阅读说明：本技术 一种适用于链霉菌的强启动子及其应用 (Strong promoter suitable for streptomycete and application thereof ) 是由 林双君 郭文丽 黄婷婷 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种适用于链霉菌的强启动子及其应用,涉及基因工程和微生物代谢工程领域,强启动子的核苷酸序列包括(a)序列、与(a)序列具有75％以上一致性,且具有启动子功能的(b)序列或者在高严谨条件下与(a)序列或(b)序列杂交且具有启动子功能的(c)序列；包含该强启动子的质粒载体；包含该质粒载体的宿主细胞包括白色链霉菌、变铅青链霉菌、天蓝色链霉菌和委内瑞拉链霉菌；包含该强启动子、质粒载体和宿主细胞在启动目的基因表达的应用。该强启动子可以应用于常用的链霉菌模式菌株,对来源于放线菌的重要蛋白质包括酶和重要代谢产物的高产具有重要意义。(The invention discloses a strong promoter suitable for streptomycete and application thereof, relating to the field of genetic engineering and microbial metabolic engineering, wherein the nucleotide sequence of the strong promoter comprises a (a) sequence, a (b) sequence which has more than 75% of consistency with the (a) sequence and has the function of the promoter, or a (c) sequence which is hybridized with the (a) sequence or the (b) sequence and has the function of the promoter under high-stringency conditions; a plasmid vector comprising the strong promoter; host cells containing the plasmid vector comprise streptomyces albus, streptomyces lividans, streptomyces coelicolor and streptomyces venezuelae; the strong promoter, the plasmid vector and the host cell are used for promoting the expression of target genes. The strong promoter can be applied to common streptomycete model strains, and has important significance for high yield of important proteins including enzymes and important metabolites from actinomycetes.)

1. A strong promoter suitable for streptomycete is characterized in that a nucleotide sequence of the strong promoter comprises a sequence (a), (b) or (c), wherein the sequence (a) is shown as a sequence table SEQ ID NO: 1;

the sequence of (b) is as follows: a nucleotide sequence having 75% or more identity to the sequence of (a) and having the function of the strong promoter;

the sequence of (c) is as follows: the nucleotide sequence which is hybridized with the (a) sequence or the (b) sequence under high-stringency conditions and has the function of the strong promoter.

2. The strong promoter according to claim 1, wherein the sequence of (a) is obtained by amplification using Streptomyces villosus (Streptomyces fluccculus) as a template; the streptomyces villosus comprises streptomyces villosus CGMCC 4.1223; the amplified primer pair is a primer stnK4p-F and a primer stnK4 p-R; the nucleotide sequence of the primer stnK4p-F is shown in a sequence table SEQ ID NO. 2; the nucleotide sequence of the primer stnK4p-R is shown in a sequence table SEQ ID NO. 3.

3. The strong promoter of claim 1, wherein said sequence of (b) comprises said nucleotide sequence that is 85%, 90%, or 95% or more identical to the nucleotide sequence of said sequence of (a) and functions as said strong promoter.

4. A plasmid vector comprising the strong promoter according to any one of claims 1, 2 or 3, wherein the plasmid vector is an episomal vector or an integrative vector, and the episomal vector is a broad-host shuttle plasmid vector comprising replicon and oriT genes that are recognizable by gram-positive bacteria; the integrative vector is a broad host shuttle plasmid vector containing a bacteriophage integrase gene int site and an oriT gene.

5. The plasmid vector of claim 4, wherein the episomal vector comprises a pJTU1278 plasmid vector and the integrating vector comprises a pSET152 plasmid vector.

6. A host cell containing the plasmid vector of any one of claims 4 or 5, wherein the host cell is a Streptomyces comprising Streptomyces albus (Streptomyces albus), Streptomyces lividans (Streptomyces lividans), Streptomyces coelicolor, or Streptomyces venezuelae (Streptomyces venezuelae).

7. Use of a strong promoter according to any one of claims 1, 2 or 3 for promoting expression of a gene of interest.

8. The use of claim 7, wherein the gene of interest comprises a biosynthetic gene of 3-hydroxyanthranilic acid; inserting the strong promoter in front of the biosynthetic gene of 3-hydroxyanthranilic acid to drive the expression of the biosynthetic gene of 3-hydroxyanthranilic acid.

9. Use of a plasmid vector according to any of claims 4 or 5 for promoting expression of a gene of interest.

10. Use of a host cell according to claim 6 for promoting expression of a gene of interest.

Technical Field

The invention relates to the fields of genetic engineering and microbial metabolic engineering, in particular to a strong promoter suitable for streptomycete and application thereof.

Background

The promoter is a special DNA sequence located at the upstream of a gene transcription unit, has the function of promoting gene transcription, and plays an important role in regulating the gene transcription level. Metabolic engineering often requires the expression of foreign genes or the regulation of the expression of endogenous genes, and the choice of promoter is crucial to the regulation of gene expression. The promoter affects the transcription level of genes, influences the coordination among the genes in an artificial synthesis way or an origin way, and then influences the metabolic function of the strain. In order to increase the yield of the target product, a commonly used means in metabolic engineering is to overexpress the genes encoding the key enzymes in the anabolic pathway using strong promoters. There are two types of promoters, inducible and constitutive. The inducible promoter needs to be added with an inducer in the process of inducing expression, but the inducer is usually expensive and is not suitable for large-scale production, and cells have non-uniformity in induction of the inducer and may have certain toxicity to the cells. The constitutive promoter can continuously express the exogenous gene in the survival period of the thallus without inducing and other special conditions, so that the operation process is simplified, and the constitutive promoter has relatively high safety, and is more suitable for being applied in practical production.

Streptomyces, a gram-positive bacterium, belongs to the bacterial domain (kingdom Prokaryotae), Theobromycetes, Actinomycetes subclasses, Actinomycetales, Streptomycetaceae. Streptomycete has wide metabolism and biotransformation capacity, can decompose and utilize various organic matters in soil, plays an important role in the material circulation of the nature, and is also an important industrial microorganism. About 60% of antibiotics for commercial and medical use are produced by streptomyces, and antitumor bleomycin, mitomycin, antifungal nystatin, antitubercular streptomycin, antimycin a used as an anti-mite agent, validamycin which can effectively prevent and treat rice sheath blight, etc. are secondary metabolites of streptomyces.

One of the biggest characteristics of streptomycete genome is that the streptomycete genome is rich in secondary metabolic gene clusters and regulatory genes, and genes related to a specific secondary metabolic pathway in the streptomycete usually exist on a linear chromosome in the form of gene clusters. The process of transferring the whole or partial secondary metabolic biosynthesis gene into host bacteria which are different from the producing bacteria (i.e. heterologous), expressing the function of the host bacteria and producing the secondary metabolite or partial structure of the secondary metabolite is called heterologous expression. Commonly used heterologous expression systems include Streptomyces albus (Streptomyces albus), Streptomyces lividans (Streptomyces lividans), Streptomyces coelicolor (Streptomyces coelicolor), Streptomyces venezuelae (Streptomyces venezuelae), and the like. The strains are widely applied to the development of streptomycete genetic tools, have good growth capacity and sporulation capacity, have relatively clear metabolic background and are beneficial to the identification of heterologous expression products. The streptomycete genome has higher CG content and complex transcriptional regulation, and the promoter of the streptomycete also shows great heterogeneity, namely the promoter which is generally available in other microorganisms often does not show activity in the streptomycete. Because the gene cluster can not be normally expressed in a host due to the cross-species heterologous expression frequently existing in streptomyces, the common solution is to introduce a compatible promoter at the same time of introducing an exogenous gene cluster so as to induce the expression of the exogenous gene cluster. In the development process of heterologous expression of streptomyces to produce active products, only a limited number of engineered promoters can be widely used. The constitutive promoters ermEp and kasOp are currently most widely used. The use of the promoter ermEp to drive expression of the jadomycin biosynthetic gene cluster is the first case of the reconstitution of the biosynthetic pathway using heterologous promoters (Zheng et al, 2007). Jadomycins are produced by Streptomyces venezuelae ISP5230 and belong to the polyketide derivative angucycline class of antibiotics with broad-spectrum cytotoxicity. The original host can only produce a very small amount of jadomycins under the standard culture condition, and the yield reaches 84.3mg/L under the expression condition of a heterologous strain. Heterologous expression of the BTM gene cluster in Streptomyces coelicolor and Streptomyces albus resulted in very low yields of 1. mu.g/L and 4. mu.g/L, respectively, with a 20-fold increase in yield following replacement of the native promoter by the promoter ermEp (Huo et al, 2012). The four genes of the hopene biosynthetic gene cluster from the s.peuceteus strain (hopA, hopB, hopD and hopE) were heterologously expressed hopene in streptomyces venezuelae under the influence of ermEp (Ghimire et al, 2015). The promoter kasOp was inserted before the combamide gene cluster, and was able to produce 1.5-3mg/L of combamide (Liu et al, 2018). The above examples illustrate that in the successful expression of a heterologous gene cluster, the selection of an appropriate promoter is crucial.

Streptomycete has important development potential as the main source of bioactive metabolite, and has been vigorously developed and widely applied in medical treatment and agriculture. Among them, the promoter is identified as the main expression element for establishing a gene high-efficiency expression tool, and promoter modification is the first strategy for optimizing protein expression level and metabolite metabolic pathway technology. However, very few promoters known to date that can be applied to Streptomyces are available, with only ermEp, SF14p and kasOp being constitutive promoters and tipAp and nitAP being inducible promoters. With the prospect of synthetic biological development today, the need for good constitutive promoters becomes very urgent.

Therefore, those skilled in the art are working on developing a constitutive strong promoter capable of efficiently working in various streptomycetes modes, and providing more choices for the existing streptomycete promoter engineering.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to modify or obtain a promoter, thereby developing a technology capable of optimizing the protein expression level and metabolic pathways of metabolites.

In order to achieve the aim, the invention provides a constitutive strong promoter capable of efficiently working in streptomyces with various modes, wherein the nucleotide sequence of the strong promoter comprises (a), (b) or (c), and the (a) sequence is shown as a sequence table SEQ ID NO. 1; (b) the sequence is as follows: a nucleotide sequence having 75% or more identity to the sequence of (a) and having a promoter function; (c) the sequence is as follows: a nucleotide sequence which can be hybridized with the (a) sequence or the (b) sequence under high-stringency conditions and has the function of a promoter.

Further, (a) the sequence is obtained by amplification with streptomyces villosus (streptomyces fluccculus) as a template; the Streptomyces villosus comprises Streptomyces villosus CGMCC 4.1223; the amplified primer pair is a primer stnK4p-F and a primer stnK4 p-R; the nucleotide sequence of the primer stnK4p-F is shown in a sequence table SEQ ID NO. 2; the nucleotide sequence of the primer stnK4p-R is shown in a sequence table SEQ ID NO. 3.

Further, the streptomyces villosus CGMCC4.1223 is purchased from China general microbiological culture collection center, and the constitutive strong promoter is obtained by separating and cloning the streptomyces villosus.

The invention also provides a plasmid vector containing the strong promoter, wherein the plasmid vector is an episomal vector or an integrative vector, and the episomal vector is a shuttle plasmid vector of a wide host containing replicons and oriT genes which can be recognized by gram-positive bacteria; the integrative vector is a broad host shuttle plasmid vector containing a bacteriophage integrase gene int site and an oriT gene.

Further, the episomal vector comprises pJTU1278 plasmid vector and the integrative vector comprises pSET152 plasmid vector.

The invention also provides a host cell containing the plasmid vector, wherein the host cell is Streptomyces, and the Streptomyces comprises Streptomyces albus (Streptomyces albus), Streptomyces lividans (Streptomyces lividans), Streptomyces coelicolor and Streptomyces venezuelae (Streptomyces venezuelae).

Further, the methods of directed evolution and point mutation mutate the promoter nucleotide sequence of the present invention. The nucleotide which is artificially modified to have 75% or more identity to the nucleotide sequence of the promoter isolated in the present invention is derived from the nucleotide sequence of the present invention and is identical to the sequence of the present invention as long as the promoter activity for expressing the target gene is maintained.

Further, "identity" originally means the similarity of a natural nucleic acid sequence, and "identity" in the present invention means a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more similarity to the promoter (a) sequence of the present invention, the nucleotide sequence being represented by SEQ ID NO:1 of the sequence Listing; identity can be assessed visually or by computer software; using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The invention also provides application of the strong promoter in promoting expression of target genes.

Further, the strong promoter is applied to the heterologous high-yield strain with high protein expression and important metabolites.

Further, the target gene includes a biosynthesis gene of 3-hydroxyanthranilic acid; a strong promoter is inserted in front of the biosynthetic gene of 3-hydroxyanthranilic acid to drive the expression of the biosynthetic gene of 3-hydroxyanthranilic acid.

The invention also provides application of the plasmid vector in starting target gene expression.

Further, the plasmid vector containing the strong promoter is applied to the heterologous high-yield strain with high protein expression and important metabolites.

Further, the transformant containing the plasmid vector is applied to a high-protein expression and important metabolite heterologous high-yield strain.

The invention also provides application of the host cell in promoting the expression of a target gene.

Further, the application of the host cell containing the plasmid vector in the high protein expression and important metabolite heterologous high-producing strains.

Further, it was found that a strong promoter stnK4p which can be efficiently expressed in various Streptomyces patterns such as Streptomyces albus (Streptomyces albus), Streptomyces lividans (Streptomyces lividans), Streptomyces coelicolor (Streptomyces coelicolor), and Streptomyces venezuelae (Streptomyces venezuelae) has xylE as a reporter gene, and the promoter activity of stnK4p is higher than that of ermEp and kasOp in all of the four patterns; the promoter stnK4p is inserted in front of the biosynthesis gene of the 3-hydroxy anthranilic acid, so that the mass expression of the 3-hydroxy anthranilic acid in a heterologous expression mode strain streptomyces coelicolor M1154 can be realized.

In the preferred embodiment of the present invention, the test of host adaptability of the strong promoter stnK4p and the results thereof are detailed;

in another preferred embodiment of the present invention, the construction of a plasmid vector containing a promoter is described in detail;

in another preferred embodiment of the present invention, the construction of a 3-hydroxyanthranilic acid highly productive strain of the promoter stnK4p is explained in detail.

In another preferred embodiment of the present invention, the detection of the fermentation product of 3-hydroxyanthranilic acid strain with high productivity from the promoter stnK4p is detailed.

The invention provides a strong promoter, a sequence containing the strong promoter, a plasmid vector and high-yield application of the strong promoter in streptomycete. Streptomyces villosus (Streptomyces fluccculus) CGMCC4.1223 is taken as a template, a strong promoter sequence is obtained by amplification, and xylE is taken as a reporter gene to evaluate the promoter. The stnK4p promoter activity was higher than ermEp and kasOp in all four model strains. The strong promoter can be efficiently expressed in various types of Streptomyces such as Streptomyces albus, Streptomyces lividans, Streptomyces coelicolor and Streptomyces venezuelae. The promoter stnK4p is inserted in front of the biosynthesis gene of the 3-hydroxy anthranilic acid, so that the mass expression of the 3-hydroxy anthranilic acid in a heterologous expression mode strain streptomyces coelicolor M1154 can be realized. The newly characterized promoter provides more choices for the existing streptomyces promoter engineering, provides a main expression element for establishing a gene high-efficiency expression tool, and has important significance for developing and optimizing protein expression level and metabolic pathway technologies.

The conception, specific results, and technical effects of the present invention will be further described in conjunction with the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a diagram showing the expression effect of a promoter according to a preferred embodiment 1 of the present invention in various strains;

FIG. 2 is a map of a plasmid vector pSET152-stnK4p-stnM1/stnM2/stnN/stnM3 according to a preferred embodiment 2 of the present invention;

FIG. 3 is a graph showing quantitative analysis of 3-hydroxyanthranilic acid production from a promoter according to a preferred embodiment 4 of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified. The materials, reagents and the like used in this example are all available from normal commercial sources unless otherwise specified.

Example 1 testing of host Adaptation for the Strong promoter stnK4p

Streptomyces villosus (Streptomyces floccculus) CGMCC4.1223 is taken as a template, a strong promoter stnK4p is obtained by amplification, and the promoter is evaluated by taking xylE as a reporter gene. This strong promoter can be applied to four commonly used streptomyces model strains. As shown in FIG. 1, the four commonly used Streptomyces model strains are Streptomyces albus (Streptomyces albus), Streptomyces lividans (Streptomyces lividans), Streptomyces coelicolor (Streptomyces coelicolor) and Streptomyces venezuelae (Streptomyces venezuelae), the control promoters are ermEp and kasOp, and the strong promoter stnK4p has a higher expression level of the reporter gene xylE than the corresponding expression levels of the control promoters ermEp and kasOp in each commonly used Streptomyces model strain.

Example 2 construction of plasmid vector containing promoter

(1) Taking streptomyces villosus (Streptomyces fluccculus) CGMCC4.1223 genome as a template, utilizing primer pairs of stnK4p-F and stnK4p-R, wherein the nucleotide sequence of the primer stnK4p-F is shown in a sequence table SEQ ID NO: 2; the nucleotide sequence of the primer stnK4p-R is shown in a sequence table SEQ ID NO. 3; PCR amplification is carried out by using high-fidelity enzyme to obtain a promoter stnK4p fragment, and the purified stnK4p fragment is obtained after electrophoresis verification and electrophoretic gel recovery.

(2) Taking streptomyces villosus (Streptomyces fluccculus) CGMCC4.1223 genome as a template, and utilizing primer pairs 3HAA-F and 3HAA-R, wherein the nucleotide sequence of the primer 3HAA-F is shown in a sequence table SEQ ID NO. 4; the nucleotide sequence of the primer 3HAA-R is shown as a sequence table SEQ ID NO. 5; PCR amplification is carried out by using high-fidelity enzyme to obtain the stnM1/M2/N/M3 gene segment, and the purified stnM1/M2/N/M3 gene segment is obtained after electrophoresis verification and electrophoretic gel recovery.

(3) Digesting the plasmid pSET152 by using fast cutting enzymes Xba I and BamH I to obtain a linearized plasmid pSET152, carrying out electrophoretic verification, recovering electrophoretic gel to obtain a purified linearized vector pSET152

(4) The purified stnK4p fragment, stnM1/M2/N/M3 fragment and linearized vector pSET152 were ligated using a one-step cloning kit (Novozam, C113-01) into a recombinant plasmid pSET152-stnK4p-stnM1/stnM2/stnN/stnM3, which was mapped as shown in FIG. 2 with a base length of 11012 bp.

(5) The ligation product is transformed into Escherichia coli DH5 alpha, spread on an LB solid plate containing 50mg/L apramycin, cultured at 37 ℃ for 12-16h, subjected to colony PCR detection, sent to the prokaryote for sequencing, and the obtained positive bacterium is named as E.coli/pSET152-stnK4p-stnM1/M2/N/M3 after the sequencing is correct. Plasmid pSET152-stnK4p-stnM1/M2/N/M3 was extracted with a plasmid kit for use, and the plasmid map is shown in FIG. 2. The nucleotide sequence of the promoter stnK4p is shown in SEQ ID NO. 1.

Example 3 construction of 3-hydroxyanthranilic acid highly productive Strain having promoter stnK4p

(1) The recombinant plasmid pSET152-stnK4p-stnM1/M2/N/M3 was transformed into E.coli ET12567/pUZ8002, plated on LB solid plates containing 50mg/L apramycin, 50mg/L kanamycin and 25mg/L chloramphenicol, and single clones were picked up and cultured at 37 ℃ for 7-8h in LB medium containing 50mg/L apramycin, 50mg/L kanamycin and 25mg/L chloramphenicol. And centrifuging the bacterial liquid, removing supernatant, resuspending the thalli by using a proper amount of 20% glycerol, and storing the thalli in a refrigerator at the temperature of minus 80 ℃ for later use.

(2) Escherichia coli ET12567/pUZ8002 (containing recombinant plasmid) was inoculated into 4mL LB medium containing 50mg/L apramycin, 50mg/L kanamycin and 25mg/L chloramphenicol, and cultured overnight at 37 ℃. Then, 50. mu.l of the bacterial suspension was aspirated and added to 5ml of LB medium containing 50mg/L apramycin, 50mg/L kanamycin, and 25mg/L chloramphenicol, and cultured at 37 ℃ until OD600 reached 0.6.

(3) Streptomyces coelicolor M1154 was inoculated into YEME medium and cultured at 30 ℃ until logarithmic growth phase (about 36 h).

(4) Carrying out mycelium joint transfer: the E.coli and Streptomyces cultures were centrifuged (4000rpm,10min), the supernatant was removed to obtain cells, and the cells were washed with LB for 3 times. After centrifugation, the cells were resuspended in 500ul LB, mixed well and spread on a tray containing MgCl₂(final concentration 10mM) MS plates (containing 25ml of medium) were dried and incubated at 30 ℃. After 10h-14h, cover with sterile water containing apramycin and trimethoprim (900. mu.l sterile water plus 25. mu.l of 50mg/L apramycin and 50. mu.l of 100mg/L apramycin). After covering, incubation at 30 ℃ was continued for about 4-5 days, and outgrowth of the zygote was observed. The developed zygotes were picked up, and the genome was extracted and verified by PCR. The positive strain was obtained and named Streptomyces coelicolor M1154/pSET152-stnK4p-stnM 1/M2/N/M3.

Example 4 detection of the high yield of 3-hydroxyanthranilic acid Strain fermentation product from promoter stnK4p

The high-yield 3-hydroxy anthranilic acid strain with the promoter stnK4p is fermented by liquid, and a seed culture medium is a YEME culture medium. Transferring the spore to 25ml YEME culture medium, and culturing for 1-2 days until mycelium grows to fine viscous state. Inoculating 500 μ l to 50ml of fermentation medium with 1% inoculum size, and culturing for 4-5 days. Then the fermentation culture is centrifuged to separate the thalli and the supernatant, and the supernatant is directly used for detecting the product by high performance liquid chromatography.

The specification of the high performance liquid chromatography chromatographic column for analysis and detection is Agilent-C18, 5um and 150 multiplied by 4.6mm, the detection flow rate is 0.6ml/min, and the detection ultraviolet wave bands are UV210, 254, 280, 310 and 375. The sample amount is 20 mul, and the mobile phase adopts ultrapure water containing 0.1 percent of formic acid in the phase A; phase B is 100% acetonitrile. The elution method comprises the following steps: 10% B phase to 70% phase (linear gradient, 0-40min), 70% B phase to 100% B phase (linear gradient, 41-45min), 100% B (constant gradient 45-50 min). The flow rate of the LC-MS analysis and detection conditions is changed to 0.3ml/min, the ultraviolet band is unchanged, the mobile phase and the sample amount are unchanged, and the elution method is unchanged.

As shown in FIG. 3, where part A is a schematic representation of the reaction principle, StnM3 is homologous to the phenazine biosynthesis gene DAHP synthase PhzC, catalyzing the first step of the shikimate pathway in chorismate synthesis, and StnM3 is responsible for providing sufficient biosynthetic precursors. The enzymes that convert DAHP to chorismate undertake these biosynthetic steps by the primary metabolic enzymes of the host. StnM1 is homologous to anthracanilate synthsase PhzE and is responsible for catalyzing the catalysis of chorismic acids to ADIC. StnM2 belongs to isochorismatase PhzD and is responsible for the hydrolysis of ADIC to pyruvate and DHHA. StnN bioinformatics analysis was 2, 3-bishydroxybenzoic acid-2, 3-dehydrogenase. stnM1/M2/N/M3 is located in an operon which is co-transcribed and is jointly responsible for the synthesis of 3-hydroxyanthranilic acid. The part B is a high performance liquid chromatogram, the part I is a strain Streptomyces coelicolor M1154-pSET152-stnK4p-stnM1/M2/N/M3, and a stnM1/M2/N/M3 operon can generate a large amount of 3HAA under the drive of a promoter stnK4 p; II, strain Streptomyces coelicolor M1154-pSET152-stnM3p-stnM1/M2/N/M3, stnM1/M2/N/M3 operon driven by an original promoter, and 3HAA production cannot be detected at a high performance liquid chromatography level; III is a strain Streptomyces coelicolor M1154-pSET152 which is a negative control strain; IV is compound 3HAA standard control. Wherein part C is Streptomyces coelicolor M1154/pSET152-stnK4p-stnM1/M2/N/M3 strain fermentation broth supernatant 3-hydroxy anthranilic acid yield quantitative analysis, and the strain fermentation time is suitable for 5 days.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

<110> Shanghai university of transportation

<120> strong promoter suitable for streptomycete and application thereof

<130> 20210801

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 430

<212> DNA

<213> unknown comment

<400> 1

tgtgcgagca taacctctgc cgccgggtcg gggtaactca ctgcggtgcc gactcgccta 60

cggcatacgg tggttgtacg cgctattcac ggcgccttcg cattctcgcg cagcacaccc 120

atcacgccca tggtgaatgc cggtggcggg ccgaggcggc gaatacgggg cggtcgccgc 180

cggccgccgg ccgtggcggg gcagggagcg gcgggggacg gcatcgtccg catccggtcc 240

gcgaaggatg gccggaacct tctccatgag gtcgccgcgg cgggcatgct tggcgtgcga 300

cggctagcct gctagcatgc tcacatgact gctgaagagg tgaggcagtt caacgtctac 360

ctgccgggag acctgatccg gcaggtgaag cacttcgcca tcgaccgcga actgtcgctg 420

tcggcgctgg 430

<210> 2

<211> 59

<212> DNA

<213> unknown comment

<400> 2

acggccagtg ccaagcttgg gctgcaggtc gactctagat gtgcgagcat aacctctgc 59

<210> 3

<211> 59

<212> DNA

<213> unknown comment

<400> 3

gccctcaccg cactgtgtca ggtgacggtt ttcctcatcc cagcgccgac agcgacagt 59

<210> 4

<211> 59

<212> DNA

<213> unknown comment

<400> 4

acttcgccat cgaccgcgaa ctgtcgctgt cggcgctggg atgaggaaaa ccgtcacct 59

<210> 5

<211> 59

<212> DNA

<213> unknown comment

<400> 5

catgattacg aattcgatat cgcgcgcggc cgcggatcct caccggccca gggcttcgc 59