CN108424945A

CN108424945A - The method for improving oxytetracycline yield based on rapA1A2-like bi-component regulating systems

Info

Publication number: CN108424945A
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: 2018-08-21
Anticipated expiration: 2038-01-30
Also published as: CN108424945B

Abstract

The present invention relates to the methods for improving oxytetracycline yield based on rapA1A2 like bi-component regulating systems.Present invention firstly provides rapA1A2 like bi-components regulating systems in terramycin production bacterium and the oxytetracycline yield of the production bacterium are closely related, block the bi-component regulating system, and in culture using glycine or aspartic acid as only nitrogen source, the oxytetracycline yield of terramycin production bacterium can be extremely significantly increased.Therefore, rapA1A2 like bi-components regulating system or adjust the system substance and method can be applied to realize terramycin production bacterium improvement, enhance the yield of terramycin.

Description

The method for improving oxytetracycline yield based on rapA1A2-like bi-component regulating systems

Technical field

The invention belongs to biotechnologies；More particularly it relates to be adjusted based on rapA1A2-like bi-components System improves method and the recombinant production bacterium of oxytetracycline yield.

Background technology

Streptomyces rimosus (Streptomyces rimosus) is a kind of Gram-positive, oxygen consumption filiform actinomyces, as The production bacterial strain of terramycin (oxytetracycline, OTC) is reported by Finlay et al. in nineteen fifty for the first time.Nowadays, Streptomyces rimosus have become one of the main bacterial strain of industrial OTC productions.OTC is a kind of broad-spectrum antibiotic, It is played a role by inhibiting the synthesis of mycoprotein, there is prodigious market to need for the extensive use in clinical and fishery It asks.

A kind of secondary metabolite that terramycin is synthesized as streptomyces rimosus, it is special that synthesis regulation is related to approach Property regulatory factor and the global regulation factor between complicated interaction, and then reinforce or inhibit antibiotic biosynthesis base The expression of cause.The most typical in the approach specific regulatory control factor is exactly streptomycete antibiotic albumen (Streptomyces Antibiotic regulatory proteins, SARP) family.This family has more typical feature, molecular weight big About 25kDa, including two typical structural domains, the ends N include the DNA binding domain of an OmpR type, and C-terminal is that a transcription swashs Domain (bacterial transcriptional activation domain, BTAD) living.It has also been sent out in streptomyces rimosus An approach specific regulatory control factor is showed, it is also to belong to SARP families, is located at OTC gene clusters, and then transport protein Gene otrB, is named as otcR.It can be directly with the oxy genes in gene cluster promoter bind directly, to regulate and control The synthesis of OTC.

And two-component regulating system (two-component Regulatory system, TCS) is the overall situation of streptomycete Regulator control system participates in various physiology generations such as intracellular osmotic pressure adjusting, metabolism, cell growth and thalli morphology differentiation During thanking.Typical protokaryon type two-component system, mainly consists of two parts：Histidine kinase (histidine Kinase, HK), also known as induction albumen and response regulator (response regulator, RR).When the certain kinds in environment The signaling molecule of type is attached on the induction of signal domain of HK albumen, by ATP as phosphodonor, in conjunction with ATP enzyme domain so that HK The signal of albumen transmits the histidine residue on domain and realizes self-phosphorylation, and phosphate group is transferred to the letter of RR albumen Asparagicacid residue in number acceptance region, and the phosphorylation of aspartic acid leads to the conformational change of RR effect structures, to real The transmission of existing signal.

As the streptomyces coelicolor (S.coelicolor) of streptomycete pattern bacterium, the biology letter of whole genome sequence Breath credit analysis is found that the RR genes of the HK genes and 80 hypothesis of 84 hypothesis, wherein including 67 typical two-component systems System.In streptomyces coelicolor bacterium, there are a pair of of two-component system rapA1A2, have been reported to streptomyces coelicolor Actinorhodin (ACT) and a kind of atypical polyketone object in grade metabolite have positive regulation effect, its effect side Formula is not elucidated with also temporarily.

So far, only a small number of two-component systems, which have been defined in antibiotics production regulation and control, works, and most of The mechanism of action of TCS is unknown.In streptomyces rimosus, similar research is still in space state so far.Also, in view of chain The cometabolism regulated and control network of mould is sufficiently complex, and navigate to has the gene of regulating and controlling effect still really for oxytetracycline yield It is extremely difficult.

Invention content

The purpose of the present invention is to provide one kind improving oxytetracycline yield based on rapA1A2-like bi-component regulating systems Method and recombinant production bacterium.

In the first aspect of the present invention, a kind of method improving oxytetracycline yield is provided, the method includes：Lower soil Mycin produces the activity of rapA1A2-like bi-component regulating systems in bacterium, and with glycine or aspartic acid for unique nitrogen Terramycin production bacterium is cultivated in source, to improve oxytetracycline yield.

In a preference, described lowers rapA1A2-like bi-component regulating systems in terramycin production bacterium Activity includes：(a) gene of knockout or silence rapA1A2-like bi-component regulating systems in terramycin produces bacterium；(b) will The lower adjustment for lowering rapA1A2-like bi-component regulating systems is transferred in terramycin production bacterium；Or (c) adjust terramycin production The stream signal access or upstream gene of rapA1A2-like bi-components regulating system in bacterium, to lower in terramycin production bacterium RapA1A2-like bi-component regulating systems.

In another preferred example, in (a), by the method for gene knockout, rapA1A2- in terramycin production bacterium is lowered The activity of like bi-component regulating systems；Preferably, knocking out response regulator gene rapA1 or induction protein gene rapA2。

In another preferred example, in (b), the lower adjustment is that specificity interference rapA1A2-like bi-components are adjusted The disturbing molecule of the expression of gene in system；Preferably, the disturbing molecule is adjusted with rapA1A2-like bi-components Gene or its transcript in system are dsRNA, antisense nucleic acid, siRNA, the Microrna of inhibition or silence target, or It can express or be formed the construction of the dsRNA, antisense nucleic acid, siRNA, Microrna.

In another preferred example, terramycin production bacterium is streptomyces rimosus.

In another aspect of this invention, the polypeptide of separation is provided, which is selected from the group：(a)SEQ ID NO:1 or 2 institutes Show the polypeptide of amino acid sequence；(b) by SEQ ID NO:Amino acid sequence shown in 1 or 2 is by one or more (such as 1-20； Preferably 1-15；More preferably 1-10, such as 5,3) replacing, missing or adding for amino acid residue and formed, and have There is the polypeptide derived from (a) of the polypeptide identical function of (a)；Or (c) have with (a) protein sequence limited 85% or more homologous Property and with (a) protein function the albumen derived from (a).

In another aspect of this invention, the polynucleotides of separation, the coding polypeptide are provided.

In another aspect of this invention, the purposes that the polypeptide is provided, the soil for producing bacterium as regulation and control terramycin The target of mould yield.

In another aspect of this invention, a kind of genetically engineered terramycin production bacterium is provided, which produces in bacterium The gene of rapA1A2-like bi-component regulating systems is lowered.

In a preference, which produces in bacterium, response regulator gene rapA1 or induction protein gene RapA2 is knocked or silence.

In another aspect of this invention, the purposes that the genetically engineered terramycin production bacterium is provided, for producing Terramycin.

In another aspect of this invention, provide it is a kind of producing the kit of terramycin, wherein including：The hereditary work The terramycin of journey produces bacterium；Or lower the lower adjustment of rapA1A2-like bi-component regulating systems in terramycin production bacterium (such as Expression plasmid or rnai reagent for knocking out the regulating system).

Further include the culture medium that bacterium is produced for cultivating the terramycin in a preference, in the kit, it should Culture medium is using glycine or aspartic acid as only nitrogen source.

The other aspects of the present invention are apparent to those skilled in the art due to this disclosure 's.

Description of the drawings

The growth of Fig. 1, M4018 (rectangular) and M4018 Δs rap (circle) under MM+Gly and MM+Asn condition of culture is bent Line and terramycin synthesis.

A, MM+Asn condition of culture；

B, MM+Gly condition of culture.

Fig. 2, go out the production terramycin level of bacterium germination M4018 and its each mutant strain under the conditions of MM+50mM Gly.

The growth curve (A) and soil of Fig. 3, M4018 (rectangular) and M4018 Δs rap (circle) under MM+Asp condition of culture Mycin synthesizes (B).

Specific implementation mode

The present inventor passes through in-depth study, finds rapA1A2-like bi-component regulating systems in terramycin production bacterium It is closely related with the oxytetracycline yield of the production bacterium, the regulating system is lowered, and in culture with glycine or aspartic acid For only nitrogen source, the oxytetracycline yield of terramycin production bacterium can be extremely significantly increased.Therefore, bis- groups of rapA1A2-like Point regulating system adjusts the substance of the system and method can be applied to realize the improvement of terramycin production bacterium, enhances terramycin Yield.

In the present invention, the rapA1A2-like bi-component regulating systems include response regulator rapA1 and induction Protein gene rapA2.

As described herein, response regulator can be directed to by lowering rapA1A2-like bi-components regulating system RapA1 implements to regulate and control, and can also implement to regulate and control for induction protein gene rapA2.

In the present invention, " rapA1 polypeptides (albumen) " refers to SEQ ID NO:The polypeptide of 1 sequence, further include have with RapA1 polypeptide identical functions, SEQ ID NO:The variant form of 1 sequence.These variant forms include (but being not limited to)： Several (such as 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more preferably such as 1-8,1-5) Missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal addition or lack it is one or several (be usually More preferably it is within 5 within preferably 10 within 20) amino acid.

In the present invention, " rapA2 polypeptides (albumen) " refers to SEQ ID NO:The polypeptide of 2 sequences, further include have with RapA2 polypeptide identical functions, SEQ ID NO:The variant form of 2 sequences.These variant forms include (but being not limited to)： Several (such as 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more preferably such as 1-8,1-5) Missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal addition or lack it is one or several (be usually More preferably it is within 5 within preferably 10 within 20) amino acid.

It is any with the rapA1 polypeptides or rapA2 peptides homologous it is high (such as with SEQ ID NO:Shown in 1 or 2 The homology of sequence is 85% or higher；Preferably, homology is 90% or higher；It is furthermore preferred that homology is 95% or more Height, such as homology 98% or 99%) and albumen that have rapA1 polypeptides or rapA2 polypeptide identical functions be also included within this hair In bright.

Although in specific embodiments of the present invention, listing specific response regulator rapA1 and induction albumen RapA2, however, it is understood that since terramycin produces bacterium there are some different mutation, sequence is highly conserved between them, comes It should be also included in the present invention from the homeopeptide in these native mould production bacterium, to these homeopeptides such as this hair Bright similar or identical regulation and control method should be also included in the present invention.The Method and kit for of the aligned sequences phase same sex is also this Field is known, such as BLAST.

As the preferred embodiment of the present invention, terramycin production bacterium bag includes streptomyces rimosus.

The invention further relates to encode response regulator rapA1 of the present invention and induction albumen rapA2 or its conservative variation The polynucleotide sequence of polypeptide.The polynucleotides can be DNA form or rna form.DNA form includes cDNA, gene Group DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.It compiles The coding region sequence of code mature polypeptide can be with SEQ ID NO:The corresponding native sequence nucleic acid of polypeptide shown in 1 or 2 it is identical or It is the variant of degeneracy.As used herein, " variant of degeneracy ", which refers to coding in the present invention, has SEQ ID NO:1 or 2 Protein, but the coding region sequence differentiated nucleic acid sequence natural with it.

The present invention determines that there are rapA1A2-like bi-component regulating systems in terramycin production bacterium for the first time, and finds it It is closely related with the oxytetracycline yield of the production bacterium.Based on the new discovery, the present invention provides a kind of raising oxytetracycline yields Method, the method includes：The activity of rapA1A2-like bi-component regulating systems in downward terramycin production bacterium, and with Glycine and/or aspartic acid are only nitrogen source culture terramycin production bacterium, to improve oxytetracycline yield.

After the regulating and controlling effect for knowing the rapA1A2-like bi-component regulating systems, this field may be used A variety of methods known to personnel adjust (downward) rapA1A2-like bi-component regulating systems.Including but not limited to：(a) exist Terramycin produces the gene of knockout or silence rapA1A2-like bi-component regulating systems in bacterium；(b) rapA1A2- will be lowered The lower adjustment of like bi-component regulating systems is transferred in terramycin production bacterium；Or (c) adjust rapA1A2- in terramycin production bacterium The stream signal access or upstream gene of like bi-component regulating systems, to lower rapA1A2-like in terramycin production bacterium Bi-component regulating system.

RapA1A2-like bi-component regulating systems include response regulator gene rapA1 or induction albumen rapA2. Therefore, it is a kind of preferred method for lowering rapA1A2-like bi-component regulating systems which lower.

RapA1 or rapA2 is lowered in terramycin produces bacterial strain, and a variety of methods known in the art may be used, including Gene silencing, gene disruption, gene knockout, gene inhibition etc..These methods are included in the present invention.

For example, can by the gene based on homologous recombination be inserted into interrupter technique come in modifying gene group rapA1 or RapA2 genes, so that rapA1 or rapA2 genes are blocked；The design interference of rapA1 or rapA2 genes can also be directed to Property RNA or GEM 132 make rapA1 or rapA2 gene expression inhibitions or silence.

It is a kind of lower rapA1 or rapA2 genes method be Gene silence, in the preferred embodiment of the present invention In, external structure rapA1 or rapA2 gene disruption plasmid, by the method for homologous recombination, in terramycin production bacterial strain dyeing Other independent elements are inserted into body rapA1 or rapA2 gene, so that rapA1 the or rapA2 genes on chromosome are no longer It is capable of the protein of encoding active.When carrying out gene disruption, the selection of independent element is that those skilled in the art are readily selected It arrives, such as using some resistant genes.A kind of method of gene disruption (knockout) see, for example, Genetic Manipulation of Streptomyces:Recorded in a Laboratory Manual.Preferably, the present invention is implemented In example, the independent element includes the subelement in pKC1139 plasmids, such as ori T, Ori pSG5, Apr.In addition, RapA1 or rapA2 genes are subjected to missing knockout, are allowed to lack the key area functioned to be also a kind of feasible downward The strategy of gene.

The two-component system consists of two parts, and as the preferred embodiment of the present invention, only lowers rapA1, this feelings Under condition, no longer need to carry out any genetic manipulation to rapA2 to can be realized under rapA1A2-like bi-component regulating systems It adjusts.Since rapA1 and rapA2 are coexpressions, when transcription is by rapA1 to rapA2, so the blocking of rapA1 so that RapA2 also can not correct transcriptional expression.

In the construction designed for carrying out gene disruption or knockout, at the same include resistance screening gene be it is preferred, To be conducive to subsequently to filter out the bacterial strain that producer is blocked or knocks out.

In a specific embodiment of the present invention, the present inventor, which constructs, blocks plasmid pKC1139-rap and covering plasmid PIB-KA-rap is blocked, covered and is overexpressed bacterial strain by engaging transfer screening.By the strain culturing of acquisition with sweet Propylhomoserin is to find that the oxytetracycline yield for blocking bacterial strain is obviously improved 60% compared to bacterium germination is gone out in the MM culture mediums of only nitrogen source More than, and be overexpressed the oxytetracycline yield in bacterial strain while being remarkably decreased, fully demonstrate rapA1A2- in streptomyces rimosus Like bi-components regulating system acts on the performance negative regulation that terramycin synthesizes.

The present invention also provides the terramycin for lowering rapA1A2-like bi-component regulating systems to produce bacterium, more particularly It is to be inserted into blocking-up method using gene the terramycin production bacterium obtained after rapA1 or rapA2 genes, the bacterial strain is blocked not to express RapA1 or rapA2 genes or expression quantity conspicuousness reduce.The invention further relates to the purposes of the bacterial strain, mould for high yield soil Element.In a specific embodiment of the present invention, by building the blocking bacterial strain of streptomyces rimosus response regulator gene rapA1, It is horizontal to investigate its OTC synthesis under Incubation Condition, finds that bacterium is blocked to add glycine or aspartic acid as unique nitrogen Under the conditions of source, 60% or more bacterium germination will be higher by out by producing plain level.

Work based on the present inventor, the present invention also provides the kits of production terramycin, wherein including：The present invention The genetically engineered terramycin produces bacterium.

The present invention also provides the kits of production terramycin, wherein including：Lower rapA1A2- in terramycin production bacterium The lower adjustment of like bi-component regulating systems；The lower adjustment is, for example, the expression plasmid for knocking out the regulating system, or Rnai reagent.

Can also include other application in the reagent of In The Oxytetracycline Production System in the kit of the described production terramycin, Such as the basal medium (such as MM culture mediums) of terramycin production bacterium, glycine and/or aspartic acid.

It can also include operation instructions in the kit of the production terramycin, illustrate to cultivate the terramycin life The method for producing bacterium, or explanation lower rapA1A2-like bi-component regulating systems in terramycin production bacterium using the lower adjustment Method.

One and the relevant two-component system of terramycin synthesis regulation that the present invention is found in streptomysin produces bacterium for the first time System, and it is particular in that, although the rapA1A2-like bi-components regulating system through the sequence alignment present invention and sky blue There are certain homology (about 75%) for rapA1A2-like bi-components regulating system in streptomycete, but unlike, relatively In rapA1A2 to the positive regulating and controlling effect of streptomyces coelicolor production actinorhodin (ACT), terramycin produces rapA1A2- in bacterium Like bi-components regulating system is negative regulation to the synthesis of terramycin.

Present invention is disclosed terramycin synthesis regulation modes in new streptomyces rimosus, have filled up two-component system in tortoise Split the blank studied in streptomycete.Using two-component system rapA1A2-like in streptomyces rimosus, builds its response and adjust egg The blocking bacterium of white gene rapA1, blocks using glycine or aspartic acid under the MM culture medium conditions of only nitrogen source, to find The terramycin production element of bacterium greatly improves horizontally relative to going out bacterium germination, it was demonstrated that two-component system rapA1A2-like is mould for soil The synthesis of element has negative regulation effect, and a kind of new terramycin synthesis regulation mechanism is thus found that in streptomyces rimosus.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine Condition such as J. Pehanorm Brookers etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the item described in 2002 Part, or according to the normal condition proposed by manufacturer.

Material and method

Strain, plasmid and primer

The thalline and plasmid used in the present invention is as shown in table 1.

Table 1

The sequence of the primer used in the present invention such as table 2.

Table 2

Culture medium

Bran mass (7.5% wheat bran, 2.5% agar), is mainly used for the Spore cultivation of streptomyces rimosus.

MM culture mediums (refer to streptomycete operation manual), and the only nitrogen source in initial MM culture mediums is asparagine It is substituted for the glycine (glycine, Gly) or aspartic acid (Asp) of 50mM in the present invention by (Asparagine, asn), It is mainly used for the measurement of streptomyces rimosus growth and terramycin content.

TSB (Oxoid, USA) culture medium：Mainly for the preparation of mycelium, for genome extraction etc..

LB culture mediums：It is mainly used for cultivating Escherichia coli, MS is used for the engagement transfer operation of Escherichia coli and streptomycete.

Basic molecule manipulation

Plasmid extraction, digestion verification, the basic molecule manipulation reference molecule operation manual such as connection, streptomycete engagement transfer Operation refers to streptomycete operation manual.

S.rimosus is cultivated into the mycelium obtained after 24-36h in TSB and can be used for extracting genome, concrete operations The operational manual that reference reagent supplier provides.

Dry weight and terramycin assay

Zymotic fluid 1mL is taken, suitable 9M hydrochloric acid is added and is acidified, adjusts PH to 1.5~1.7, vibrates mixing, is placed After 5min, 12000rpm centrifuges 5min, by 0.22 μm of water phase membrane filtration of supernatant, completes sample preparation.Using Shimadzu LC-20A high-pressure liquid phase systems and Kromasil C18 columns (4.6 × 200nm) are analyzed.Mobile phase is ultra-pure water 60%, first Alcohol 10%, acetonitrile 20%, the mixed solution of 0.2M phosphatase 11s 0%, flow velocity 0.8mL/min, 10 μ L of liquid inlet volume, Detection wavelength 350nm, 35 DEG C of column temperature will appear a significant terramycin characteristic peak in 3-4min or so with this condition.Fetch earth mycin Powder mark product, prepare 10000U mark product solution, with 0.01M hydrochloric acids to concentration be respectively 0U, 20U, 40U, 60U, 80U, 100U, for drawing standard curve.

In fermentation process, the growing state of thalline is understood by measuring dry cell weight, after fermentation process sampling, room temperature Under, 12000rpm centrifuges 10min, abandons supernatant, remaining wet thallus is placed in 105 DEG C of baking ovens to drying to constant weight, weighs and survey Paint growth curve.

The acquisition of embodiment 1, rapA1A2-like

The present inventor carries out genome sequencing to streptomyces rimosus S.rimosus M4018, therefrom functional-analytical group Cause.By further investigation, the full gene sequence of the two-component system rapA1A2-like in S.rimosus M4018 is obtained. Complete rapA1A2-like gene order length is 2059bp, and HK portion genes (rapA2) length is 1404bp, and amino acid is big Small is 467aa, and RR portion genes (rapA1) length is 663bp, and amino acid size is 220aa, and the two gene order has 8bp's Overlapping.

Following (5 ' -3 ') (SEQ ID NO of sequence of complete rapA1A2 genes: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 ') (the SEQ ID NO of rapA1:3,663bp)：

ATGCGCCTGTTGATCGTGGAGGACGAGAAGCGACTGGCCTTGTCCCTGGCCGGAGGACTGC GGGCCGAGGGATACGCGGTCGATGTGGTGCACGACGGCCTGGAGGGCCTGCACCGGGCGG GCGAGGGCACGTACGACCTGGTGATCCTGGACATCATGCTGCCGGGCATGAACGGCTACCG CGTGTGCGGCGCCCTGCGTGCCGCGGGCAACGAGGTGCCGGTGCTGATGCTGACGGCCAAG GACGGCGAGTACGACGAGGCCGAGGGGCTGGACACCGGCGCGGACGACTATCTGACGAAG CCGTTCTCGTACGTGGTGCTGGTGGCCCGCGTGAAGGCGCTGCTGCGCCGCCGCGGACGGA CGGCGCTGCCCGTGCTGCGCGTGGGAGAGCTGAGCATCGACCAGGGGGCGCACCGCGTGGA GCGCGCCGGCGTGGAGGTGACGCTGACCGCCAAGGAGTTCGCCGTGCTGGAGCAACTGGCG CTGCGCGCGGGCGAGGTGGTGTCCAAGGCCGAGATCCTGGAGCACGTGTGGGACTTCGCGT ACGAGGGCGACAACAACATCGTCGAGGTGTACGTGAGCGCGCTGCGCCGCAAGCTGGGCG CCGCGGCGATCCAGACGGTGCGCGGCGCCGGGTACCGGCTGGTGCCGGGTGCGTAG

RapA1 polypeptide sequences (SEQ ID NO:1,220aa)：

MRLLIVEDEKRLALSLAGGLRAEGYAVDVVHDGLEGLHRAGEGTYDLVILDIMLPGMNGYRV CGALRAAGNEVPVLMLTAKDGEYDEAEGLDTGADDYLTKPFSYVVLVARVKALLRRRGRTAL PVLRVGELSIDQGAHRVERAGVEVTLTAKEFAVLEQLALRAGEVVSKAEILEHVWDFAYEGDN NIVEVYVSALRRKLGAAAIQTVRGAGYRLVPGA

Encode gene order (5 ' -3 ') (the SEQ ID NO of rapA2:4,1404bp)：

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 sequences (SEQ ID NO:2,467aa)：

VRRVIGKVRVRAALGASLVVAVALVAAGTALLLVLKNNLVDQADLQAENTAREVATQIATGK PYEKLDLPDGDDRPVVVLSEDGRVLAAGDDVRAVDGKAVTARQTPPAGQPHDSDDDDDDHEI KPGEVEGKARHTGGTATVGHRTADYRFATVEAKDTQGGKAVVRAGAPLAAEREAVGSVRTA MLIGLPCLLLVVAGVTWLVTRRALRPVEGIRREMAAITASTDLSRRVPEPGSRDEIDRLARTTNE TLGALQESVERQRRFVADASHELRSPIASLRTQLEVGIAHPELLDAPGAVEDAVRLQNLAADLL LLARLDAGERPADARIDLAALVREEVSQRVGDRIAVQVGELAGVEVAGSRSQLGRVLGNLLDN AQRHARESVRASVAREGEWAVLRVEDDGPGVPPEERERIFERFVRLDDARSRDDGGAGLGLAI ARDVAGRHGGTLAVRTGSVFELRLPVA

The foundation for the bacterial strain that embodiment 2, rapA1A2-like bi-component regulating systems are lowered

1, mutant strain builds

By the two-component system rapA1A2- on S.rimosus M4018 genomes first by way of single-swap Response regulator gene rapA1 on like is blocked.Concrete operations mode be by primer DrapF and DrapR from Amplified fragments are connected to plasmid pMD19-TS by the Partial Fragment that rapA1 genes are expanded on S.rimosus M4018 genomes In, it recycles Hind III and Xba I by this segment digestion, this segment is finally connected to the Hind III of plasmid pKC1139 With Xba I sites, to obtain pKC1139-rap.By the method for Conjugative tiansfer, by recombinant plasmid pKC1139-rap from S.rimosus M4018 are transferred in E.coli ET12567, because temperature sensitive type plasmid pKC1139 is under the conditions of higher than 34 DEG C It can not replicate, so by all possible joint element culture under the conditions of 37 DEG C, and A Pula resistance screenings are utilized simultaneously.So Afterwards, the gene for the joint element that extracting screening obtains, utilizes primer rap-single-P1/rap-single-P2, aprF/aprR Further verification determines, the final mutant strain S.rimosus M4018 Δs rap for obtaining rapA1 gene disruptions.

Using plasmid pET-28a as template, kan resistance fragments, design primer pKANTTF, pKANTTR, by kan bases are expanded Because the promoter region of the upstream 100bp of code area and the transcript termination regions of downstream 100bp or so expand together, And NheI restriction enzyme sites are introduced, respectively by pIB139 and the amplified fragments of recycling NheI single endonuclease digestions, by resistance fragments after recycling It is connected with plasmid, after importing DH5 α competence, picking individual colonies, extraction plasmid NheI single endonuclease digestions verification is proved to be successful, illustrates Plasmid construction success, is named as pIB-KA.

Because integrative plasmid pIB-KA can express two kinds of resistances of kanamycins and apramycin, using this plasmid as base Plinth, structure covering plasmid.Using primer rap-pIBF and rap-pIBR by complete rapA1A2-like genes from M4018 bases Because being expanded in group, the downstream of erythromycin promoter on plasmid pIB-KA is inserted into realize the composing type table of the gene It reaches, is named as pIB-KA-rap.Successful plasmid pIB-KA-rap will be built to import in the method that same engagement is shifted In S.rimosus M4018 Δs rap, possible covering is obtained by selecting A Pula and the Double joint element of kanamycins Bacterial strain.Because covering complete rapA1A2-like two-components system there are one containing in bacterial strain, primer rapL/rapR can be used Preliminary identification.

It is M4018 Δs rap (pIB-KA-rap) by the Strain Designation being proved to be successful.Analogize in this approach, by plasmid integration Enter in S.rimosus M4018, then what is built is to be overexpressed bacterial strain M4018 (pIB-KA-rap).

Empty plasmid pIB139-KA is directed respectively into S.rimosus M4018 and S.rimosus M4018 Δs rap, Structure is the empty control plasmid bacterial strain M4018 (pIB-KA) and M4018 Δs rap (pIB-KA) of the two.

Because pIB139 plasmids are inserted into a manner of site-specific integration in streptomyces gene group, occur in the sites att It is more than specificity recombination, design primer attLf and attLr amplification verification attL segments (401bp), primer attRf and attRr Amplification verification attR segments (502bp), come whether mutual authentication plasmid pIB-KA and pIB-KA-rap are integrated into streptomycete with this Genome.

2, fermentation process

By bacterial strain obtained as above respectively in culture on solid bran mass, 30 DEG C, culture medium is collected after 5~7d On spore.The spore count for being generated each mutant strain being collected into using viable plate count method, in inoculation, so that it may with Inoculum concentration is fixed on 1.0 × 10⁶A spore/mL.Culture medium used is using 50mM glycine as the MM liquid of only nitrogen source Culture medium dispenses 50 mL in each 250mL shaking flasks.After unified inoculation, shaking flask is placed on shaking table, 30 DEG C, 220rpm cultures 5~7 days, according to experiment demand sampling.Three shaking flasks of each condition setting are parallel, every to be sampled for 24 hours for measuring dry weight and soil Mycin content.

Embodiment 3, culture bacterial strain and production terramycin

1, the dry weight and production element difference for going out bacterium germination and blocking bacterium under MM+Gly condition of culture

Whether the blocking in order to verify rapA1 genes, which is known as the production of streptomyces rimosus, is influenced, by M4018 and M4018 Δ rap cultures are fermented in liquid MM+50mM Gly culture mediums.It generates within second day enough thalline to start to sample later, for surveying Determine dry weight and terramycin content, and the oxytetracycline yield of unit of account dry weight.In addition, with the asparagine (Asn) of 50mM for MM The only nitrogen source condition of culture medium as a contrast, is compared.

From Figure 1A it is found that when Asn is only nitrogen source, goes out bacterium germination and blocks the dry weight of both bacterium substantially without difference, But under the conditions of Gly, the dry weight outline of bacterial strain is blocked to be higher than bacterium germination.Simultaneously it is found that under the conditions of 50mM Asn, the two Per dry wt production element substantially also without difference, these the result shows that：In the MM culture mediums that Asn is only nitrogen source, bacterium is blocked Strain in terms of growing and producing terramycin with go out bacterium germination and there is no difference.But under the conditions of 50mM Gly, bacterial strain M4018- Δs are blocked The production element of rap is horizontal to be begun to be higher by out bacterium germination at second day, when fermenting to the 5th day (120h), in M4018- Δs rap Per dry wt production element is horizontal to be higher by out bacterium germination M4018 at least 60% or more, such as Figure 1B.

So far, a preliminary conclusion can be obtained, the bacterial strain after rapA1 gene disruptions is only in specific amino acid Under the conditions of can just show the significant difference of oxytetracycline yield, and the production element level of bacterial strain is blocked far to be higher by out bacterium germination, demonstrate,proved It is bright at least using glycine as under the MM culture medium conditions of only nitrogen source, two-component system rapA1A2-like should be negative tune Control the terramycin synthesis in streptomyces rimosus.

2, dry weight and production plain difference of each mutant strain under MM+Gly condition of culture

On the basis of the above experiment, bacterium germination M4018 will be gone out, block bacterium M4018 Δ rap, bacterium is blocked to compare M4018 Δs Rap (pIB-KA), covering bacterium M4018 Δs rap (pIB-KA-rap) go out bacterium germination control M4018 (pIB-KA) and are overexpressed M4018 (pIB-KA-rap) is introduced simultaneously, and same method is inoculated into the liquid MM culture mediums containing 50mM glycine, into One step confirms the influence that this two-component system synthesizes terramycin, while introducing the empty control plasmid of each mutant strain, to arrange Except the polarity effect being likely to occur.

As a result such as Fig. 2, first it can be found that between M4018 and its empty control plasmid M4018 (pIB-KA), M4018- Production element between Δ rap and its empty control plasmid M4018- Δs rap (pIB-KA) is horizontal substantially without difference, also with regard to explanation The importing of plasmid can't cause the difference of thalline production element, and the plain difference of terramycin production is strictly due to two-component system gene Caused by operation.Meanwhile last experimental result, M4018- Δ rap and M4018- Δs rap being also repeated again (pIB-KA) 50% or more M4018 is still higher by when the production horizontal 120h of element in.In addition, being overexpressed bacterial strain M4018 (pIB-KA-rap) it is found in, produces that element is horizontal significantly to have dropped 45% (120h) compared to going out bacterium germination.

By a series of production element of this bacterial strain as a result, more comprehensively having confirmed two-component system rapA1A2-like's The really synthesis of negative regulation terramycin.By lowering two-component system rapA1A2-like, then terramycin can be significantly improved Yield.

3, the dry weight and production element difference for going out bacterium germination and blocking bacterium under MM+Asp condition of culture

The present inventor is used as only nitrogen source, other conditions and " 1 " portion in the present embodiment using aspartic acid (Asp, 50mM) Divide constant, to determine bacterium germination and block bacterium under MM+Asp condition of culture dry weight and production element difference.

As a result see Fig. 3.It is found that going out bacterium germination and blocking the dry weight difference of bacterium between the two little from Fig. 3 A, but unit There were significant differences for the oxytetracycline yield of dry weight.From Fig. 3 B it is found that at the 5th day, the per dry wt production element of M4018 Δs rap is higher by out Bacterium germination 34% or so.

All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, people in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Sequence table

<110>East China University of Science

<120>The method for improving oxytetracycline yield based on rapA1A2-like bi-component regulating systems

<130> 180687

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 220

<212> PRT

<213>Streptomyces rimosus (S. 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 (S. 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 (S. 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 (S. 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 (S. 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>Primer (Primer)

<400> 6

cccaagctta cacctcgacg atgttgttg 29

<210> 7

<211> 28

<212> DNA

<213>Primer (Primer)

<400> 7

tgctctagaa tacgcggtcg atgtggtg 28

<210> 8

<211> 21

<212> DNA

<213>Primer (Primer)

<400> 8

gtgcaatacg aatggcgaaa a 21

<210> 9

<211> 19

<212> DNA

<213>Primer (Primer)

<400> 9

tcagccaatc gactggcga 19

<210> 10

<211> 23

<212> DNA

<213>Primer (Primer)

<400> 10

aaccggtagt ccgccgtacg gtg 23

<210> 11

<211> 26

<212> DNA

<213>Primer (Primer)

<400> 11

aggttgagaa gctgaccgat gagctc 26

<210> 12

<211> 30

<212> DNA

<213>Primer (Primer)

<400> 12

ctagctagcc tcagtggaac gaaaactcac 30

<210> 13

<211> 31

<212> DNA

<213>Primer (Primer)

<400> 13

ctagctagca caatttcagg tggcactttt c 31

<210> 14

<211> 28

<212> DNA

<213>Primer (Primer)

<400> 14

cggaattcct acgccaccgg caggcgta 28

<210> 15

<211> 31

<212> DNA

<213>Primer (Primer)

<400> 15

ggaattccat atgcgcctgt tgatcgtgga g 31

<210> 16

<211> 24

<212> DNA

<213>Primer (Primer)

<400> 16

ctacgccacc ggcaggcgta gttc 24

<210> 17

<211> 24

<212> DNA

<213>Primer (Primer)

<400> 17

atgcgcctgt tgatcgtgga ggac 24

<210> 18

<211> 21

<212> DNA

<213>Primer (Primer)

<400> 18

gttcacccac agctggaggc c 21

<210> 19

<211> 21

<212> DNA

<213>Primer (Primer)

<400> 19

gctcgacttc gcgctgaagg t 21

<210> 20

<211> 21

<212> DNA

<213>Primer (Primer)

<400> 20

gctataatga ccccgaagca g 21

<210> 21

<211> 17

<212> DNA

<213>Primer (Primer)

<400> 21

tcgtcatgcc ccgcagt 17

Claims

1. a kind of method improving oxytetracycline yield, which is characterized in that the method includes：It lowers in terramycin production bacterium The activity of rapA1A2-like bi-component regulating systems, and using glycine or aspartic acid as the only nitrogen source culture terramycin Bacterium is produced, to improve oxytetracycline yield.

2. the method as described in claim 1, which is characterized in that rapA1A2-like is bis- in the downward terramycin production bacterium The activity of composition regulation system includes：

(a) gene of knockout or silence rapA1A2-like bi-component regulating systems in terramycin produces bacterium；

(b) the lower adjustment for lowering rapA1A2-like bi-component regulating systems is transferred in terramycin production bacterium；Or

(c) the stream signal access or upstream gene of rapA1A2-like bi-component regulating systems in terramycin production bacterium are adjusted, To lower rapA1A2-like bi-component regulating systems in terramycin production bacterium.

3. method as claimed in claim 2, which is characterized in that in (a), by the method for gene knockout, lower terramycin life Produce the activity of rapA1A2-like bi-component regulating systems in bacterium；Preferably, knocking out response regulator gene rapA1 or induction Protein gene rapA2.

4. method as claimed in claim 2, which is characterized in that in (b), the lower adjustment is specificity interference rapA1A2- The disturbing molecule of the expression of gene in like bi-component regulating systems；Preferably, the disturbing molecule is with rapA1A2- Gene or its transcript in like bi-component regulating systems are inhibition or the dsRNA of silence target, antisense nucleic acid, small interference RNA, Microrna, or can express or be formed the construction of the dsRNA, antisense nucleic acid, siRNA, Microrna.

5. the method as described in claim 1, which is characterized in that the terramycin production bacterium is streptomyces rimosus.

6. the polypeptide of separation, the polypeptide are selected from the group：

(a)SEQ ID NO:The polypeptide of amino acid sequence shown in 1 or 2；

(b) by SEQ ID NO:Amino acid sequence shown in 1 or 2 passes through the substitution of one or more amino acid residues, lacks or add Add and formed, and the polypeptide derived from (a) of the polypeptide identical function with (a)；Or

(c) there is 85% or more homology with (a) protein sequence limited and there is the albumen derived from (a) of (a) protein function.

7. the polynucleotides of separation encode the polypeptide described in claim 6.

8. the purposes of the polypeptide described in claim 6 or 7, the target of the native mould yield for producing bacterium as regulation and control terramycin Mark.

9. a kind of genetically engineered terramycin produces bacterium, which is characterized in that it is bis- that the terramycin produces rapA1A2-like in bacterium The gene of composition regulation system is lowered.

10. terramycin as claimed in claim 9 produces bacterium, which is characterized in that the terramycin produces in bacterium, response regulator Gene rapA1 or induction protein gene rapA2 are knocked or silence.

11. the purposes of the genetically engineered terramycin production bacterium described in claim 9 or 10, for producing terramycin.

12. a kind of kit producing terramycin, which is characterized in that wherein include：

Genetically engineered terramycin described in claim 10 or 11 produces bacterium；Or

Lower the lower adjustment of rapA1A2-like bi-component regulating systems in terramycin production bacterium.

13. kit as claimed in claim 12, which is characterized in that further include mould for cultivating the soil in the kit The culture medium of element production bacterium, the culture medium is using glycine or aspartic acid as only nitrogen source.