggcgctccgg gaggggtcga 60

acatgtgcgg gcactcgaag gcgctcgtgt cgtggttggc cgcggcgatc aacggcacga 120

tgccgtcgcc cgccttgatg agctgcccgc cgatctcgac gtcctcgacg gccacccgca 180

gggacacgat gtcggcgacc gagtggaagc gcagggtctc ctccaccgcc cggtcgtcac 240

cgatccactg ggggttccgc agcagcgtga cgacgcccag cgcgatgttg ttggccgtgg 300

tctcgtggcc ggcgatgagc agcagcatca gcacgcccga cagctcgtgc ggtgcgatcg 360

aaccggcggc cagcagccgg ctgatgaggt cgtcgccggg ccacttcgcc ttgatgccca 420

ccagccgctt gatgtagcgc agcagctcct tgacggcggt ctcgcgctgc gcgtcggtcg 480

aggcgcggag cgagaccagg gtgcgggtcc gggactcgaa gaagtcgcgg tcggagggcg 540

gcacgccgag cagtgtcgag atcaccaggg acggcacggg cagtgcgtag tccgcgacga 600

ggtcggcgga gttgcccgcg gccagcatcg cgtcgaggcg ctcctccacc gtgcgctcga 660

tcgccgggcg caacccccgg atgcgccgca cggtgaattc ggggatgagg gccttgcgga 720

accggtcgtg ttcgggggag tccaacccca cgaaccagcc ggggatctgc tcctgcgtgg 780

gcacgcccac cgtcccgacg ttggggaagc ccttgtgcga ggggttggag ctgatcctgg 840

agtcggtcag cacggcgcgc acgtcctcgt gccgggtgac cagccacgcc cgggcgccgt 900

tgggcaggcg cgagaggacc agcccctcgt ggtcgcggta cccgtcgtag tcgggcggcg 960

ggaagggcac gccgggtttg cgggtgggga agtccaccac caccgggtcc gagtgcgtca 1020

tgaacggaat ttcctctctc cgacggggtc gatcaacgga cgccgtagaa ggcccggatc 1080

ttgctgacga tgaagtcctg gtcggcgggg gacaggtcgg tgtgcgtggg caggtagagc 1140

ccgtcctcgg cgaactcgct ggccttgagc gacggccagt cggggtcgaa gtacatgggc 1200

tgccggctca tcggcttgaa gaagagccgg gtctcgatgg agtggtcggc caggaacgcc 1260

tggagctccg cccggcgttc cgcccggagg tcgtacatcc acagcacgtc ccgcgggggc 1320

atcagcgtga tgccggggac gtcggccagc ccctcgtcgt agtgcttctc gatcctgcgg 1380

cgcacttcga ggatctcgtc gaggcgttcg gtctgcgcca gggccacggc ggcctgcatg 1440

gccgtgatgc ggtagttgta cgccaacttc ttctaga 1477

<210> 11

<211> 26

<212> DNA

<213> Artificial sequence C340A-tetrG-L-F (Artificial sequence)

<400> 11

gaattcacac cacgaccagt tcgcag 26

<210> 12

<211> 26

<212> DNA

<213> Artificial sequence C340A-tetrG-L-R (Artificial sequence)

<400> 12

aagctttggg gcagaacctg gtgcgg 26

<210> 13

<211> 26

<212> DNA

<213> Artificial sequence C340A-tetrG-R-F (Artificial sequence)

<400> 13

aagcttggtg gatcccgtag ccgaac 26

<210> 14

<211> 26

<212> DNA

<213> Artificial sequence C340A-tetrG-R (Artificial sequence)

<400> 14

tctagaagaa gttggcgtac aactac 26

<210> 15

<211> 1176

<212> DNA

<213> Strain Streptomyces hygroscopicus Beijing variant tetrG gene (Streptomyces hygrospinosus var. beijingensis tetrG)

<400> 15

atgacgcact cggacccggt ggtggtggac ttccccaccc gcaaacccgg cgtgcccttc 60

ccgccgcccg actacgacgg gtaccgcgac cacgaggggc tggtcctctc gcgcctgccc 120

aacggcgccc gggcgtggct ggtcacccgg cacgaggacg tgcgcgccgt gctgaccgac 180

tccaggatca gctccaaccc ctcgcacaag ggcttcccca acgtcgggac ggtgggcgtg 240

cccacgcagg agcagatccc cggctggttc gtggggttgg actcccccga acacgaccgg 300

ttccgcaagg ccctcatccc cgaattcacc gtgcggcgca tccgggggtt gcgcccggcg 360

atcgagcgca cggtggagga gcgcctcgac gcgatgctgg ccgcgggcaa ctccgccgac 420

ctcgtcgcgg actacgcact gcccgtgccg tccctggtga tctcgacact gctcggcgtg 480

ccgccctccg accgcgactt cttcgagtcc cggacccgca ccctggtctc gctccgcgcc 540

tcgaccgacg cgcagcgcga gaccgccgtc aaggagctgc tgcgctacat caagcggctg 600

gtgggcatca aggcgaagtg gcccggcgac gacctcatca gccggctgct ggccgccggt 660

tcgatcgcac cgcacgagct gtcgggcgtg ctgatgctgc tgctcatcgc cggccacgag 720

accacggcca acaacatcgc gctgggcgtc gtcacgctgc tgcggaaccc ccagtggatc 780

ggtgacgacc gggcggtgga ggagaccctg cgcttccact cggtcgccga catcgtgtcc 840

ctgcgggtgg ccgtcgagga cgtcgagatc ggcgggcagc tcatcaaggc gggcgacggc 900

atcgtgccgt tgatcgccgc ggccaaccac gacacgagcg ccttcgagtg cccgcacatg 960

ttcgacccct cccggagcgc ccgtcatcac gtggcgttcg gctacgggat ccaccagtgc 1020

ctggggcaga acctggtgcg ggtcgagatg gagatcgcct accgcaagct cttcgagcgc 1080

attcccgaca tccaactcgc cgttcccgat gagggtttga gcatcaagta cgacggcgtg 1140

ctctacggcc tggagcagct tcccgtccgc tggtag 1176

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence V-C340A-tetrG-F (Artificial sequence)

<400> 16

gaccaggacg aacggaaggt 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence V-C340A-tetrG-R (Artificial sequence)

<400> 17

gtgctgatgc tgctgctcat 20

<210> 18

<211> 1318

<212> DNA

<213> Artificial sequence KasOp _ -tetrK (Artificial sequence)

<400> 18

tctagaggta cccaattgtg ttcacattcg aacggtctct gctttgacaa catgctgtgc 60

ggtgttgtaa agtcgtggcc aggagaatac gacagcgtgc aggactgggg gagttatgac 120

ctccccccac cgtgatctgc cgtccctcga cctcgaaacg cccgccctac tgcgcgtcag 180

cccgctcctg cgggacctcc aggaacgggg gccggtctgc ctggtgcgca cccccgccgg 240

ggacgagggc tggctggtga cccgccactc cgtgctcaag cagctgctga acgacgaacg 300

catcggccat tcgcatccgg acccggcgaa cgcggcgcag tacgtgcgca acccgttcct 360

ggacctgatg atcgccgaca ccgacgccga gaccgcccgt cgcacgcaca ccgagtcccg 420

ccggctcctg gccccgatgt tctccgcccg gcgggtgcgc gagatggagc cgcgggtagc 480

ggcggtcgtg gacgccgtgc tggacgactt caccgcccag gagccgcccg gcgacctgca 540

cggcggcgtg tccgtgccgg tggcgaggac ggtgctctgc gacatcatcg gcgtgccgcc 600

gcagaaccgc gaacacctca cggcgctgct gtcccagacg gccgtgctcg gggaccgcga 660

gggggtgcag cgcacgcagc gcgacctgta cgccttcgtc ggggggttgg tcgagcacaa 720

gcgggccgca ccgggccagg acatcatcac ccgcctggcc gagggcgggc tgtccgacga 780

gcgcgtgacg cacctggccg tgggcctgct gttcgccggg ctggacagcg tcgtgaccat 840

catggaccac ggggtggtgc tgctggcgac ccaccccgag cagcgggcgg cggcactggc 900

ggacccggac gtgatgacgc acgccgtcga ggaagtgctg cgggccgcga aagccggcgg 960

gtcgatcctg ccgcgctacg ccaccgagga cctgacggtc ggcggagaga cgatccgggc 1020

cggggacctg gtcctgttcg acttcagtct ccccaacttc gacgagcggg ccttcgacga 1080

gccggagcgg ttcgacgtca cccggagtcc caacccgcat ctgaccttcg cgcacgggat 1140

gtggcactgc atcggcgcac ccctggcgcg gatcgagctg aacacggtct tcacccagtt 1200

gttcacgcgc ctgcccgacc tgcgactggc gctgccggcg ggcgaactgg cggagaacga 1260

aggccggttg tccggcgggc tcagtgagct gccggtgacc tggtagctta agggatcc 1318

<210> 19

<211> 301

<212> DNA

<213> Artificial sequence KasOp _ -tetrF (Artificial sequence)

<400> 19

cttaagtgtt cacattcgaa cggtctctgc tttgacaaca tgctgtgcgg tgttgtaaag 60

tcgtggccag gagaatacga cagcgtgcag gactggggga gttatgcgta tcaccatcga 120

ttcggagcgc tgcgtgggcg ccggccagtg cgtgctgtcc gcgccacagg tcttcgacca 180

ggacgaggac gggatcgtca ccctgctcgc cgcgcccggc cccgacgagg cggccaaggt 240

ccgcatcgcg gcggcgctgt gcccctccgg ggcgatcacc gcacacgagg gctgaggatc 300

c 301

<210> 20

<211> 1918

<212> DNA

<213> Artificial Sequence ermEp-accA 2(Artificial Sequence)

<400> 20

tctagaggta ccgctagccg cggtcgatct tgacggctgg cgagaggtgc ggggaggatc 60

tgaccgacgc ggtccacacg tggcaccgcg atgctgttgt gggcacaatc gtgccggttg 120

gtaggatcca gcggtgcgca aggtgctcat cgccaatcgt ggcgaaatcg ctgtccgcgt 180

ggcccgggcc tgccgggacg ccgggatcgc gagcgtggcc gtctacgcgg atccggaccg 240

ggacgcgttg cacgtccgtg ccgctgatga ggcgttcgcc ctgggtggtg acacccccgc 300

gaccagctat ctggacatcg ccaaggtcct caaagccgcg cgcgagtcgg gcgcggacgc 360

catccacccc ggctacgggt tcctctcgga gaacgccgag ttcgcgcagg cggtcctgga 420

cgccggcctg atctggatcg gcccgccccc gcacgccatc cgcgacctgg gcgacaaggt 480

cgccgcccgc cacatcgccc agcgggccgg cgcccccctg gtcgccggca cccccgaccc 540

cgtctccggc gcggacgagg tcgtcgcctt cgccaaggag cacggcctgc ccatcgccat 600

caaggccgcc ttcggcggcg gcgggcgcgg cctcaaggtc gcccgcaccc tcgaagaggt 660

gccggagctg tacgactccg ccgtccgcga ggccgtggcc gccttcggcc gcggggagtg 720

cttcgtcgag cgctacctcg acaagccccg ccacgtggag acccagtgcc tggccgacac 780

ccacggcaac gtggtcgtcg tctccacccg cgactgctcc ctccagcgcc gccaccaaaa 840

gctcgtcgag gaggcccccg cgcccttcct ctccgaggcc cagacggagc agctgtactc 900

atcctccaag gccatcctga aggaggccgg ctacgtcggc gccggcaccg tggagttcct 960

cgtcggcatg gacggcacga tctccttcct ggaggtcaac acccgcctcc aggtcgagca 1020

cccggtcacc gaggaagtcg ccggcatcga cctggtccgc gagatgttcc gcatcgccga 1080

cggcgaggaa ctcggctacg acgaccccgc cctgcgcggc cactccttcg agttccgcat 1140

caacggcgag gaccccggcc gcggcttcct gcccgccccc ggcaccgtca ccctcttcga 1200

cgcgcccacc ggccccggcg tccgcctgga cgccggcgtc gagtccggct ccgtcatcgg 1260

ccccgcctgg gactccctcc tcgccaaact gatcgtcacc ggccgcaccc gcgccgaggc 1320

actccagcgc gcggcccgcg ccctggacga gttcaccgtc gagggcatgg ccaccgccat 1380

ccccttccac cgcacggtcg tccgcgaccc ggccttcgcc cccgaactca ccggctccac 1440

ggaccccttc accgtccaca cccggtggat cgagacggag ttcgtcaacg agatcaagcc 1500

cttcaccacg cccgccgaca ccgagacgga cgaggagtcg ggccgggaga cggtcgtcgt 1560

cgaggtcggc ggcaagcgcc tggaagtctc cctcccctcc agcctgggca tgtccctggc 1620

ccgcaccggc ctggccgccg gggcccgccc caagcgccgc gcggccaaga agtccggccc 1680

cgccgcctcg ggcgacaccc tcgcctcccc gatgcagggc acgatcgtca agatcgccgt 1740

cgaggagggc caggaagtcc aggaaggcga cctcatcgtc gtactcgagg cgatgaagat 1800

ggaacagccc ctcaacgccc acaggtccgg caccatcaag ggcctcaccg ccgaggtcgg 1860

cgcctccctc acctccggcg ccgccatctg cgagatcaag gactgaagat ctccatgg 1918

<210> 21

<211> 1944

<212> DNA

<213> Artificial sequence ermEp-accBE (Artificial sequence)

<400> 21

agatctgcta gccgcggtcg atcttgacgg ctggcgagag gtgcggggag gatctgaccg 60

acgcggtcca cacgtggcac cgcgatgctg ttgtgggcac aatcgtgccg gttggtagga 120

tccagcgatg accgttttgg atgaggcgcc gggcgagccg acggacgcgc gcgggcgggt 180

ggccgagctg cacgggatcc gtgcagcggc gctcgccggg ccgagtgaga aggcgacggc 240

ggcgcagcac gccaagggca agctgacggc acgtgagcgc atcgagctgc tcctggaccc 300

cggctccttc cgcgaggtcg agcagctgcg ccggcaccgg gcgaccgggt tcggcctgga 360

ggccaagaag ccgtacaccg acggtgtcat caccggctgg ggcacggtcg agggccgcac 420

ggtcttcgtc tacgcccacg acttccggat cttcggcggc gcgctgggcg aggcccacgc 480

cacgaagatc cacaagatca tggacatggc catcgcggcc ggtgccccgc tggtgtcgct 540

gaacgacggc gccggcgccc gtatccagga gggcgtcagc gcgctcgccg ggtacggcgg 600

catcttccag cgcaacacca aggcgtccgg cgtcatcccg cagatcagcg tgatgctcgg 660

cccctgcgcg ggcggcgcgg cctacagccc cgccctcacc gacttcgtct tcatggtccg 720

cgacacctcg cagatgttca tcacgggccc ggacgtcgtc aaggcggtca ccggcgagga 780

gatcacgcag aacggtctgg gcggcgccga cgtgcacgcc gagacgtccg gcgtgtgcca 840

cttcgcctac gacgacgagg agacctgcct cgccgaggtc cgctacctcc tctccctcct 900

cccgcagaac aaccgggaga acccgccccg cgccgagtcc tccgaccccg tggaccgccg 960

ctcggacacc ctcctcgacc tggtcccggc ggacggcaac cgcccgtacg acatgaccaa 1020

ggtcatcgag gaactcgtcg acgagggcga gtacctggag gtccacgagc gttgggcccg 1080

caacatcatc tgcgcgctgg cccgtctcga cgggcgggtc gtgggcatcg tcgccaacca 1140

gccgcaggcc ctggccggtg tcctggacat cgaggcgtcg gagaaggcgg cccgcttcgt 1200

ccagatgtgc gacgccttca acatcccgat catcactctt ctggacgtac ccggcttcct 1260

gcccggcgtc gaccaggagc acggcgggat catccgccac ggcgccaagc tgctctacgc 1320

gtactgcaac gcgaccgtgc cccggatctc gctgatcctg cgcaaggcgt acggaggtgc 1380

ttacatcgtc atggacagcc agtccatcgg cgccgacctc acctacgcct ggccgaccaa 1440

cgagatcgcc gtcatgggcg cggaaggtgc cgcgaacgtc atcttccgcc ggcagatcgc 1500

cgacgccgag gaccccgagg ccatgcgggc gcgcatggtc aaggagtaca agtccgagct 1560

gatgcacccc tactacgcgg ccgaacgcgg tctggtcgac gacgtcatcg accccgccga 1620

aacccgcgag gtgctgatca cgtccctggc gatgctccac accaagcacg ccgacctgcc 1680

ctcccgcaag cacggcaacc cgccgcagtg acccgaggag ccacttccat gtcccctgcc 1740

gacatccgcg tcgagaaggg ccacgccgag cccgaggaag tcgccgccat cacggccctc 1800

ctcctggccc gcgccgccgc ccgccccgcc gagatcgcgc cgacccacgg cggcggccgc 1860

gcccgcgccg gctggcgccg cctggaacgc gagccgggct tccgcgcccc gcacagctgg 1920

cgctgacaat tgtctagacc atgg 1944

<210> 22

<211> 38

<212> DNA

<213> Artificial sequence KasOp _ -tetrK-1-F (Artificial sequence)

<400> 22

tctagaggta cccaattgtg ttcacattcg aacggtct 38

<210> 23

<211> 40

<212> DNA

<213> Artificial sequence KasOp _ -tetrK-1-R (Artificial sequence)

<400> 23

tcacggtggg gggaggtcat aactccccca gtcctgcacg 40

<210> 24

<211> 40

<212> DNA

<213> Artificial sequence KasOp _ -tetrK-2-F (Artificial sequence)

<400> 24

cgtgcaggac tgggggagtt atgacctccc cccaccgtga 40

<210> 25

<211> 32

<212> DNA

<213> Artificial sequence KasOp _ -tetrK-2-R (Artificial sequence)

<400> 25

ggatccctta agctaccagg tcaccggcag ct 32

<210> 26

<211> 26

<212> DNA

<213> Artificial sequence KasOp _ -tetrF-1-F (Artificial sequence)

<400> 26

cttaagtgtt cacattcgaa cggtct 26

<210> 27

<211> 40

<212> DNA

<213> Artificial sequence KasOp _ -tetrF-1-R (Artificial sequence)

<400> 27

gaatcgatgg tgatacgcat aactccccca gtcctgcacg 40

<210> 28

<211> 40

<212> DNA

<213> Artificial sequence KasOp _ -tetrF-2-F (Artificial sequence)

<400> 28

cgtgcaggac tgggggagtt atgcgtatca ccatcgattc 40

<210> 29

<211> 26

<212> DNA

<213> Artificial sequence KasOp _ -tetrF-2-R (Artificial sequence)

<400> 29

ggatcctcag ccctcgtgtg cggtga 26

<210> 30

<211> 32

<212> DNA

<213> Artificial sequence ermEp-accA 2-1-F (artificial sequence)

<400> 30

tctagaggta ccgctagccg cggtcgatct tg 32

<210> 31

<211> 40

<212> DNA

<213> Artificial sequence ermEp-accA 2-1-R (artificial sequence)

<400> 31

gcgatgagca ccttgcgcac cgctggatcc taccaaccgg 40

<210> 32

<211> 40

<212> DNA

<213> Artificial sequence ermEp-accA 2-2-F (artificial sequence)

<400> 32

ccggttggta ggatccagcg gtgcgcaagg tgctcatcgc 40

<210> 33

<211> 32

<212> DNA

<213> Artificial sequence ermEp-accA 2-2-R (artificial sequence)

<400> 33

ccatggagat cttcagtcct tgatctcgca ga 32

<210> 34

<211> 26

<212> DNA

<213> Artificial sequence ermEp-accBE-1-F (artificial sequence)

<400> 34

agatctgcta gccgcggtcg atcttg 26

<210> 35

<211> 40

<212> DNA

<213> Artificial sequence ermEp-accBE-1-F (artificial sequence)

<400> 35

gcctcatcca aaacggtcat cgctggatcc taccaaccgg 40

<210> 36

<211> 40

<212> DNA

<213> Artificial sequence ermEp-accBE-2-F (artificial sequence)

<400> 36

ccggttggta ggatccagcg atgaccgttt tggatgaggc 40

<210> 37

<211> 38

<212> DNA

<213> Artificial sequence ermEp-accBE-2-R (artificial sequence)

<400> 37

ccatggtcta gacaattgtc agcgccagct gtgcgggg 38

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence V-ermEp-accA 2-F (artificial sequence)

<400> 38

aaacgacggc cagtgccaag 20

<210> 39

<211> 22

<212> DNA

<213> Artificial sequence V-ermEp-accA 2-R (artificial sequence)

<400> 39

ttctccgaga ggaacccgta gc 22

<210> 40

<211> 1773

<212> DNA

<213> Streptomyces coelicolor M152 accA2 gene (Streptomyces coelicolor)

<400> 40

gtgcgcaagg tgctcatcgc caatcgtggc gaaatcgctg tccgcgtggc ccgggcctgc 60

cgggacgccg ggatcgcgag cgtggccgtc tacgcggatc cggaccggga cgcgttgcac 120

gtccgtgccg ctgatgaggc gttcgccctg ggtggtgaca cccccgcgac cagctatctg 180

gacatcgcca aggtcctcaa agccgcgcgc gagtcgggcg cggacgccat ccaccccggc 240

tacgggttcc tctcggagaa cgccgagttc gcgcaggcgg tcctggacgc cggcctgatc 300

tggatcggcc cgcccccgca cgccatccgc gacctgggcg acaaggtcgc cgcccgccac 360

atcgcccagc gggccggcgc ccccctggtc gccggcaccc ccgaccccgt ctccggcgcg 420

gacgaggtcg tcgccttcgc caaggagcac ggcctgccca tcgccatcaa ggccgccttc 480

ggcggcggcg ggcgcggcct caaggtcgcc cgcaccctcg aagaggtgcc ggagctgtac 540

gactccgccg tccgcgaggc cgtggccgcc ttcggccgcg gggagtgctt cgtcgagcgc 600

tacctcgaca agccccgcca cgtggagacc cagtgcctgg ccgacaccca cggcaacgtg 660

gtcgtcgtct ccacccgcga ctgctccctc cagcgccgcc accaaaagct cgtcgaggag 720

gcccccgcgc ccttcctctc cgaggcccag acggagcagc tgtactcatc ctccaaggcc 780

atcctgaagg aggccggcta cgtcggcgcc ggcaccgtgg agttcctcgt cggcatggac 840

ggcacgatct ccttcctgga ggtcaacacc cgcctccagg tcgagcaccc ggtcaccgag 900

gaagtcgccg gcatcgacct ggtccgcgag atgttccgca tcgccgacgg cgaggaactc 960

ggctacgacg accccgccct gcgcggccac tccttcgagt tccgcatcaa cggcgaggac 1020

cccggccgcg gcttcctgcc cgcccccggc accgtcaccc tcttcgacgc gcccaccggc 1080

cccggcgtcc gcctggacgc cggcgtcgag tccggctccg tcatcggccc cgcctgggac 1140

tccctcctcg ccaaactgat cgtcaccggc cgcacccgcg ccgaggcact ccagcgcgcg 1200

gcccgcgccc tggacgagtt caccgtcgag ggcatggcca ccgccatccc cttccaccgc 1260

acggtcgtcc gcgacccggc cttcgccccc gaactcaccg gctccacgga ccccttcacc 1320

gtccacaccc ggtggatcga gacggagttc gtcaacgaga tcaagccctt caccacgccc 1380

gccgacaccg agacggacga ggagtcgggc cgggagacgg tcgtcgtcga ggtcggcggc 1440

aagcgcctgg aagtctccct cccctccagc ctgggcatgt ccctggcccg caccggcctg 1500

gccgccgggg cccgccccaa gcgccgcgcg gccaagaagt ccggccccgc cgcctcgggc 1560

gacaccctcg cctccccgat gcagggcacg atcgtcaaga tcgccgtcga ggagggccag 1620

gaagtccagg aaggcgacct catcgtcgta ctcgaggcga tgaagatgga acagcccctc 1680

aacgcccaca ggtccggcac catcaagggc ctcaccgccg aggtcggcgc ctccctcacc 1740

tccggcgccg ccatctgcga gatcaaggac tga 1773

<210> 41

<211> 1584

<212> DNA

<213> Streptomyces coelicolor M152 accB gene (Streptomyces coelicolor)

<400> 41

atgaccgttt tggatgaggc gccgggcgag ccgacggacg cgcgcgggcg ggtggccgag 60

ctgcacggga tccgtgcagc ggcgctcgcc gggccgagtg agaaggcgac ggcggcgcag 120

cacgccaagg gcaagctgac ggcacgtgag cgcatcgagc tgctcctgga ccccggctcc 180

ttccgcgagg tcgagcagct gcgccggcac cgggcgaccg ggttcggcct ggaggccaag 240

aagccgtaca ccgacggtgt catcaccggc tggggcacgg tcgagggccg cacggtcttc 300

gtctacgccc acgacttccg gatcttcggc ggcgcgctgg gcgaggccca cgccacgaag 360

atccacaaga tcatggacat ggccatcgcg gccggtgccc cgctggtgtc gctgaacgac 420

ggcgccggcg cccgtatcca ggagggcgtc agcgcgctcg ccgggtacgg cggcatcttc 480

cagcgcaaca ccaaggcgtc cggcgtcatc ccgcagatca gcgtgatgct cggcccctgc 540

gcgggcggcg cggcctacag ccccgccctc accgacttcg tcttcatggt ccgcgacacc 600

tcgcagatgt tcatcacggg cccggacgtc gtcaaggcgg tcaccggcga ggagatcacg 660

cagaacggtc tgggcggcgc cgacgtgcac gccgagacgt ccggcgtgtg ccacttcgcc 720

tacgacgacg aggagacctg cctcgccgag gtccgctacc tcctctccct cctcccgcag 780

aacaaccggg agaacccgcc ccgcgccgag tcctccgacc ccgtggaccg ccgctcggac 840

accctcctcg acctggtccc ggcggacggc aaccgcccgt acgacatgac caaggtcatc 900

gaggaactcg tcgacgaggg cgagtacctg gaggtccacg agcgttgggc ccgcaacatc 960

atctgcgcgc tggcccgtct cgacgggcgg gtcgtgggca tcgtcgccaa ccagccgcag 1020

gccctggccg gtgtcctgga catcgaggcg tcggagaagg cggcccgctt cgtccagatg 1080

tgcgacgcct tcaacatccc gatcatcact cttctggacg tacccggctt cctgcccggc 1140

gtcgaccagg agcacggcgg gatcatccgc cacggcgcca agctgctcta cgcgtactgc 1200

aacgcgaccg tgccccggat ctcgctgatc ctgcgcaagg cgtacggagg tgcttacatc 1260

gtcatggaca gccagtccat cggcgccgac ctcacctacg cctggccgac caacgagatc 1320

gccgtcatgg gcgcggaagg tgccgcgaac gtcatcttcc gccggcagat cgccgacgcc 1380

gaggaccccg aggccatgcg ggcgcgcatg gtcaaggagt acaagtccga gctgatgcac 1440

ccctactacg cggccgaacg cggtctggtc gacgacgtca tcgaccccgc cgaaacccgc 1500

gaggtgctga tcacgtccct ggcgatgctc cacaccaagc acgccgacct gccctcccgc 1560

aagcacggca acccgccgca gtga 1584

<210> 42

<211> 198

<212> DNA

<213> Streptomyces coelicolor M152 accE gene (Streptomyces coelicolor)

<400> 42

atgtcccctg ccgacatccg cgtcgagaag ggccacgccg agcccgagga agtcgccgcc 60

atcacggccc tcctcctggc ccgcgccgcc gcccgccccg ccgagatcgc gccgacccac 120

ggcggcggcc gcgcccgcgc cggctggcgc cgcctggaac gcgagccggg cttccgcgcc 180

ccgcacagct ggcgctga 198

<210> 43

<211> 1191

<212> DNA

<213> Streptomyces hygroscopicus Beijing variant tetrK gene (Streptomyces hygrospinus var. beijingensis tetrK)

<400> 43

atgacctccc cccaccgtga tctgccgtcc ctcgacctcg aaacgcccgc cctactgcgc 60

gtcagcccgc tcctgcggga cctccaggaa cgggggccgg tctgcctggt gcgcaccccc 120

gccggggacg agggctggct ggtgacccgc cactccgtgc tcaagcagct gctgaacgac 180

gaacgcatcg gccattcgca tccggacccg gcgaacgcgg cgcagtacgt gcgcaacccg 240

ttcctggacc tgatgatcgc cgacaccgac gccgagaccg cccgtcgcac gcacaccgag 300

tcccgccggc tcctggcccc gatgttctcc gcccggcggg tgcgcgagat ggagccgcgg 360

gtagcggcgg tcgtggacgc cgtgctggac gacttcaccg cccaggagcc gcccggcgac 420

ctgcacggcg gcgtgtccgt gccggtggcg aggacggtgc tctgcgacat catcggcgtg 480

ccgccgcaga accgcgaaca cctcacggcg ctgctgtccc agacggccgt gctcggggac 540

cgcgaggggg tgcagcgcac gcagcgcgac ctgtacgcct tcgtcggggg gttggtcgag 600

cacaagcggg ccgcaccggg ccaggacatc atcacccgcc tggccgaggg cgggctgtcc 660

gacgagcgcg tgacgcacct ggccgtgggc ctgctgttcg ccgggctgga cagcgtcgtg 720

accatcatgg accacggggt ggtgctgctg gcgacccacc ccgagcagcg ggcggcggca 780

ctggcggacc cggacgtgat gacgcacgcc gtcgaggaag tgctgcgggc cgcgaaagcc 840

ggcgggtcga tcctgccgcg ctacgccacc gaggacctga cggtcggcgg agagacgatc 900

cgggccgggg acctggtcct gttcgacttc agtctcccca acttcgacga gcgggccttc 960

gacgagccgg agcggttcga cgtcacccgg agtcccaacc cgcatctgac cttcgcgcac 1020

gggatgtggc actgcatcgg cgcacccctg gcgcggatcg agctgaacac ggtcttcacc 1080

cagttgttca cgcgcctgcc cgacctgcga ctggcgctgc cggcgggcga actggcggag 1140

aacgaaggcc ggttgtccgg cgggctcagt gagctgccgg tgacctggta g 1191

<210> 44

<211> 192

<212> DNA

<213> Streptomyces hygroscopicus Beijing variant tetrF gene (Streptomyces hygrospinus var. beijingensis tetrF)

<400> 44

atgcgtatca ccatcgattc ggagcgctgc gtgggcgccg gccagtgcgt gctgtccgcg 60

ccacaggtct tcgaccagga cgaggacggg atcgtcaccc tgctcgccgc gcccggcccc 120

gacgaggcgg ccaaggtccg catcgcggcg gcgctgtgcc cctccggggc gatcaccgca 180

cacgagggct ga 192

Claims

1. A high-efficiency low-toxicity tetramycin B derivative with molecular formula of C₃₅H₅₅NO₁₂The chemical structural formula is as follows:

2. a metabolic engineering method for high yield of highly potent and low toxicity tetramycin B derivatives according to claim 1, which comprises the steps of:

s3, obtaining a mutant strain with high yield of the tetramycin B derivative by enhancing the supply of malonyl coenzyme A serving as a precursor of a polyketide chain extension unit and the expression of tetrK and tetrF which are modified after a tetramycin biosynthesis gene cluster in the mutant strain with the inactivated tetrG gene;

s4, fermenting and culturing the mutant strain of the high-yield tetramycin B derivative to obtain the high-efficiency low-toxicity tetramycin B derivative;

step S2 specifically includes the following steps:

s23, carrying out PCR verification on the genomic DNA extracted from the mutant strain obtained in the step S22 to obtain a mutant strain with inactivated P450 monooxygenase gene tetrG in a tetramycin biosynthesis gene cluster;

step S3 includes the following steps:

s32, integrating a plasmid pJQK512 to a chromosome of the gene tetrG inactivated mutant strain through the intergeneric conjugative transfer of streptomyces and escherichia coli to obtain a mutant strain with high yield of the tetramycin B derivative;

in step S4, the fermentation culture is to inoculate the spore suspension of the mutant strain with high yield of the tetramycin B derivative into a TBSY seed culture medium, culture the mutant strain at 220rpm and 30 ℃ for 24h, transfer the mutant strain to the fermentation culture medium in a proportion of 5 percent, and continuously culture the mutant strain at 220rpm and 30 ℃ for 120h to obtain a fermentation culture solution.

3. The high-yield metabolic engineering method of high-efficiency low-toxicity tetramycin B derivatives according to claim 2, wherein in step S31, the acc gene is derived from Streptomyces coelicolor M152 and consists of three genes accA2, accB and accE, wherein the accB and accE genes share one promoter in Streptomyces coelicolor M152; wherein, the sequence of the accA2 gene is shown in SEQ ID NO. 40; the accB gene sequence is shown in SEQ ID NO. 41; the accE gene sequence is shown in SEQ ID NO. 42.

4. The high-yield metabolic engineering method of the high-efficiency low-toxicity tetramycin B derivative according to claim 3, wherein the heterologous expression of the acc gene is realized by respectively placing the accA2 and accBE genes under a strong promoter PermE.

5. The high-yield metabolic engineering method of the high-efficiency low-toxicity tetramycin B derivative according to claim 2, wherein in step S31, the post-modifier tetrK gene sequence is shown in SEQ ID No. 43.

6. The high-yield metabolic engineering method of the high-efficiency low-toxicity tetramycin B derivative according to claim 2, wherein in step S31, the gene sequence of the post-modifier tetRF is shown in SEQ ID No. 44.

7. The method for metabolic engineering of high productivity of highly potent and low toxic tetramycin B derivatives according to claim 2, wherein the enhancing of the tetrK and tetrF modification genes after the tetramycin biosynthesis gene cluster in step S31 is performed by placing both tetrK and tetrF genes under the strong promoter KasOp.

8. A highly potent and low-toxic tetramycin B derivative producing strain as claimed in claim 1, which is Streptomyces hygroscopicus var beijing (Beijijingningsis) SY05 with the accession number of CGMCC NO. 18370.