CN108424945B

CN108424945B - Method for improving terramycin yield based on rapA1A2-like dual-component regulation system

Info

Publication number: CN108424945B
Application number: CN201810089282.5A
Authority: CN
Inventors: 郭美锦; 倪辉; 庄英萍; 储炬; 曹君书; 张风新
Original assignee: Datong Tongxing Antibiotic Co ltd; East China University of Science and Technology
Current assignee: Datong Tongxing Antibiotic Co ltd; East China University of Science and Technology
Priority date: 2018-01-30
Filing date: 2018-01-30
Publication date: 2021-10-15
Anticipated expiration: 2038-01-30
Also published as: CN108424945A

Abstract

The invention relates to a method for improving terramycin yield based on a rapA1A2-like two-component regulation system. The invention firstly provides that the rapA1A2-like two-component regulation system in the terramycin producing strain is closely related to the terramycin yield of the producing strain, the two-component regulation system is blocked, and glycine or aspartic acid is used as a unique nitrogen source during culture, so that the terramycin yield of the terramycin producing strain can be remarkably improved. Therefore, the rapA1A2-like two-component regulation system or the substance and the method for regulating the system can be applied to realize the improvement of the terramycin producing strain and the enhancement of the yield of the terramycin.

Description

Method for improving terramycin yield based on rapA1A2-like dual-component regulation system

Technical Field

The invention belongs to the field of biotechnology; more particularly, the invention relates to a method for improving terramycin yield based on a rapA1A2-like double-component regulation system and a recombinant production bacterium.

Background

Streptomyces rimosus (Streptomyces rimosus) is a gram-positive, oxygen-consuming filamentous actinomycete, which is the first reported by Finlay et al in 1950 as a producing strain of Oxytetracycline (OTC). Streptomyces rimosus has become one of the major strains of OTC production in industry today. OTC is a broad-spectrum antibiotic, plays a role by inhibiting the synthesis of mycoprotein, is widely applied to clinical and fishery industries, and has great market demand.

Terramycin is used as a secondary metabolite synthesized by streptomyces rimosus, and the synthesis regulation of terramycin relates to complex interaction between pathway specific regulation factors and global regulation factors, so that the expression of antibiotic biosynthesis genes is enhanced or inhibited. The most typical of pathway-specific regulators is the Streptomyces Antibiotica Regulatory Proteins (SARP) family. This family is typically characterized by a molecular weight of approximately 25kDa and comprises two typical domains, the N-terminus comprising an OmpR-type DNA binding domain and the C-terminus a transcriptional activation domain (BTAD). A pathway-specific regulatory factor has also been found in S.rimosus, which also belongs to the SARP family, is located in the OTC gene cluster next to the transporter gene otrB and is named otcR. It can be directly combined with the promoter of the oxy gene on the gene cluster, thereby regulating and controlling the synthesis of OTC.

The two-component regulation system (TCS) is a whole local regulation system of streptomycete and participates in various physiological metabolic processes such as intracellular osmotic pressure regulation, metabolism, cell growth, thallus morphological differentiation and the like. A typical prokaryotic two-component system is mainly composed of two parts: histidine Kinase (HK), also known as a sensor protein and Response Regulator (RR). When a specific type of signal molecule in the environment binds to the signal sensing domain of the HK protein, ATP serves as a phosphate donor, and binds to the atpase domain, so that autophosphorylation of the conserved histidine residue in the signal transmission domain of the HK protein is achieved, the phosphate group is transferred to the aspartic acid residue in the signal receiving domain of the RR protein, and phosphorylation of aspartic acid causes a conformational change in the RR effector structure, thereby achieving signal transmission.

Bioinformatics analysis of the whole genome sequence of streptomyces coelicolor, found 84 putative HK genes and 80 putative RR genes, including 67 typical two-component systems. In Streptomyces coelicolor, a pair of two-component system rapA1A2 exists, which has been reported to have positive regulation effect on Actinorhodin (ACT) in a secondary metabolite of Streptomyces coelicolor and an atypical polyketide, and the action mode of the positive regulation effect is not elucidated for a while.

To date, only a few two-component systems have been identified to play a role in the regulation of antibiotic production, and the mechanism of action of most TCSs is unknown. In S.rimosus, similar studies have so far been left blank. Moreover, given the complexity of the secondary metabolic regulatory network of streptomyces, it is still extremely difficult to target genes that do have a regulatory effect on oxytetracycline production.

Disclosure of Invention

The invention aims to provide a method for improving the yield of oxytetracycline based on a rapA1A2-like two-component regulation system and a recombinant production strain.

In a first aspect of the present invention, there is provided a method for increasing the yield of oxytetracycline, the method comprising: the activity of a rapA1A2-like two-component regulation system in the terramycin producing strain is reduced, and glycine or aspartic acid is used as a unique nitrogen source to culture the terramycin producing strain, so that the terramycin yield is improved.

In a preferred embodiment, the activity of the rapA1A2-like two-component regulation system in the down-regulated terramycin producing strain comprises: (a) knocking out or silencing a gene of a rapA1A2-like two-component regulation system in terramycin producing bacteria; (b) transferring a down-regulator of a down-regulated rapA1A2-like two-component regulating system into oxytetracycline producing bacteria; or (c) regulating an upstream signal path or an upstream gene of a rapA1A2-like two-component regulation system in the terramycin producing strain to down-regulate the rapA1A2-like two-component regulation system in the terramycin producing strain.

In another preferred embodiment, (a) the activity of rapA1A2-like two-component regulatory system in terramycin producing bacteria is down-regulated by a gene knockout method; preferably, the knockout response regulatory protein gene rapA1 or the sensor protein gene rapA 2.

In another preferred embodiment, (b) the down-regulator is an interfering molecule that specifically interferes with the expression of a gene in the rapA1A2-like two-component regulatory system; preferably, the interfering molecule is dsRNA, antisense nucleic acid, small interfering RNA, micro RNA or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, micro RNA, which is used as a target for inhibiting or silencing a gene or a transcript thereof in a rapA1A2-like two-component regulatory system.

In another preferred embodiment, the oxytetracycline-producing bacteria is Streptomyces rimosus.

In another aspect of the invention, there is provided an isolated polypeptide selected from the group consisting of: (a) 1 or 2; (b) a polypeptide derived from (a) wherein the amino acid sequence shown in SEQ ID NO 1 or 2 is substituted, deleted or added with one or more (e.g., 1 to 20; preferably 1 to 15; more preferably 1 to 10; e.g., 5, 3) amino acid residues, and which has the same function as the polypeptide of (a); or (c) protein which has more than 85% of homology with the protein sequence limited by the (a) and has the function of the protein of the (a) and is derived from the (a).

In another aspect of the invention, there is provided an isolated polynucleotide encoding the polypeptide.

In another aspect of the invention, the polypeptide is provided for use as a target for regulating the yield of terramycin from a terramycin producing strain.

In another aspect of the invention, a genetically engineered terramycin producing bacterium is provided in which the gene of the rapA1A2-like two-component regulatory system is down-regulated.

In a preferred embodiment, the terramycin producing bacterium has the response regulatory protein gene rapA1 or the sensor protein gene rapA2 knocked out or silenced.

In another aspect of the invention, the genetically engineered terramycin producing strain is provided for use in producing terramycin.

In another aspect of the present invention, there is provided a kit for producing oxytetracycline, comprising: the genetically engineered terramycin producing strain; or a down-regulator for down-regulating rapA1A2-like two-component regulatory system in terramycin producing bacteria (such as an expression plasmid for knocking out the regulatory system, or an RNA interference agent).

In a preferred embodiment, the kit further comprises a culture medium for culturing the terramycin producing strain, wherein the culture medium uses glycine or aspartic acid as a unique nitrogen source.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, growth curves and oxytetracycline synthesis under MM + Gly and MM + Asn culture conditions for M4018 (squares) and M4018 Δ rap (circles).

A. MM + Asn culture conditions;

B. MM + Gly culture conditions.

FIG. 2, the terramycin production level of the original M4018 and its respective mutants under MM +50mM Gly.

FIG. 3, growth curves (A) and oxytetracycline synthesis (B) for M4018 (squares) and M4018 Δ rap (circles) in MM + Asp culture conditions.

Detailed Description

Through intensive research, the inventor finds that a rapA1A2-like two-component regulation system in the terramycin producing strain is closely related to the terramycin yield of the producing strain, the regulation system is regulated, glycine or aspartic acid is used as a unique nitrogen source during culture, and the terramycin yield of the terramycin producing strain can be remarkably improved. Therefore, the rapA1A2-like two-component regulation system or the substance and the method for regulating the system can be applied to realize the improvement of the terramycin producing strain and the enhancement of the yield of the terramycin.

In the invention, the rapA1A2-like two-component regulation system comprises response regulatory protein rapA1 and sensor protein gene rapA 2.

According to the invention, the down-regulation rapA1A2-like two-component regulation system can regulate and control response regulatory protein rapA1 and also regulate and control sensor protein gene rapA 2.

In the present invention, "rapA 1 polypeptide (protein)" refers to a polypeptide having the sequence of SEQ ID NO:1 and also includes a variant of the sequence of SEQ ID NO:1 having the same function as rapA1 polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of several (e.g., 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually, up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus.

In the present invention, "rapA 2 polypeptide (protein)" refers to a polypeptide having the sequence of SEQ ID NO:2, and also includes a variant of the sequence of SEQ ID NO:2 having the same function as rapA2 polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of several (e.g., 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually, up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus.

Any protein having high homology (e.g., 85% or more homology to the sequence shown in SEQ ID NO:1 or 2; preferably 90% or more homology; more preferably 95% or more homology, e.g., 98% or 99%) with the rapA1 polypeptide or rapA2 polypeptide and having the same function as the rapA1 polypeptide or rapA2 polypeptide is also included in the present invention.

Although specific response regulatory protein rapA1 and sensor protein rapA2 are exemplified in the specific examples of the present invention, it is to be understood that since terramycin producing bacteria exist in several different varieties which are highly conserved in sequence among them, homologous polypeptides from these terramycin producing bacteria should also be included in the present invention, and similar or identical regulatory methods as those of the present invention for these homologous polypeptides should also be included in the present invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

As a preferred mode of the invention, the terramycin producing strain comprises Streptomyces rimosus.

The invention also relates to polynucleotide sequences encoding the response regulatory protein rapA1 and the sensor protein rapA2 or conservative variants thereof. The polynucleotide may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the natural nucleic acid sequence corresponding to the polypeptide shown in SEQ ID NO. 1 or 2 or may be a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes a protein having SEQ ID NO 1 or 2, but differs from its native coding region sequence.

The invention firstly determines that the rapA1A2-like two-component regulation system exists in the terramycin producing strain, and finds that the two-component regulation system is closely related to the terramycin yield of the producing strain. Based on the new finding, the invention provides a method for improving the yield of oxytetracycline, which comprises the following steps: the activity of a rapA1A2-like two-component regulation system in the terramycin producing strain is reduced, and the terramycin producing strain is cultured by taking glycine and/or aspartic acid as a unique nitrogen source, so that the terramycin yield is improved.

After learning the regulatory role of the rapA1A2-like two-component regulatory system, the rapA1A2-like two-component regulatory system can be modulated (down-regulated) by a variety of methods well known to those skilled in the art. Including but not limited to: (a) knocking out or silencing a gene of a rapA1A2-like dual-component regulation system in terramycin producing bacteria; (b) transferring a down-regulator of a down-regulated rapA1A2-like dual-component regulating system into terramycin producing bacteria; or (c) regulating an upstream signal path or an upstream gene of a rapA1A2-like two-component regulation system in the terramycin producing strain so as to down-regulate the rapA1A2-like two-component regulation system in the terramycin producing strain.

The rapA1A2-like two-component regulatory system comprises the response regulatory protein gene rapA1 or the sensor protein rapA 2. Thus, down-regulation of both genes is a preferred method of down-regulating the rapA1A2-like two-component regulatory system.

Down-regulation of rapA1 or rapA2 in a terramycin producing strain can employ a variety of methods known in the art, including gene silencing, gene blocking, gene knockout, gene suppression, and the like. These methods are all included in the present invention.

For example, the rapA1 or rapA2 gene in the genome can be engineered by gene insertion blocking technology based on homologous recombination, such that the rapA1 or rapA2 gene is blocked; interfering RNA or antisense nucleotides can also be designed against the rapA1 or rapA2 gene to inhibit or silence rapA1 or rapA2 gene expression.

One method for down-regulating the rapA1 or rapA2 gene is gene disruption technology, in a preferred embodiment of the invention, rapA1 or rapA2 gene disruption plasmids are constructed in vitro, and other unrelated elements are inserted into the rapA1 or rapA2 gene of the terramycin producing strain by homologous recombination, so that the rapA1 or rapA2 gene on the chromosome can no longer encode an active protein. When gene blocking is performed, the choice of the irrelevant element is readily selectable by the person skilled in the art, for example by using certain resistance genes. A method of gene disruption (knock-out) is described in Genetic management of Streptomyces a Laboratory Manual, for example. Preferably, in the present embodiment, the unrelated element includes a part of the pKC1139 plasmid, such as Ori T, Ori pSG5, Apr. In addition, deletion of rapA1 or rapA2 gene to lack critical regions for function is a feasible strategy for down-regulation of genes.

The two-component system consists of two parts, and as a preferred mode of the invention, only rapA1 is down-regulated, and in this case, the down-regulation of the rapA1A2-like two-component regulation system can be realized without any gene manipulation on rapA 2. Since rapA1 and rapA2 are co-expressed and transcription proceeds from rapA1 to rapA2, rapA1 is blocked, so that rapA2 is not expressed by proper transcription.

In designing a construct for gene disruption or knockout, it is preferable to include a resistance selection gene at the same time, thereby facilitating subsequent selection of strains in which gene disruption or knockout has occurred.

In the specific embodiment of the invention, the inventor constructs a blocking plasmid pKC1139-rap and a anaplerosis plasmid pIB-KA-rap, and obtains blocking, anaplerosis and overexpression strains through conjugative transfer screening. The obtained strain is cultured in an MM culture medium with glycine as a unique nitrogen source, the terramycin yield of the blocking strain is found to be remarkably improved by over 60 percent compared with that of the original strain, the terramycin yield in the over-expression strain is remarkably reduced at the same time, and the negative regulation and control effect of a rapA1A2-like two-component regulation system in streptomyces rimosus on terramycin synthesis is fully proved.

The invention also provides terramycin producing bacteria for down-regulating rapA1A2-like dual-component regulation system, more particularly terramycin producing bacteria obtained after blocking rapA1 or rapA2 gene by adopting a gene insertion blocking method, wherein the strain does not express rapA1 or rapA2 gene or the expression quantity is obviously reduced. The invention also relates to the application of the strain in high-yield oxytetracycline. In the specific embodiment of the invention, the OTC synthesis level of the streptomyces cheloni response regulatory protein gene rapA1 is examined by constructing a blocking strain of the streptomyces cheloni response regulatory protein gene rapA1 under specific culture conditions, and the production level of the blocking strain is higher than that of the initiating strain by more than 60 percent under the condition that glycine or aspartic acid is added as a unique nitrogen source.

Based on the work of the present inventors, the present invention also provides a kit for producing oxytetracycline, comprising: the genetically engineered terramycin producing strain of the invention.

The present invention also provides a kit for producing oxytetracycline, comprising: a down-regulator for down-regulating rapA1A2-like two-component regulation system in terramycin producing bacteria; the down-regulator is for example an expression plasmid for knocking out the regulatory system, or an RNA interference agent.

The kit for producing terramycin can also comprise other reagents applied to the terramycin production process, such as a basal culture medium (such as MM culture medium) of terramycin producing bacteria, glycine and/or aspartic acid.

The kit for producing the terramycin can also comprise an instruction manual for culturing the terramycin producing strain or a method for down-regulating a rapA1A2-like two-component regulation system in the terramycin producing strain by utilizing the down-regulating agent.

The invention finds a two-component system related to terramycin synthesis regulation in streptomycin producing bacteria for the first time, and is characterized in that although certain homology (about 75%) exists between the rapA1A2-like two-component regulation system and the rapA1A2-like two-component regulation system in streptomyces coelicolor through sequence comparison, the difference is that the synthesis of terramycin by the rapA1A2-like two-component regulation system in the terramycin producing bacteria is negative regulation relative to the positive regulation effect of the rapA1A2 on Actinorhodin (ACT) produced by streptomyces coelicolor.

The invention discloses a novel method for regulating and controlling terramycin synthesis in streptomyces rimosus, and fills the blank of research of a two-component system in streptomyces rimosus. A two-component system rapA1A2-like in streptomyces rimosus is utilized to construct a blocking bacterium responding to regulatory protein gene rapA1, under the condition of an MM culture medium taking glycine or aspartic acid as a unique nitrogen source, the oxytetracycline production level of the blocking bacterium is greatly improved relative to that of a growing bacterium, the negative regulation and control effect of the two-component system rapA1A2-like on the synthesis of oxytetracycline is proved, and therefore a novel oxytetracycline synthesis regulation and control mechanism is found in streptomyces rimosus.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are examples in which specific conditions are not specified, typically according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to manufacturer's recommendations.

Materials and methods

Strains, plasmids and primers

The cells and plasmids used in the present invention are shown in Table 1.

TABLE 1

The sequences of the primers used in the present invention are shown in Table 2.

TABLE 2

Culture medium

Bran culture medium (7.5% bran, 2.5% agar) is mainly used for spore culture of streptomyces rimosus.

MM medium (refer to Streptomyces operation manual), the only nitrogen source in the original MM medium is Asparagine (Asparagine, asn), which is replaced by glycine (glycine, Gly) or aspartic acid (Asp) with the concentration of 50mM in the invention, and the method is mainly used for the growth of Streptomyces rimosus and the determination of terramycin content.

TSB (Oxoid, USA) medium: the method is mainly used for preparing mycelium and extracting genome.

LB culture medium: the method is mainly used for culturing the Escherichia coli, and the MS is used for the conjugation transfer operation of the Escherichia coli and the streptomycete.

Basic molecular manipulation

And (3) plasmid extraction, enzyme digestion verification, connection and other basic molecular operation reference molecular operation manuals, and streptomycete conjugal transfer operation reference streptomycete operation manuals.

Mycelium obtained after culturing S.rimosus in TSB for 24-36h can be used for extracting genome, and the specific operation refers to the operation instruction provided by reagent supplier.

Determination of Dry weight and Terramycin content

Taking 1mL of fermentation liquor, adding a proper amount of 9M hydrochloric acid for acidification, adjusting the pH value to 1.5-1.7, oscillating, uniformly mixing, standing for 5min, centrifuging at 12000rpm for 5min, and filtering the supernatant with a 0.22-micron water phase filter membrane to complete sample preparation. The analysis was carried out using Shimadzu LC-20A high pressure liquid system and Kromasil C18 column (4.6X 200 nm). The mobile phase is a mixed solution of 60% of ultrapure water, 10% of methanol, 20% of acetonitrile and 10% of 0.2M phosphoric acid, the flow rate is 0.8mL/min, the liquid inlet amount is 10 mu L, the detection wavelength is 350nm, the column temperature is 35 ℃, and a remarkable oxytetracycline characteristic peak can appear in about 3-4min under the condition. Taking oxytetracycline powder samples, preparing 10000U sample solution, diluting with 0.01M hydrochloric acid until the concentration is 0U, 20U, 40U, 60U, 80U and 100U respectively, and drawing a standard curve.

During the fermentation process, the growth condition of the thalli is known by measuring the dry weight of the thalli, after sampling in the fermentation process, centrifuging at 12000rpm for 10min at normal temperature, discarding supernatant, placing the residual wet thalli in a drying oven at 105 ℃ to dry to constant weight, weighing and mapping the growth curve.

Example 1 preparation of rapA1A2-like

The present inventors performed whole genome sequencing of S.rimosus M4018 of S.rimosus, from which functional genes were analyzed. Through intensive research, the whole gene sequence of the two-component system rapA1A2-like in the streptomyces rimosus M4018 is obtained. The length of the complete rapA1A2-like gene sequence is 2059bp, the length of the HK partial gene (rapA2) is 1404bp, the size of the amino acid is 467aa, the length of the RR partial gene (rapA1) is 663bp, the size of the amino acid is 220aa, and the gene sequences of the complete rapA1A2-like gene sequence and the HK partial gene sequence are overlapped by 8 bp.

The sequence of the complete rapA1A2 gene is as follows (5 '-3') (SEQ ID NO: 5):

ATGCGCCTGTTGATCGTGGAGGACGAGAAGCGACTGGCCTTGTCCCTGGCCGGAGGACTGC GGGCCGAGGGATACGCGGTCGATGTGGTGCACGACGGCCTGGAGGGCCTGCACCGGGCGG GCGAGGGCACGTACGACCTGGTGATCCTGGACATCATGCTGCCGGGCATGAACGGCTACCG CGTGTGCGGCGCCCTGCGTGCCGCGGGCAACGAGGTGCCGGTGCTGATGCTGACGGCCAAG GACGGCGAGTACGACGAGGCCGAGGGGCTGGACACCGGCGCGGACGACTATCTGACGAAG CCGTTCTCGTACGTGGTGCTGGTGGCCCGCGTGAAGGCGCTGCTGCGCCGCCGCGGACGGA CGGCGCTGCCCGTGCTGCGCGTGGGAGAGCTGAGCATCGACCAGGGGGCGCACCGCGTGGA GCGCGCCGGCGTGGAGGTGACGCTGACCGCCAAGGAGTTCGCCGTGCTGGAGCAACTGGCG CTGCGCGCGGGCGAGGTGGTGTCCAAGGCCGAGATCCTGGAGCACGTGTGGGACTTCGCGT ACGAGGGCGACAACAACATCGTCGAGGTGTACGTGAGCGCGCTGCGCCGCAAGCTGGGCG CCGCGGCGATCCAGACGGTGCGCGGCGCCGGGTACCGGCTGGTGCCGGGTGCGTAGGGTCA TCGGAAAGGTACGGGTCCGGGCGGCGCTGGGCGCCTCCCTCGTAGTGGCGGTCGCCCTGGT CGCCGCGGGCACTGCCCTCCTCCTGGTCCTCAAGAACAATCTCGTGGACCAGGCCGACCTCC AGGCGGAGAACACCGCCCGCGAGGTCGCCACGCAGATCGCGACCGGCAAGCCGTACGAGA AGCTGGACCTCCCCGACGGCGACGACCGCCCGGTGGTGGTGCTGTCCGAGGACGGCCGGGT GCTGGCGGCGGGCGACGACGTACGGGCGGTGGACGGCAAGGCCGTCACCGCGCGGCAGAC GCCGCCCGCCGGGCAGCCGCACGACTCCGACGACGATGACGACGACCACGAGATCAAGCC GGGCGAGGTCGAGGGCAAGGCGCGGCACACCGGCGGTACGGCGACGGTCGGCCACCGTAC GGCGGACTACCGGTTCGCCACCGTCGAGGCCAAGGACACCCAGGGCGGCAAGGCCGTCGTA CGGGCCGGGGCACCGCTGGCGGCCGAGCGGGAGGCGGTGGGCTCGGTGCGCACCGCGATG CTGATCGGGCTGCCCTGCCTGCTGCTGGTCGTGGCCGGGGTGACCTGGCTGGTCACGCGGCG GGCGCTGCGCCCGGTGGAGGGCATCCGCCGGGAGATGGCGGCGATCACGGCCAGTACGGA TCTGTCGCGGCGGGTGCCGGAGCCGGGCTCGCGGGACGAGATCGACCGGCTGGCCCGTACG ACCAACGAGACGCTGGGCGCGCTCCAGGAGTCGGTGGAGCGGCAGCGGCGGTTCGTCGCG GACGCCTCGCACGAGCTGCGTAGCCCGATCGCGAGCCTGCGGACGCAGCTGGAGGTGGGCA TCGCGCATCCGGAGCTGCTGGACGCGCCGGGCGCCGTGGAGGACGCCGTACGGCTGCAGAA CCTGGCGGCGGACCTGTTGCTGCTGGCGCGGCTGGACGCGGGGGAGCGGCCGGCGGACGCG CGGATCGACCTGGCGGCACTGGTGCGCGAGGAGGTCTCGCAGCGGGTGGGCGACCGGATCG CCGTGCAGGTGGGCGAGCTGGCGGGCGTGGAGGTCGCCGGGTCGCGGAGCCAGCTCGGGC GGGTGCTGGGGAATCTGCTGGACAATGCGCAGCGGCACGCGCGGGAGTCCGTACGGGCGA GTGTGGCGCGCGAGGGGGAGTGGGCCGTGCTGCGGGTCGAGGACGACGGGCCCGGGGTGC CGCCGGAGGAACGGGAGCGGATCTTCGAGCGGTTCGTCCGGCTCGACGACGCCCGCAGCCG TGACGACGGCGGGGCCGGACTGGGCCTCGCCATCGCCCGCGACGTGGCCGGGCGGCACGG GGGCACACTGGCCGTCCGCACGGGCTCGGTCTTCGAACTACGCCTGCCGGTGGCGTAG

gene (5 '-3') of rapA1 (SEQ ID NO:3, 663 bp):

ATGCGCCTGTTGATCGTGGAGGACGAGAAGCGACTGGCCTTGTCCCTGGCCGGAGGACTGC GGGCCGAGGGATACGCGGTCGATGTGGTGCACGACGGCCTGGAGGGCCTGCACCGGGCGG GCGAGGGCACGTACGACCTGGTGATCCTGGACATCATGCTGCCGGGCATGAACGGCTACCG CGTGTGCGGCGCCCTGCGTGCCGCGGGCAACGAGGTGCCGGTGCTGATGCTGACGGCCAAG GACGGCGAGTACGACGAGGCCGAGGGGCTGGACACCGGCGCGGACGACTATCTGACGAAG CCGTTCTCGTACGTGGTGCTGGTGGCCCGCGTGAAGGCGCTGCTGCGCCGCCGCGGACGGA CGGCGCTGCCCGTGCTGCGCGTGGGAGAGCTGAGCATCGACCAGGGGGCGCACCGCGTGGA GCGCGCCGGCGTGGAGGTGACGCTGACCGCCAAGGAGTTCGCCGTGCTGGAGCAACTGGCG CTGCGCGCGGGCGAGGTGGTGTCCAAGGCCGAGATCCTGGAGCACGTGTGGGACTTCGCGT ACGAGGGCGACAACAACATCGTCGAGGTGTACGTGAGCGCGCTGCGCCGCAAGCTGGGCG CCGCGGCGATCCAGACGGTGCGCGGCGCCGGGTACCGGCTGGTGCCGGGTGCGTAG

rapA1 polypeptide sequence (SEQ ID NO:1, 220 aa):

MRLLIVEDEKRLALSLAGGLRAEGYAVDVVHDGLEGLHRAGEGTYDLVILDIMLPGMNGYRV CGALRAAGNEVPVLMLTAKDGEYDEAEGLDTGADDYLTKPFSYVVLVARVKALLRRRGRTAL PVLRVGELSIDQGAHRVERAGVEVTLTAKEFAVLEQLALRAGEVVSKAEILEHVWDFAYEGDN NIVEVYVSALRRKLGAAAIQTVRGAGYRLVPGA

the gene sequence (5 '-3') encoding rapA2 (SEQ ID NO:4, 1404 bp):

GTGCGTAGGGTCATCGGAAAGGTACGGGTCCGGGCGGCGCTGGGCGCCTCCCTCGTAGTGG CGGTCGCCCTGGTCGCCGCGGGCACTGCCCTCCTCCTGGTCCTCAAGAACAATCTCGTGGAC CAGGCCGACCTCCAGGCGGAGAACACCGCCCGCGAGGTCGCCACGCAGATCGCGACCGGC AAGCCGTACGAGAAGCTGGACCTCCCCGACGGCGACGACCGCCCGGTGGTGGTGCTGTCCG AGGACGGCCGGGTGCTGGCGGCGGGCGACGACGTACGGGCGGTGGACGGCAAGGCCGTCA CCGCGCGGCAGACGCCGCCCGCCGGGCAGCCGCACGACTCCGACGACGATGACGACGACC ACGAGATCAAGCCGGGCGAGGTCGAGGGCAAGGCGCGGCACACCGGCGGTACGGCGACGG TCGGCCACCGTACGGCGGACTACCGGTTCGCCACCGTCGAGGCCAAGGACACCCAGGGCGG CAAGGCCGTCGTACGGGCCGGGGCACCGCTGGCGGCCGAGCGGGAGGCGGTGGGCTCGGT GCGCACCGCGATGCTGATCGGGCTGCCCTGCCTGCTGCTGGTCGTGGCCGGGGTGACCTGG CTGGTCACGCGGCGGGCGCTGCGCCCGGTGGAGGGCATCCGCCGGGAGATGGCGGCGATCA CGGCCAGTACGGATCTGTCGCGGCGGGTGCCGGAGCCGGGCTCGCGGGACGAGATCGACCG GCTGGCCCGTACGACCAACGAGACGCTGGGCGCGCTCCAGGAGTCGGTGGAGCGGCAGCG GCGGTTCGTCGCGGACGCCTCGCACGAGCTGCGTAGCCCGATCGCGAGCCTGCGGACGCAG CTGGAGGTGGGCATCGCGCATCCGGAGCTGCTGGACGCGCCGGGCGCCGTGGAGGACGCCG TACGGCTGCAGAACCTGGCGGCGGACCTGTTGCTGCTGGCGCGGCTGGACGCGGGGGAGCG GCCGGCGGACGCGCGGATCGACCTGGCGGCACTGGTGCGCGAGGAGGTCTCGCAGCGGGT GGGCGACCGGATCGCCGTGCAGGTGGGCGAGCTGGCGGGCGTGGAGGTCGCCGGGTCGCG GAGCCAGCTCGGGCGGGTGCTGGGGAATCTGCTGGACAATGCGCAGCGGCACGCGCGGGA GTCCGTACGGGCGAGTGTGGCGCGCGAGGGGGAGTGGGCCGTGCTGCGGGTCGAGGACGA CGGGCCCGGGGTGCCGCCGGAGGAACGGGAGCGGATCTTCGAGCGGTTCGTCCGGCTCGAC GACGCCCGCAGCCGTGACGACGGCGGGGCCGGACTGGGCCTCGCCATCGCCCGCGACGTGG CCGGGCGGCACGGGGGCACACTGGCCGTCCGCACGGGCTCGGTCTTCGAACTACGCCTGCC GGTGGCGTAG

rapA2 polypeptide sequence (SEQ ID NO:2, 467 aa):

VRRVIGKVRVRAALGASLVVAVALVAAGTALLLVLKNNLVDQADLQAENTAREVATQIATGK PYEKLDLPDGDDRPVVVLSEDGRVLAAGDDVRAVDGKAVTARQTPPAGQPHDSDDDDDDHEI KPGEVEGKARHTGGTATVGHRTADYRFATVEAKDTQGGKAVVRAGAPLAAEREAVGSVRTA MLIGLPCLLLVVAGVTWLVTRRALRPVEGIRREMAAITASTDLSRRVPEPGSRDEIDRLARTTNE TLGALQESVERQRRFVADASHELRSPIASLRTQLEVGIAHPELLDAPGAVEDAVRLQNLAADLL LLARLDAGERPADARIDLAALVREEVSQRVGDRIAVQVGELAGVEVAGSRSQLGRVLGNLLDN AQRHARESVRASVAREGEWAVLRVEDDGPGVPPEERERIFERFVRLDDARSRDDGGAGLGLAI ARDVAGRHGGTLAVRTGSVFELRLPVA

example 2 establishment of strains with a Down-regulated rapA1A2-like two-component regulatory System

1. Construction of mutant strains

Response regulatory protein gene rapA1 on the two-component system rapA1A2-like on the S.rimosus M4018 genome was first blocked by means of a single crossover. The specific operation mode is that a partial fragment of rapA1 gene is amplified from S.rimosus M4018 genome through primers Drapf and Drapr, the amplified fragment is connected to plasmid pMD19-TS, then enzyme cutting is carried out on the fragment by using Hind III and Xba I, and finally the fragment is connected to Hind III and Xba I sites of plasmid pKC1139, thereby obtaining pKC 1139-rap. The recombinant plasmid pKC1139-rap was transferred from E.coli ET12567 to S.rimosus M4018 by a combined transfer method, since the thermo-sensitive plasmid pKC1139 cannot replicate above 34 ℃, all possible zygotes were cultured at 37 ℃ and simultaneously screened for resistance to Applela. Then, the genes of the obtained zygotes are extracted and screened, and primers rap-single-P1/rap-single-P2 and aprF/aprR are used for further verification and determination, and finally, a mutant strain S.rimosus M4018 delta rap with rapA1 gene blockage is obtained.

Amplifying a kan resistant fragment by taking a plasmid pET-28a as a template, designing primers pKANTF and pKANTTR, amplifying a promoter region of 100bp at the upstream of a kan gene coding region and a transcription termination region of about 100bp at the downstream of the kan gene coding region together, introducing NheI enzyme digestion sites, performing single enzyme digestion on pIB139 and the recovered amplified fragment by NheI respectively, connecting the resistant fragment and the plasmid after recovery, introducing DH5 alpha competence, selecting a single colony, extracting the plasmid NheI single enzyme digestion verification, and if the verification is successful, indicating that the plasmid construction work is realized, and naming the plasmid construction work as pIB-KA.

Because the integrative plasmid pIB-KA can express two resistances of kanamycin and apramycin, and a complementation plasmid is constructed on the basis of the plasmid. The complete rapA1A2-like gene is amplified from the M4018 genome by using primers rap-pIBF and rap-pIBR, and is inserted into the downstream of the erythromycin promoter on the plasmid pIB-KA so as to realize the constitutive expression of the gene, and the gene is named as pIB-KA-rap. The successfully constructed plasmid pIB-KA-rap was introduced into S.rimosus M4018. delta. rap by the same conjugative transfer method, and possible anaplerotic strains were obtained by selecting for the double resistant adaptors, Aphana and kanamycin. Since the complementation strain contains a complete rapA1A2-like two-component system, the primer rapL/rapR can be used for preliminary verification.

The successfully verified strain was named M4018. delta. rap (pIB-KA-rap). By analogy with this method, the plasmid was integrated into s.rimosus M4018, and the over-expression strain M4018(pIB-KA-rap) was constructed.

Empty plasmids pIB139-KA were introduced into S.rimosus M4018 and S.rimosus M4018. delta. rap, respectively, and empty plasmid control strains M4018(pIB-KA) and M4018. delta. rap (pIB-KA) were constructed as both.

Because the pIB139 plasmid is inserted into the streptomycete genome in a site-specific integration mode, specific recombination occurs at att sites, primers attLf and attLr are designed to amplify and verify attL fragments (401bp), and primers attRf and attRr amplify and verify attR fragments (502bp), so that whether the plasmids pIB-KA and pIB-KA-rap are integrated into the streptomycete genome or not is verified together.

2. Fermentation process

Respectively culturing the obtained strains on a solid bran culture medium, and harvesting after 5-7 days at 30 DEG CCollecting spores on the culture medium. Counting spores generated by each mutant strain collected by using a flat viable count method, and fixing the inoculation amount at 1.0X 10 during inoculation⁶spores/mL. The medium used was MM liquid medium with 50mM glycine as the sole nitrogen source, 50mL each in 250mL shake flasks. After uniform inoculation, the shake flask is placed on a shaking table, cultured for 5-7 days at 30 ℃ and 220rpm, and sampled according to the experimental requirements. Three flasks were set in parallel for each condition and samples were taken every 24h for determination of dry weight and oxytetracycline content.

Example 3 culturing of the Strain and production of oxytetracycline

1. Difference between dry weight and yield of developing bacteria and blocking bacteria under MM + Gly culture condition

To ascertain whether blockade of the rapA1 gene had an effect on the production of S.rimosus, M4018 and M4018 Δ rap were grown in liquid MM +50mM Gly medium for fermentation. Sampling was started the next day after sufficient cells were produced for determination of dry weight and oxytetracycline content, and oxytetracycline production per dry weight was calculated. In addition, the alignment was performed using 50mM asparagine (Asn) as the sole nitrogen source condition of MM medium.

As can be seen from fig. 1A, there was essentially no difference in dry weight between the outgrowth and the blocker when Asn was the sole nitrogen source, although the dry weight of the blocker was slightly higher than that of the outgrowth under Gly conditions. Meanwhile, the yield per dry weight of the two genes is basically not different under the condition of 50mM Asn, and the results show that: in the MM culture medium with Asn as the only nitrogen source, the blocking strain has no difference from the original strain in terms of growth and oxytetracycline production. However, under the condition of 50mM Gly, the production level of the blocking strain M4018-delta rap is higher than that of the starter on the next day, and the production level per unit dry weight in the M4018-delta rap is higher than that of the starter M4018 by at least 60% by fermentation to the 5 th day (120h), as shown in figure 1B.

So far, a preliminary conclusion can be drawn that the strain with rapA1 gene blocked can show a significant difference of terramycin yield only under the condition of specific amino acid, and the production level of the blocked strain is far higher than that of the developed strain, which proves that at least under the condition of MM culture medium with glycine as a unique nitrogen source, the two-component system rapA1A2-like should negatively regulate terramycin synthesis in streptomyces rimosus.

2. Differences in dry weight and yield of each mutant strain in MM + Gly culture conditions

On the basis of the above experiments, a starting bacterium M4018, a blocking bacterium M4018 delta rap, a blocking bacterium control M4018 delta rap (pIB-KA), a anaplerotic bacterium M4018 delta rap (pIB-KA-rap), a starting bacterium control M4018(pIB-KA) and an overexpression M4018(pIB-KA-rap) are introduced simultaneously, and inoculated into a liquid MM culture medium containing 50mM glycine by the same method, so that the influence of the two-component system on terramycin synthesis is further confirmed, and simultaneously, an empty plasmid control of each mutant strain is introduced to eliminate a possible polarization effect.

As shown in FIG. 2, it can be found that there is almost no difference in the production level between M4018 and its empty plasmid control M4018(pIB-KA), and between M4018- Δ rap and its empty plasmid control M4018- Δ rap (pIB-KA), which means that the introduction of plasmid does not cause the difference in the production of mycoprotein, and the oxytetracycline production difference is caused by the gene manipulation of the two-component system. Meanwhile, the previous experimental result is repeated again, and the production level of the M4018-delta rap and the M4018-delta rap (pIB-KA) is higher than that of the M401850 percent in 120 hours. In addition, the production level of the gene is greatly reduced by 45 percent (120h) compared with that of the original strain in an overexpression strain M4018 (pIB-KA-rap).

Through the production results of the series of strains, the fact that the two-component system rapA1A2-like is the synthesis of the negative regulation terramycin is comprehensively proved. By down-regulating the two-component system rapA1A2-like, the terramycin yield can be obviously improved.

3. Differences in dry weight and Productivity of developing and blocking bacteria under MM + Asp culture conditions

The inventors determined the difference in dry weight and productivity of the developing and blocking bacteria under MM + Asp culture conditions using aspartic acid (Asp, 50mM) as the sole nitrogen source, otherwise unchanged from section "1" in this example.

The results are shown in FIG. 3. As can be seen from FIG. 3A, the difference in dry weight between the developing bacteria and the blocking bacteria was not large, but the oxytetracycline production per dry weight was significantly different. As can be seen from FIG. 3B, on day 5, the productivity per dry weight of M4018. delta. rap was higher than that of the original strain by about 34%.

All documents referred to herein are incorporated by reference into this application as if each document were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> university of east China's college of science

<120> method for improving terramycin yield based on rapA1A2-like two-component regulation system

<130> 180687

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 220

<212> PRT

<213> Streptomyces rimosus

<400> 1

Met Arg Leu Leu Ile Val Glu Asp Glu Lys Arg Leu Ala Leu Ser Leu

1 5 10 15

Ala Gly Gly Leu Arg Ala Glu Gly Tyr Ala Val Asp Val Val His Asp

20 25 30

Gly Leu Glu Gly Leu His Arg Ala Gly Glu Gly Thr Tyr Asp Leu Val

35 40 45

Ile Leu Asp Ile Met Leu Pro Gly Met Asn Gly Tyr Arg Val Cys Gly

50 55 60

Ala Leu Arg Ala Ala Gly Asn Glu Val Pro Val Leu Met Leu Thr Ala

65 70 75 80

Lys Asp Gly Glu Tyr Asp Glu Ala Glu Gly Leu Asp Thr Gly Ala Asp

85 90 95

Asp Tyr Leu Thr Lys Pro Phe Ser Tyr Val Val Leu Val Ala Arg Val

100 105 110

Lys Ala Leu Leu Arg Arg Arg Gly Arg Thr Ala Leu Pro Val Leu Arg

115 120 125

Val Gly Glu Leu Ser Ile Asp Gln Gly Ala His Arg Val Glu Arg Ala

130 135 140

Gly Val Glu Val Thr Leu Thr Ala Lys Glu Phe Ala Val Leu Glu Gln

145 150 155 160

Leu Ala Leu Arg Ala Gly Glu Val Val Ser Lys Ala Glu Ile Leu Glu

165 170 175

His Val Trp Asp Phe Ala Tyr Glu Gly Asp Asn Asn Ile Val Glu Val

180 185 190

Tyr Val Ser Ala Leu Arg Arg Lys Leu Gly Ala Ala Ala Ile Gln Thr

195 200 205

Val Arg Gly Ala Gly Tyr Arg Leu Val Pro Gly Ala

210 215 220

<210> 2

<211> 467

<212> PRT

<213> Streptomyces rimosus

<400> 2

Val Arg Arg Val Ile Gly Lys Val Arg Val Arg Ala Ala Leu Gly Ala

1 5 10 15

Ser Leu Val Val Ala Val Ala Leu Val Ala Ala Gly Thr Ala Leu Leu

20 25 30

Leu Val Leu Lys Asn Asn Leu Val Asp Gln Ala Asp Leu Gln Ala Glu

35 40 45

Asn Thr Ala Arg Glu Val Ala Thr Gln Ile Ala Thr Gly Lys Pro Tyr

50 55 60

Glu Lys Leu Asp Leu Pro Asp Gly Asp Asp Arg Pro Val Val Val Leu

65 70 75 80

Ser Glu Asp Gly Arg Val Leu Ala Ala Gly Asp Asp Val Arg Ala Val

85 90 95

Asp Gly Lys Ala Val Thr Ala Arg Gln Thr Pro Pro Ala Gly Gln Pro

100 105 110

His Asp Ser Asp Asp Asp Asp Asp Asp His Glu Ile Lys Pro Gly Glu

115 120 125

Val Glu Gly Lys Ala Arg His Thr Gly Gly Thr Ala Thr Val Gly His

130 135 140

Arg Thr Ala Asp Tyr Arg Phe Ala Thr Val Glu Ala Lys Asp Thr Gln

145 150 155 160

Gly Gly Lys Ala Val Val Arg Ala Gly Ala Pro Leu Ala Ala Glu Arg

165 170 175

Glu Ala Val Gly Ser Val Arg Thr Ala Met Leu Ile Gly Leu Pro Cys

180 185 190

Leu Leu Leu Val Val Ala Gly Val Thr Trp Leu Val Thr Arg Arg Ala

195 200 205

Leu Arg Pro Val Glu Gly Ile Arg Arg Glu Met Ala Ala Ile Thr Ala

210 215 220

Ser Thr Asp Leu Ser Arg Arg Val Pro Glu Pro Gly Ser Arg Asp Glu

225 230 235 240

Ile Asp Arg Leu Ala Arg Thr Thr Asn Glu Thr Leu Gly Ala Leu Gln

245 250 255

Glu Ser Val Glu Arg Gln Arg Arg Phe Val Ala Asp Ala Ser His Glu

260 265 270

Leu Arg Ser Pro Ile Ala Ser Leu Arg Thr Gln Leu Glu Val Gly Ile

275 280 285

Ala His Pro Glu Leu Leu Asp Ala Pro Gly Ala Val Glu Asp Ala Val

290 295 300

Arg Leu Gln Asn Leu Ala Ala Asp Leu Leu Leu Leu Ala Arg Leu Asp

305 310 315 320

Ala Gly Glu Arg Pro Ala Asp Ala Arg Ile Asp Leu Ala Ala Leu Val

325 330 335

Arg Glu Glu Val Ser Gln Arg Val Gly Asp Arg Ile Ala Val Gln Val

340 345 350

Gly Glu Leu Ala Gly Val Glu Val Ala Gly Ser Arg Ser Gln Leu Gly

355 360 365

Arg Val Leu Gly Asn Leu Leu Asp Asn Ala Gln Arg His Ala Arg Glu

370 375 380

Ser Val Arg Ala Ser Val Ala Arg Glu Gly Glu Trp Ala Val Leu Arg

385 390 395 400

Val Glu Asp Asp Gly Pro Gly Val Pro Pro Glu Glu Arg Glu Arg Ile

405 410 415

Phe Glu Arg Phe Val Arg Leu Asp Asp Ala Arg Ser Arg Asp Asp Gly

420 425 430

Gly Ala Gly Leu Gly Leu Ala Ile Ala Arg Asp Val Ala Gly Arg His

435 440 445

Gly Gly Thr Leu Ala Val Arg Thr Gly Ser Val Phe Glu Leu Arg Leu

450 455 460

Pro Val Ala

465

<210> 3

<211> 663

<212> DNA

<213> Streptomyces rimosus

<400> 3

atgcgcctgt tgatcgtgga ggacgagaag cgactggcct tgtccctggc cggaggactg 60

cgggccgagg gatacgcggt cgatgtggtg cacgacggcc tggagggcct gcaccgggcg 120

ggcgagggca cgtacgacct ggtgatcctg gacatcatgc tgccgggcat gaacggctac 180

cgcgtgtgcg gcgccctgcg tgccgcgggc aacgaggtgc cggtgctgat gctgacggcc 240

aaggacggcg agtacgacga ggccgagggg ctggacaccg gcgcggacga ctatctgacg 300

aagccgttct cgtacgtggt gctggtggcc cgcgtgaagg cgctgctgcg ccgccgcgga 360

cggacggcgc tgcccgtgct gcgcgtggga gagctgagca tcgaccaggg ggcgcaccgc 420

gtggagcgcg ccggcgtgga ggtgacgctg accgccaagg agttcgccgt gctggagcaa 480

ctggcgctgc gcgcgggcga ggtggtgtcc aaggccgaga tcctggagca cgtgtgggac 540

ttcgcgtacg agggcgacaa caacatcgtc gaggtgtacg tgagcgcgct gcgccgcaag 600

ctgggcgccg cggcgatcca gacggtgcgc ggcgccgggt accggctggt gccgggtgcg 660

tag 663

<210> 4

<211> 1404

<212> DNA

<213> Streptomyces rimosus

<400> 4

gtgcgtaggg tcatcggaaa ggtacgggtc cgggcggcgc tgggcgcctc cctcgtagtg 60

gcggtcgccc tggtcgccgc gggcactgcc ctcctcctgg tcctcaagaa caatctcgtg 120

gaccaggccg acctccaggc ggagaacacc gcccgcgagg tcgccacgca gatcgcgacc 180

ggcaagccgt acgagaagct ggacctcccc gacggcgacg accgcccggt ggtggtgctg 240

tccgaggacg gccgggtgct ggcggcgggc gacgacgtac gggcggtgga cggcaaggcc 300

gtcaccgcgc ggcagacgcc gcccgccggg cagccgcacg actccgacga cgatgacgac 360

gaccacgaga tcaagccggg cgaggtcgag ggcaaggcgc ggcacaccgg cggtacggcg 420

acggtcggcc accgtacggc ggactaccgg ttcgccaccg tcgaggccaa ggacacccag 480

ggcggcaagg ccgtcgtacg ggccggggca ccgctggcgg ccgagcggga ggcggtgggc 540

tcggtgcgca ccgcgatgct gatcgggctg ccctgcctgc tgctggtcgt ggccggggtg 600

acctggctgg tcacgcggcg ggcgctgcgc ccggtggagg gcatccgccg ggagatggcg 660

gcgatcacgg ccagtacgga tctgtcgcgg cgggtgccgg agccgggctc gcgggacgag 720

atcgaccggc tggcccgtac gaccaacgag acgctgggcg cgctccagga gtcggtggag 780

cggcagcggc ggttcgtcgc ggacgcctcg cacgagctgc gtagcccgat cgcgagcctg 840

cggacgcagc tggaggtggg catcgcgcat ccggagctgc tggacgcgcc gggcgccgtg 900

gaggacgccg tacggctgca gaacctggcg gcggacctgt tgctgctggc gcggctggac 960

gcgggggagc ggccggcgga cgcgcggatc gacctggcgg cactggtgcg cgaggaggtc 1020

tcgcagcggg tgggcgaccg gatcgccgtg caggtgggcg agctggcggg cgtggaggtc 1080

gccgggtcgc ggagccagct cgggcgggtg ctggggaatc tgctggacaa tgcgcagcgg 1140

cacgcgcggg agtccgtacg ggcgagtgtg gcgcgcgagg gggagtgggc cgtgctgcgg 1200

gtcgaggacg acgggcccgg ggtgccgccg gaggaacggg agcggatctt cgagcggttc 1260

gtccggctcg acgacgcccg cagccgtgac gacggcgggg ccggactggg cctcgccatc 1320

gcccgcgacg tggccgggcg gcacgggggc acactggccg tccgcacggg ctcggtcttc 1380

gaactacgcc tgccggtggc gtag 1404

<210> 5

<211> 2059

<212> DNA

<213> Streptomyces rimosus

<400> 5

atgcgcctgt tgatcgtgga ggacgagaag cgactggcct tgtccctggc cggaggactg 60

cgggccgagg gatacgcggt cgatgtggtg cacgacggcc tggagggcct gcaccgggcg 120

ggcgagggca cgtacgacct ggtgatcctg gacatcatgc tgccgggcat gaacggctac 180

cgcgtgtgcg gcgccctgcg tgccgcgggc aacgaggtgc cggtgctgat gctgacggcc 240

aaggacggcg agtacgacga ggccgagggg ctggacaccg gcgcggacga ctatctgacg 300

aagccgttct cgtacgtggt gctggtggcc cgcgtgaagg cgctgctgcg ccgccgcgga 360

cggacggcgc tgcccgtgct gcgcgtggga gagctgagca tcgaccaggg ggcgcaccgc 420

gtggagcgcg ccggcgtgga ggtgacgctg accgccaagg agttcgccgt gctggagcaa 480

ctggcgctgc gcgcgggcga ggtggtgtcc aaggccgaga tcctggagca cgtgtgggac 540

ttcgcgtacg agggcgacaa caacatcgtc gaggtgtacg tgagcgcgct gcgccgcaag 600

ctgggcgccg cggcgatcca gacggtgcgc ggcgccgggt accggctggt gccgggtgcg 660

tagggtcatc ggaaaggtac gggtccgggc ggcgctgggc gcctccctcg tagtggcggt 720

cgccctggtc gccgcgggca ctgccctcct cctggtcctc aagaacaatc tcgtggacca 780

ggccgacctc caggcggaga acaccgcccg cgaggtcgcc acgcagatcg cgaccggcaa 840

gccgtacgag aagctggacc tccccgacgg cgacgaccgc ccggtggtgg tgctgtccga 900

ggacggccgg gtgctggcgg cgggcgacga cgtacgggcg gtggacggca aggccgtcac 960

cgcgcggcag acgccgcccg ccgggcagcc gcacgactcc gacgacgatg acgacgacca 1020

cgagatcaag ccgggcgagg tcgagggcaa ggcgcggcac accggcggta cggcgacggt 1080

cggccaccgt acggcggact accggttcgc caccgtcgag gccaaggaca cccagggcgg 1140

caaggccgtc gtacgggccg gggcaccgct ggcggccgag cgggaggcgg tgggctcggt 1200

gcgcaccgcg atgctgatcg ggctgccctg cctgctgctg gtcgtggccg gggtgacctg 1260

gctggtcacg cggcgggcgc tgcgcccggt ggagggcatc cgccgggaga tggcggcgat 1320

cacggccagt acggatctgt cgcggcgggt gccggagccg ggctcgcggg acgagatcga 1380

ccggctggcc cgtacgacca acgagacgct gggcgcgctc caggagtcgg tggagcggca 1440

gcggcggttc gtcgcggacg cctcgcacga gctgcgtagc ccgatcgcga gcctgcggac 1500

gcagctggag gtgggcatcg cgcatccgga gctgctggac gcgccgggcg ccgtggagga 1560

cgccgtacgg ctgcagaacc tggcggcgga cctgttgctg ctggcgcggc tggacgcggg 1620

ggagcggccg gcggacgcgc ggatcgacct ggcggcactg gtgcgcgagg aggtctcgca 1680

gcgggtgggc gaccggatcg ccgtgcaggt gggcgagctg gcgggcgtgg aggtcgccgg 1740

gtcgcggagc cagctcgggc gggtgctggg gaatctgctg gacaatgcgc agcggcacgc 1800

gcgggagtcc gtacgggcga gtgtggcgcg cgagggggag tgggccgtgc tgcgggtcga 1860

ggacgacggg cccggggtgc cgccggagga acgggagcgg atcttcgagc ggttcgtccg 1920

gctcgacgac gcccgcagcc gtgacgacgg cggggccgga ctgggcctcg ccatcgcccg 1980

cgacgtggcc gggcggcacg ggggcacact ggccgtccgc acgggctcgg tcttcgaact 2040

acgcctgccg gtggcgtag 2059

<210> 6

<211> 29

<212> DNA

<213> primers (Primer)

<400> 6

cccaagctta cacctcgacg atgttgttg 29

<210> 7

<211> 28

<212> DNA

<213> primers (Primer)

<400> 7

tgctctagaa tacgcggtcg atgtggtg 28

<210> 8

<211> 21

<212> DNA

<213> primers (Primer)

<400> 8

gtgcaatacg aatggcgaaa a 21

<210> 9

<211> 19

<212> DNA

<213> primers (Primer)

<400> 9

tcagccaatc gactggcga 19

<210> 10

<211> 23

<212> DNA

<213> primers (Primer)

<400> 10

aaccggtagt ccgccgtacg gtg 23

<210> 11

<211> 26

<212> DNA

<213> primers (Primer)

<400> 11

aggttgagaa gctgaccgat gagctc 26

<210> 12

<211> 30

<212> DNA

<213> primers (Primer)

<400> 12

ctagctagcc tcagtggaac gaaaactcac 30

<210> 13

<211> 31

<212> DNA

<213> primers (Primer)

<400> 13

ctagctagca caatttcagg tggcactttt c 31

<210> 14

<211> 28

<212> DNA

<213> primers (Primer)

<400> 14

cggaattcct acgccaccgg caggcgta 28

<210> 15

<211> 31

<212> DNA

<213> primers (Primer)

<400> 15

ggaattccat atgcgcctgt tgatcgtgga g 31

<210> 16

<211> 24

<212> DNA

<213> primers (Primer)

<400> 16

ctacgccacc ggcaggcgta gttc 24

<210> 17

<211> 24

<212> DNA

<213> primers (Primer)

<400> 17

atgcgcctgt tgatcgtgga ggac 24

<210> 18

<211> 21

<212> DNA

<213> primers (Primer)

<400> 18

gttcacccac agctggaggc c 21

<210> 19

<211> 21

<212> DNA

<213> primers (Primer)

<400> 19

gctcgacttc gcgctgaagg t 21

<210> 20

<211> 21

<212> DNA

<213> primers (Primer)

<400> 20

gctataatga ccccgaagca g 21

<210> 21

<211> 17

<212> DNA

<213> primers (Primer)

<400> 21

tcgtcatgcc ccgcagt 17

Claims

1. A method for increasing oxytetracycline production, comprising: the activity of a rapA1A2-like dual-component regulation system in the terramycin producing strain is reduced, and glycine or aspartic acid is used as a unique nitrogen source to culture the terramycin producing strain, so that the terramycin yield is improved; the terramycin producing strain is streptomyces rimosus (S.) (S. rimosus) (ii) a The rapA1A2-like two-component regulation system consists of response regulatory protein rapA1 and sensor protein gene rapA2, wherein the amino acid of rapA1The sequence is shown as SEQ ID NO. 1, and the amino acid sequence of rapA2 is shown as SEQ ID NO. 2; the activity is the expression level of rapA1 and/or rapA 2.

2. The method of claim 1, wherein down-regulating the activity of a rapA1A2-like two-component regulatory system in a terramycin producing strain comprises:

(a) knockout or silencing in terramycin producing bacteriarapA1A2-genes of the like two-component regulatory system, said genes being of rapA1 and/or rapA 2; or

(b) Will be down-regulatedrapA1A2-a downregulator of the activity of the like two-component regulatory system is transferred into oxytetracycline-producing bacteria.

3. The method of claim 2, wherein in (a), the activity of rapA1A2-like two-component regulatory system in a terramycin producing strain is down-regulated by a gene knockout procedure.

4. The method of claim 3, wherein response-modulating protein is knocked outrapA1Or a sensor proteinrapA2The gene of (1).

5. The method of claim 2, wherein in (b) the down-regulating agent is a specific interferencerapA1A2Interfering molecules to the expression of genes in the like two-component regulatory system.

6. The method of claim 5, wherein said interfering molecule is a peptide of the formularapA1A2-the gene or transcript thereof in the like two-component regulatory system is a dsRNA, an antisense nucleic acid, a small interfering RNA, a microrna, or a construct capable of expressing or forming said dsRNA, antisense nucleic acid, small interfering RNA, microrna, to inhibit or silence the target.

7. The method of claim 1, wherein the terramycin producing bacterium is S.rimosus M4018.

8. The application of the down-regulating agent for down-regulating the activity of a rapA1A2-like dual-component regulating system in the terramycin producing strain is used for improving the terramycin yield of the terramycin producing strain; the down regulator down regulates the activity of a rapA1A2-like two-component regulation system in the terramycin producing strain and improves the terramycin yield when the terramycin producing strain is cultured by taking glycine or aspartic acid as a unique nitrogen source; the terramycin producing strain is streptomyces rimosus; the rapA1A2-like dual-component regulation system consists of response regulatory protein rapA1 and sensor protein gene rapA2, wherein the amino acid sequence of rapA1 is shown as SEQ ID NO. 1, and the amino acid sequence of rapA2 is shown as SEQ ID NO. 2; the activity is the expression level of rapA1 and/or rapA 2.

9. A genetically engineered terramycin producing strain characterized in that it contains terramycinrapA1A2-the activity of a gene of the like two-component regulatory system is down-regulated; the terramycin producing strain is streptomyces rimosus; the rapA1A2-like dual-component regulation system consists of response regulatory protein rapA1 and sensor protein gene rapA2, wherein the amino acid sequence of rapA1 is shown as SEQ ID NO. 1, and the amino acid sequence of rapA2 is shown as SEQ ID NO. 2; the activity is the expression level of rapA1 and/or rapA 2.

10. The terramycin producing strain of claim 9, wherein a response regulatory protein gene is present in the terramycin producing strainrapA1Or a sensor protein generapA2Is knocked out or silenced.

11. Use of the genetically engineered terramycin producing bacterium of claim 9 or 10 for the production of terramycin.

12. A kit for the production of oxytetracycline, comprising:

the genetically engineered oxytetracycline-producing bacteria of claim 10 or 11; or

Down regulation in terramycin producing bacteriarapA1A2Downregulator of the Activity of a like two-component regulatory System(ii) a The terramycin producing strain is streptomyces rimosus; the rapA1A2-like dual-component regulation system consists of response regulatory protein rapA1 and sensor protein gene rapA2, wherein the amino acid sequence of rapA1 is shown as SEQ ID NO. 1, and the amino acid sequence of rapA2 is shown as SEQ ID NO. 2; the activity is the expression level of rapA1 and/or rapA 2.

13. The kit of claim 12, further comprising a medium for culturing the oxytetracycline-producing bacteria, wherein glycine or aspartic acid is the sole nitrogen source.