CN108424946A

CN108424946A - The method for improving oxytetracycline yield based on afsQ1Q2-like bi-component regulating systems

Info

Publication number: CN108424946A
Application number: CN201810089411.0A
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: CN108424946B

Abstract

The present invention relates to the methods for improving oxytetracycline yield based on afsQ1Q2 like bi-component regulating systems.Present invention firstly provides afsQ1Q2 like bi-components regulating systems in terramycin production bacterium and the oxytetracycline yield of the production bacterium are closely related, lower 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, afsQ1Q2 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 afsQ1Q2-like bi-component regulating systems

Technical field

The invention belongs to biotechnologies；More particularly it relates to be adjusted based on afsQ1Q2-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, the extensive use in clinical and fishery has the prodigious market demand.

A kind of secondary metabolite that terramycin is synthesized as streptomyces rimosus, synthesis regulation are related to approach specificity Complicated interaction between regulatory factor and the global regulation factor, and then reinforce or inhibit antibiotic biosynthesis gene Expression.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, N-terminal include the DNA binding domain of an OmpR type, and C-terminal is a transcriptional activation Domain (Bacterial Transcriptional Activation Domain, BTAD).It is had also been discovered that in streptomyces rimosus One approach specific regulatory control factor, it is also to belong to SARP families, is located at OTC gene clusters, and then transporter gene OtrB is named as otcR.It can be directly with the oxy genes in gene cluster promoter bind directly, to regulate and control OTC's Synthesis.

And it is a kind of that two-component regulating system (Two-Component Regulatory System, TCS), which is in streptomycete, Very important global regulation's system participates in intracellular osmotic pressure adjusting, metabolism, cell growth and thalli morphology differentiation During various physiological metabolisms.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).Work as ring Certain types of signaling molecule in border is attached on the induction of signal domain of HK albumen, by ATP as phosphodonor, in conjunction with ATP Enzyme domain so that the signal of HK albumen transmits the histidine residue on domain and realizes self-phosphorylation, and phosphate group is transferred to Asparagicacid residue in the signal acceptance region of RR albumen, and the phosphorylation of aspartic acid causes the conformation of RR effect structures to change Become, to realize the transmission of signal.

As the streptomyces coelicolor (S.coelicolor) of streptomycete pattern bacterium, the biological information of whole genome sequence 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. In streptomyces coelicolor bacterium, there are a pair of of two-component system afsQ1Q2, have been reported to streptomyces coelicolor secondary generation Thanking actinorhodin (ACT) undecylprodigiosin (RED) in product and Ca-dependent antibiotic (CDA), there are positive regulation and control Effect, its mode of action is direct with the promoter of the approach specific regulatory control factor actII-ORF4, redZ and cdaR respectively In conjunction with.

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 strepto- The cometabolism regulated and control network of bacterium is sufficiently complex, and it is still pole that navigate to for oxytetracycline yield has the gene of regulating and controlling effect really It is difficult.

Invention content

The purpose of the present invention is to provide one kind improving oxytetracycline yield based on afsQ1Q2-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：It is mould to lower soil The activity of afsQ1Q2-like bi-component regulating systems in element production bacterium, and trained as only nitrogen source using glycine or aspartic acid It supports the terramycin and produces bacterium, to improve oxytetracycline yield.

In a preference, the work for lowering afsQ1Q2-like bi-component regulating systems in terramycin production bacterium Property includes：(a) gene of knockout or silence afsQ1Q2-like bi-component regulating systems in terramycin produces bacterium；(b) will under The lower adjustment of afsQ1Q2-like bi-component regulating systems is adjusted to be transferred in terramycin production bacterium；Or it (c) adjusts terramycin and produces bacterium The stream signal access or upstream gene of middle afsQ1Q2-like bi-components regulating system, to lower in terramycin production bacterium AfsQ1Q2-like bi-component regulating systems.

In another preferred example, in (a), by the method for gene knockout, afsQ1Q2- in terramycin production bacterium is lowered The activity of like bi-component regulating systems；Preferably, knocking out response regulator gene afsQ1 or induction protein gene afsQ2.

In another preferred example, in (b), the lower adjustment is that specificity interference afsQ1Q2-like bi-components adjust system The disturbing molecule of the expression of gene in system；Preferably, the disturbing molecule is to adjust to be with afsQ1Q2-like bi-components Gene or its transcript in system are dsRNA, antisense nucleic acid, siRNA, the Microrna of inhibition or silence target, or can table Reach or 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) there is 85% or more homology with (a) protein sequence limited And the albumen derived from (a) with (a) protein function.

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 afsQ1Q2-like bi-component regulating systems is lowered.

In a preference, which produces in bacterium, response regulator gene afsQ1 or induction protein gene AfsQ2 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 genetic engineering The terramycin of change produces bacterium；Or the lower adjustment of afsQ1Q2-like bi-component regulating systems in terramycin production bacterium is lowered (as used In the expression plasmid or rnai reagent that knock 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 curve of Fig. 1, M4018 (rectangular) and M4018 Δs afs (circle) under MM+Asn and MM+Gly condition of culture It is synthesized with terramycin.

A, MM+Asn condition of culture；

B, MM+Gly condition of culture.

Fig. 2, go out the production terramycin water of bacterium germination M4018 and its each modified recombinant bacterial strain under the conditions of MM+50mM Gly It is flat.

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

Specific implementation mode

The present inventor pass through in-depth study, find terramycin production bacterium in afsQ1Q2-like bi-components regulating system with The oxytetracycline yield of the production bacterium is closely related, lowers the regulating system, and be with glycine or aspartic acid in culture Only nitrogen source can extremely significantly increase the oxytetracycline yield of terramycin production bacterium.Therefore, afsQ1Q2-like bi-components tune Section system or adjust the system substance and method can be applied to realize terramycin production bacterium improvement, enhance the production of terramycin Amount.

In the present invention, the afsQ1Q2-like bi-component regulating systems include response regulator afsQ1 and induction Albumen afsQ2.

As described herein, response regulator can be directed to by lowering afsQ1Q2-like bi-components regulating system AfsQ1 implements to regulate and control, and can also implement to regulate and control for induction albumen afsQ2.

In the present invention, " afsQ1 polypeptides (albumen) " refers to SEQ ID NO:The polypeptide of 1 sequence, further include have with AfsQ1 polypeptide identical functions, SEQ ID NO:The variant form of 1 sequence.These variant forms include (but being not limited to)：If Dry (such as 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more preferably such as 1-8,1-5) amino Acid missing, insertion and/or substitution, and C-terminal and/or N-terminal addition or lack it is one or several (be usually 20 with It is interior, be more preferably within 5 within preferably 10) amino acid.

In the present invention, " afsQ2 polypeptides (albumen) " refers to SEQ ID NO:The polypeptide of 2 sequences, further include have with AfsQ2 polypeptide identical functions, SEQ ID NO:The variant form of 2 sequences.These variant forms include (but being not limited to)：If Dry (such as 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more preferably such as 1-8,1-5) amino Acid missing, insertion and/or substitution, and C-terminal and/or N-terminal addition or lack it is one or several (be usually 20 with It is interior, be more preferably within 5 within preferably 10) amino acid.

It is any with the afsQ1 polypeptides or afsQ2 peptides homologous it is high (such as with SEQ ID NO:Sequence shown in 1 or 2 The homology of row is 85% or higher；Preferably, homology is 90% or higher；It is furthermore preferred that homology is 95% or higher, Such as homology 98% or 99%) and albumen that have afsQ1 polypeptides or afsQ2 polypeptide identical functions be also included in the present invention.

Although in specific embodiments of the present invention, listing specific response regulator afsQ1 and induction albumen AfsQ2, however, it is understood that since terramycin produces bacterium there are some different mutation, sequence is highly conserved between them, comes from Homeopeptide in these native mould production bacterium should be also included in the present invention, to these homeopeptides such as class of the present invention Seemingly 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 week Know, 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 afsQ1 of the present invention and induction albumen afsQ2 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.Coding The coding region sequence of mature polypeptide can be with SEQ ID NO:The corresponding native sequence nucleic acid of polypeptide shown in 1 or 2 is identical either 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 for the first time determine terramycin production bacterium in there are afsQ1Q2-like bi-component regulating systems, and find its with The oxytetracycline yield of the production bacterium is closely related.Based on the new discovery, the present invention provides a kind of sides for improving oxytetracycline yield Method, the method includes：The activity of afsQ1Q2-like bi-component regulating systems in terramycin production bacterium is lowered, and with sweet ammonia Acid or aspartic acid are only nitrogen source culture terramycin production bacterium, to improve oxytetracycline yield.

After the regulating and controlling effect for knowing the afsQ1Q2-like bi-component regulating systems, people from this field may be used A variety of methods adjust (downward) afsQ1Q2-like bi-component regulating systems known to member.Including but not limited to：(a) mould in soil The gene of knockout or silence afsQ1Q2-like bi-component regulating systems in element production bacterium；(b) downward afsQ1Q2-like is bis- The lower adjustment of composition regulation system is transferred in terramycin production bacterium；Or (c) adjust terramycin production bacterium in afsQ1Q2-like it is bis- The stream signal access or upstream gene of composition regulation system, to lower afsQ1Q2-like bi-component tune in terramycin production bacterium Section system.

AfsQ1Q2-like bi-component regulating systems include response regulator gene afsQ1 or induction albumen afsQ2.Cause This, it is a kind of preferred method for lowering afsQ1Q2-like bi-component regulating systems which, which lower,.

AfsQ1 or afsQ2 is lowered in terramycin produces bacterial strain, and a variety of methods known in the art, including base may be used Because of silence, 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 afsQ1 or AfsQ2 genes, so that afsQ1 or afsQ2 genes are blocked；AfsQ1 or afsQ2 genes can also be directed to and design interference RNA or GEM 132 make afsQ1 or afsQ2 gene expression inhibitions or silence.

It is a kind of lower afsQ1 or afsQ2 genes method be Gene silence, in the preferred embodiment of the present invention In, external structure afsQ1 or afsQ2 gene disruption plasmid produces strain chromosome by the method for homologous recombination in terramycin Other independent elements are inserted into afsQ1 or afsQ2 genes, so that afsQ1 the or afsQ2 genes on chromosome are no longer able to The protein of encoding active.When carrying out gene disruption, the selection of independent element, which is that those skilled in the art are readily selected, 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, in the embodiment of the present invention, the nothing It includes the subelement in pKC1139 plasmids, such as ori T, Ori pSG5, Apr to close element.In addition, by afsQ1 or afsQ2 Gene carries out missing knockout, is allowed to lack the key area functioned to be also a kind of strategy of feasible down-regulated gene.

The two-component system consists of two parts, and as the preferred embodiment of the present invention, only lowers afsQ1, this feelings Under condition, no longer need to carry out any genetic manipulation to afsQ2 to can be realized under afsQ1Q2-like bi-component regulating systems It adjusts.Since afsQ1 and afsQ2 are coexpressions, when transcription is by afsQ1 to afsQ2, so the blocking of afsQ1 so that AfsQ2 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-afs and covering plasmid PIB-KA-afs is blocked by engaging transfer screening, covering and overexpression bacterial strain.By the strain culturing of acquisition with sweet ammonia In acid or the MM culture mediums that aspartic acid is only nitrogen source, find to block the oxytetracycline yield of bacterial strain significantly to carry compared to going out bacterium germination 60% or more is risen, and is overexpressed the oxytetracycline yield in bacterial strain while being remarkably decreased, is fully demonstrated in streptomyces rimosus AfsQ1Q2-like bi-components regulating system acts on the performance negative regulation that terramycin synthesizes.

The present invention also provides the terramycin for lowering afsQ1Q2-like bi-component regulating systems to produce bacterium, more particularly Being inserted into blocking-up method using gene blocks the terramycin obtained after afsQ1 or afsQ2 genes to produce bacterium, which does not express AfsQ1 or afsQ2 genes or expression quantity conspicuousness reduce.The invention further relates to the purposes of the bacterial strain, are used for high yield terramycin. In a specific embodiment of the present invention, it by building the blocking bacterial strain of streptomyces rimosus response regulator gene afsQ1, investigates Its OTC synthesis under Incubation Condition is horizontal, and discovery blocking bacterium is only nitrogen source item in addition glycine or aspartic acid Under part, 60% or more bacterium germination will be higher by out by producing plain level, and even up to 80% or more.

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

The present invention also provides the kits of production terramycin, wherein including：Lower afsQ1Q2- 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 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 afsQ1Q2-like bi-component regulating systems in terramycin production bacterium using lower adjust 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 afsQ1Q2-like bi-components regulating system through the sequence alignment present invention and sky blue There are certain homologys for afsQ1Q2-like bi-components regulating system in streptomycete, but unlike, relative to afsQ1Q2 All to streptomyces coelicolor production actinorhodin (ACT), undecylprodigiosin (RED) and Ca-dependent antibiotic (CDA) Just regulate and control, it is negative regulation to the synthesis of terramycin that terramycin, which produces afsQ1Q2-like bi-components regulating system in bacterium,.

Present invention is disclosed terramycin synthesis regulation modes in a kind of new streptomyces rimosus, have filled up two-component system and have existed The blank studied in streptomyces rimosus.Using two-component system afsQ1Q2-like in streptomyces rimosus, its response adjusting is built The blocking bacterium of protein gene afsQ1 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 afsQ1Q2-like is for terramycin Synthesis have negative regulation effect, 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 the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part is such as《Molecular Cloning:A Laboratory guide》Or《Streptomycete operation manual》Described in condition, or according to the item proposed by manufacturer Part.

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)：It is mainly used for the Spore cultivation of streptomyces rimosus.

MM culture mediums (refer to streptomycete operation manual)：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 behaviour Make to refer 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 ginseng The operational manual of reagent suppliers offer is provided.

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%, methanol 10%, acetonitrile 20%, the mixed solution of 0.2M phosphatase 11s 0%, flow velocity 0.8mL/min, liquid inlet volume 10 μ L, 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, It prepares 10000U and marks product solution, be respectively 0U, 20U, 40U, 60U, 80U, 100U with 0.01M hydrochloric acids to concentration, for painting Standard curve processed.

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 and draw Growth curve.

The acquisition of embodiment 1, afsQ1Q2-like

The present inventor carries out genome sequencing to streptomyces rimosus S.rimosus M4018, therefrom analytic function gene. By further investigation, the full gene sequence of the two-component system afsQ1Q2-like in S.rimosus M4018 is obtained.Entirely The overall length of afsQ1Q2 genes is 2639bp, and HK portion genes (afsQ2) length is 1965bp, and amino acid size is 654aa, RR Portion gene (afsQ1) length is 678bp, and amino acid size is 225aa, and two gene orders have the juxtaposition of 4bp.Completely AfsQ1Q2 genes following (the SEQ ID NO of full sequence:5,5 ' -3 ')：

GTGCCTTTCCTGTTGCTGATCGAGGACGACGACGCCATCCGCACGGCCCTCGAACTCTCGCTGTCCCGC CAGGGTCACCGTGTGGTGACCGCGGCGACGGGCGAGGACGGCCTGAAGCTGCTGCGCGAGCAGCGCCCGGACCTGAT CGTGCTGGACGTGATGCTCCCGGGCATCGACGGTTTCGAGGTGTGCCGGCGGATCAGGCGGACGGACCAGCTGCCCA TCATCCTGCTGACCGCGCGCAACGACGACATCGACGTGGTGGTCGGGCTGGAGTCGGGCGCGGACGACTACGTGGTC AAGCCGGTGCAGGGCCGGGTGCTGGACGCCCGTATCCGGGCCGTGCTGCGGCGCGGGGAGCGCGAGTCCAACGACGC GGCGACCTTCGGCTCGCTGGTCATCGACCGCTCGGCGATGACGGTCACCAAGAACGGCGAGGATCTGCAACTGACAC CCACCGAGCTGCGGTTGCTGCTGGAGCTCAGCCGCCGCCCCGGCCAGGCGCTGTCCCGGCAGCAGCTGCTGCGCCTG GTGTGGGAGCACGACTACCTGGGTGACTCGCGGCTGGTGGACGCGTGCGTGCAGCGGCTGCGCGCGAAGGTGGAGGA CGTACCGTCCTCGCCGACCTTGATCCGTACGGTCCGCGGTGTCGGCTACCGGCTGGACACCCCGCAGTGACCGAACG CTCGACCGGTCCGGACCCGGACCGTACGGACGGCCCGCGGGACACGGCCCGGCCGGGCAGGGACAGGAAGGACAGGG ACGTGGGGGGCCGTACGGGCACCGACGGCGCGGGTGCCGGGCGGACGGACGGGCGTGCGGGCGGCCGTACGGCGGAC GACACGGAGCGGACCGCCGACGGCACGGCCGACGAGGGCGCGCCCGGACAGGAGCGCTCCTCGCTGCTCGTACGGTG CGTACGGGCGCTCGGCCTGCTGCGCTGGACCAGCCTGCGGCTGCGGCTGGTCTTCGTGTTCGCGCTGGTCGCGCTGA CCGCGGCGGTCTCCGCCTCCGGCATCGCGTACTGGCTCAACCGCAACACGGTGCTGGACCGCACCCAGAACTCCGCG CTCAACGACTTCCACAAGTCGCTGGAGGACAGCACCCGCTCGCTGCCGCTGCGCCCGCACTGCGCGGAGCTTCAGGA CGCCGCGCGGCAGATGGCGGGCGGGCCGCAGAACTACGCCGTGCTGCTCGTCATGCGCGGCGCGGACGGCCGGCAGT GCGTCGCGACCACCGACAAGGACCTCTTCACGCTCAAGACGGTGCCGCAGTCGCTCCAGACCGCGGTCAACGAGAAG CGCGCGCTCGACGACGGCAACCGGGTGCCGTACCACATGTACTGGCAGCGCAAGGAGCTGCACGACACCCCGTACCT CGTCGCCGGTGCCCGGATGAACGGCGGCGGCCCGACCGGCTACATGCTCAAGTCGCTGGCCACCGAGCGTCAGGACC TGAACTCGCTGGCCTGGTCGCTGGGCATCGCCACCGCGCTGGCCCTGGTCGGCTCGGCGCTGCTGGCGCAGGCCGCC GCGACCACCGTGCTGCGGCCGGTGCAGCGCCTGGGCCACGCGGCCCGGCAGCTGGGCGAGGGGCGGCTGGACACCCG GCTGCGGGTGACCGGCACCGACGAGCTCGCCGACCTCTCCCGTACGTTCAACCGCACCGCCGAGCGGCTGGAGCAGC GGGTGGCCGAGCTGAGCGGGCGGGAGGCGGCGTCCCGCCGCTTCGTCGCCGACATGTCGCACGAGCTGCGCACGCCG CTGACGGCCATCACCGCGGTCACCGAGGTGCTGGAGGAAGAGGAGGACTCCCTCGACCCGATGATCGCGCCCGCGGT GAAGCTGGTGGTCAGCGAGACCCGGCGGCTGAACGACCTGGTGGAGAACCTCATGGAGGTCACCCGCTTCGACGCGG GGACGGCACGGCTGGTGCAGGACGACGTGGACGTGGCGGACCAGGTGACCGCGTGCATCGACGCGCGGGCCTGGCTG GACGCGGTCGAGCTGGACGCCGAGCGCGGCATCGTGGCCCGTCTGGACCCGCGCCGCCTCGACGTCATCCTCGCCAA CCTCATCGGCAACGCGCTCAAGCACGGCGGCTCGCCGGTCCGGGTGTCCGTACGGACCCAGGCCACGCCGGAGGGCG AGGACCTGCTGATCCGGGTACGCGACCACGGCCCCGGCATCCCGCAGGAGGTCCTGCCGCACGTCTTCGACCGCTTC TACAAGGCGAGCGCGTCCCGGCCGCGGTCGGAGGGCAGCGGCCTGGGCCTGTCGATCGCCCTGGAGAACGCGCACAT CCACGGTGGCGAGATCACCGCCGCGAACTCGCCGGAGGGCGGCGCGGTCTTCACGCTGCGGCTGCCGGTGGGGACCG GCGGGGCTGCGGAGGAGGGGCAGGAGGACGGGCAGGGGGCTGCGCAGGGCGTCGCGCAGGGGACCGCGCAGGAGGCG CCGAAGGCGCCGGAGACGCCGGAGACGCCGGAGACGCCGGACGGGACCGCTGTATCGGAGCGTGCGGAGGACGCGAC CCCGGGTACGGGCTCCGCACCCGGCCCCGGCTCCGACCCCGACGCCGCCCCGGACTCCGGACCCGACTCAGGCCCCC GCCGCAAGGAAGGCGGTGACCGCCGATGA

The gene order of afsQ1 is (SEQ ID NO:3,678bp, 5 ' -3 ')：

GTGCCTTTCCTGTTGCTGATCGAGGACGACGACGCCATCCGCACGGCCCTCGAACTCTCGCTGTCCCGC CAGGGTCACCGTGTGGTGACCGCGGCGACGGGCGAGGACGGCCTGAAGCTGCTGCGCGAGCAGCGCCCGGACCTGAT CGTGCTGGACGTGATGCTCCCGGGCATCGACGGTTTCGAGGTGTGCCGGCGGATCAGGCGGACGGACCAGCTGCCCA TCATCCTGCTGACCGCGCGCAACGACGACATCGACGTGGTGGTCGGGCTGGAGTCGGGCGCGGACGACTACGTGGTC AAGCCGGTGCAGGGCCGGGTGCTGGACGCCCGTATCCGGGCCGTGCTGCGGCGCGGGGAGCGCGAGTCCAACGACGC GGCGACCTTCGGCTCGCTGGTCATCGACCGCTCGGCGATGACGGTCACCAAGAACGGCGAGGATCTGCAACTGACAC CCACCGAGCTGCGGTTGCTGCTGGAGCTCAGCCGCCGCCCCGGCCAGGCGCTGTCCCGGCAGCAGCTGCTGCGCCTG GTGTGGGAGCACGACTACCTGGGTGACTCGCGGCTGGTGGACGCGTGCGTGCAGCGGCTGCGCGCGAAGGTGGAGGA CGTACCGTCCTCGCCGACCTTGATCCGTACGGTCCGCGGTGTCGGCTACCGGCTGGACACCCCGCAGTGA

The polypeptide sequence of afsQ1 is (SEQ ID NO:1,225aa)：

VPFLLLIEDDDAIRTALELSLSRQGHRVVTAATGEDGLKLLREQRPDLIVLDVMLPGIDGFEVCRRIRR TDQLPIILLTARNDDIDVVVGLESGADDYVVKPVQGRVLDARIRAVLRRGERESNDAATFGSLVIDRSAMTVTKNGE DLQLTPTELRLLLELSRRPGQALSRQQLLRLVWEHDYLGDSRLVDACVQRLRAKVEDVPSSPTLIRTVRGVGYRLDT PQ

The gene order of afsQ2 is (SEQ ID NO:4,1965bp, 5 ' -3 ')：

GTGACCGAACGCTCGACCGGTCCGGACCCGGACCGTACGGACGGCCCGCGGGACACGGCCCGGCCGGGC AGGGACAGGAAGGACAGGGACGTGGGGGGCCGTACGGGCACCGACGGCGCGGGTGCCGGGCGGACGGACGGGCGTGC GGGCGGCCGTACGGCGGACGACACGGAGCGGACCGCCGACGGCACGGCCGACGAGGGCGCGCCCGGACAGGAGCGCT CCTCGCTGCTCGTACGGTGCGTACGGGCGCTCGGCCTGCTGCGCTGGACCAGCCTGCGGCTGCGGCTGGTCTTCGTG TTCGCGCTGGTCGCGCTGACCGCGGCGGTCTCCGCCTCCGGCATCGCGTACTGGCTCAACCGCAACACGGTGCTGGA CCGCACCCAGAACTCCGCGCTCAACGACTTCCACAAGTCGCTGGAGGACAGCACCCGCTCGCTGCCGCTGCGCCCGC ACTGCGCGGAGCTTCAGGACGCCGCGCGGCAGATGGCGGGCGGGCCGCAGAACTACGCCGTGCTGCTCGTCATGCGC GGCGCGGACGGCCGGCAGTGCGTCGCGACCACCGACAAGGACCTCTTCACGCTCAAGACGGTGCCGCAGTCGCTCCA GACCGCGGTCAACGAGAAGCGCGCGCTCGACGACGGCAACCGGGTGCCGTACCACATGTACTGGCAGCGCAAGGAGC TGCACGACACCCCGTACCTCGTCGCCGGTGCCCGGATGAACGGCGGCGGCCCGACCGGCTACATGCTCAAGTCGCTG GCCACCGAGCGTCAGGACCTGAACTCGCTGGCCTGGTCGCTGGGCATCGCCACCGCGCTGGCCCTGGTCGGCTCGGC GCTGCTGGCGCAGGCCGCCGCGACCACCGTGCTGCGGCCGGTGCAGCGCCTGGGCCACGCGGCCCGGCAGCTGGGCG AGGGGCGGCTGGACACCCGGCTGCGGGTGACCGGCACCGACGAGCTCGCCGACCTCTCCCGTACGTTCAACCGCACC GCCGAGCGGCTGGAGCAGCGGGTGGCCGAGCTGAGCGGGCGGGAGGCGGCGTCCCGCCGCTTCGTCGCCGACATGTC GCACGAGCTGCGCACGCCGCTGACGGCCATCACCGCGGTCACCGAGGTGCTGGAGGAAGAGGAGGACTCCCTCGACC CGATGATCGCGCCCGCGGTGAAGCTGGTGGTCAGCGAGACCCGGCGGCTGAACGACCTGGTGGAGAACCTCATGGAG GTCACCCGCTTCGACGCGGGGACGGCACGGCTGGTGCAGGACGACGTGGACGTGGCGGACCAGGTGACCGCGTGCAT CGACGCGCGGGCCTGGCTGGACGCGGTCGAGCTGGACGCCGAGCGCGGCATCGTGGCCCGTCTGGACCCGCGCCGCC TCGACGTCATCCTCGCCAACCTCATCGGCAACGCGCTCAAGCACGGCGGCTCGCCGGTCCGGGTGTCCGTACGGACC CAGGCCACGCCGGAGGGCGAGGACCTGCTGATCCGGGTACGCGACCACGGCCCCGGCATCCCGCAGGAGGTCCTGCC GCACGTCTTCGACCGCTTCTACAAGGCGAGCGCGTCCCGGCCGCGGTCGGAGGGCAGCGGCCTGGGCCTGTCGATCG CCCTGGAGAACGCGCACATCCACGGTGGCGAGATCACCGCCGCGAACTCGCCGGAGGGCGGCGCGGTCTTCACGCTG CGGCTGCCGGTGGGGACCGGCGGGGCTGCGGAGGAGGGGCAGGAGGACGGGCAGGGGGCTGCGCAGGGCGTCGCGCA GGGGACCGCGCAGGAGGCGCCGAAGGCGCCGGAGACGCCGGAGACGCCGGAGACGCCGGACGGGACCGCTGTATCGG AGCGTGCGGAGGACGCGACCCCGGGTACGGGCTCCGCACCCGGCCCCGGCTCCGACCCCGACGCCGCCCCGGACTCC GGACCCGACTCAGGCCCCCGCCGCAAGGAAGGCGGTGACCGCCGATGA

The polypeptide sequence of afsQ2 is (SEQ ID NO:2,654aa)：

VTERSTGPDPDRTDGPRDTARPGRDRKDRDVGGRTGTDGAGAGRTDGRAGGRTADDTERTADGTADEGA PGQERSSLLVRCVRALGLLRWTSLRLRLVFVFALVALTAAVSASGIAYWLNRNTVLDRTQNSALNDFHKSLEDSTRS LPLRPHCAELQDAARQMAGGPQNYAVLLVMRGADGRQCVATTDKDLFTLKTVPQSLQTAVNEKRALDDGNRVPYHMY WQRKELHDTPYLVAGARMNGGGPTGYMLKSLATERQDLNSLAWSLGIATALALVGSALLAQAAATTVLRPVQRLGHA ARQLGEGRLDTRLRVTGTDELADLSRTFNRTAERLEQRVAELSGREAASRRFVADMSHELRTPLTAITAVTEVLEEE EDSLDPMIAPAVKLVVSETRRLNDLVENLMEVTRFDAGTARLVQDDVDVADQVTACIDARAWLDAVELDAERGIVAR LDPRRLDVILANLIGNALKHGGSPVRVSVRTQATPEGEDLLIRVRDHGPGIPQEVLPHVFDRFYKASASRPRSEGSG LGLSIALENAHIHGGEITAANSPEGGAVFTLRLPVGTGGAAEEGQEDGQGAAQGVAQGTAQEAPKAPETPETPETPD GTAVSERAEDATPGTGSAPGPGSDPDAAPDSGPDSGPRRKEGGDRR

The foundation for the bacterial strain that embodiment 2, afsQ1Q2-like bi-component regulating systems are lowered

1, mutant strain builds

By the two-component system on streptomyces rimosus (S.rimosus) M4018 genomes first by way of single-swap Response regulator gene afsQ1 on afsQ1Q2-like is blocked.Concrete operations mode is to pass through primer DafsF and DafsR Amplified fragments are connected to plasmid pMD19-TS by the Partial Fragment that afsQ1 genes are expanded from 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-afs.By the method for Conjugative tiansfer, by recombinant plasmid pKC1139-afs from S.rimosus M4018 are transferred in E.coli ET12567, because of temperature sensitive type plasmid pKC1139 nothings under the conditions of higher than 34 DEG C Method replicates, so by all possible joint element culture under the conditions of 37 DEG C, and A Pula resistance screenings are utilized simultaneously.Then, The gene for the joint element that extracting screening obtains, using primer afs-single-P1/afs-single-P2, aprF/aprR is into one Step card determines, finally obtains the mutant strain S.rimosus M4018 Δs afs of afsQ1 gene disruptions.

Using plasmid pET-28a as template, kan resistance fragments, design primer pKANTTF, pKANTTR, by kan genes are expanded The promoter region of the upstream 100bp of code area and the transcript termination regions of downstream 100bp or so expand together, and draw Enter NheI restriction enzyme sites, respectively by pIB139 and the amplified fragments of recycling NheI single endonuclease digestions, by resistance fragments and matter after recycling Grain connection, after importing DH5 α competence, picking individual colonies, extraction plasmid NheI single endonuclease digestions verification is proved to be successful, illustrates plasmid It builds successfully, 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 afs-pIBF and afs-pIBR by complete afsQ1Q2-like genes from M4018 genes It is expanded in group, is inserted into the downstream of erythromycin promoter on plasmid pIB-KA to realize the constitutive expression of the gene, It is named as pIB-KA-afs.Successful plasmid pIB-KA-afs will be built to import in the method that same engagement is shifted In S.rimosus M4018 Δs afs, possible covering bacterium is obtained by selecting A Pula and the Double joint element of kanamycins Strain.Contain in bacterial strain there are one complete afsQ1Q2-like two-components system because covering, primer afsL/afsR can be used preliminary Verification.

It is M4018 Δs afs (pIB-KA-afs) 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-afs).

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

2, fermentation process

By bacterial strain obtained as above respectively in culture on solid bran mass, 30 DEG C, collected on culture medium after 5~7d Spore.The spore count for being generated each mutant strain being collected into using viable plate count method, in inoculation, so that it may will connect Kind amount is fixed on 1.0 × 10⁶Spore/mL.Culture medium used be using 50mM glycine as the MM fluid nutrient mediums of only nitrogen source, 50mL is dispensed in each 250mL shaking flasks.After unified inoculation, shaking flask being placed on shaking table, 30 DEG C, 220rpm is cultivated 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 terramycin 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 afsQ1 genes, which is known as the production of streptomyces rimosus, is influenced, by M4018 and M4018 Δ afs 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.As a result such as Fig. 1.

It is found that when Asn is only nitrogen source from Figure 1A, afs blocks bacterial strain compared to bacterium germination is gone out, and bacterium amount is in fermentation Later stage outline is higher than bacterium germination, but the oxytetracycline yield of per dry wt is almost the same.Such as Figure 1B, under the conditions of glycine, two The dry weight of person is substantially without significant difference, but the per dry wt production element of M4018 Δs afs is substantially higher in going out bacterium germination M4018. Asn is in the MM culture mediums of only nitrogen source, and afsQ1 blocks bacterial strain and starting strain in terms of per dry wt terramycin and goes out bacterium germination There is no difference.And under the conditions of 50mM Gly, block the production element of bacterial strain M4018- Δs afs it is horizontal on day 3 when it is significantly high Go out out bacterium germination, when fermenting to the 5th day (120h), the per dry wt production element level in M4018- Δs afs is higher by out bacterium germination M4018 at least 80% or more.

So far, a preliminary conclusion can be obtained, the bacterial strain after afsQ1 gene disruptions is only in specific amino acid item The significant difference of oxytetracycline yield can be just shown under part, and the production element level of bacterial strain is blocked far to be higher by out bacterium germination, it was demonstrated that At least using glycine as under the MM culture medium conditions of only nitrogen source, two-component system afsQ1Q2-like should be negative regulation tortoise Split the terramycin synthesis in streptomycete.

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 afs, covering bacterium M4018 Δs afs (pIB-KA-afs) and overexpression bacterial strain M4018 (pIB-KA-afs) is introduced simultaneously, and same method is inoculated into containing 50mM In the liquid MM culture mediums of glycine, the influence that this two-component system synthesizes terramycin is further confirmed that.

As a result last experimental result is repeated in such as Fig. 2, the result again, and the production element of M4018- Δs afs is horizontal to exist Still M4018 64% is higher by when 120h.In addition, being overexpressed bacterial strain and covering bacterial strain discovery by introducing, it is being overexpressed bacterial strain Its production element of M4018 (pIB-KA-afs) is horizontal significantly to have dropped 20% (120h) or more compared to going out bacterium germination, though and covering bacterium Part production element can so be restored, but still outline is less than the production element level of bacterium germination.

By a series of production element of this bacterial strain as a result, more comprehensively having confirmed two-component system afsQ1Q2-like really It is the synthesis of negative regulation terramycin.By lowering two-component system afsQ1Q2-like, then terramycin production can be significantly improved Amount.

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 figure A, but unit is dry There were significant differences for the oxytetracycline yield of weight.From Fig. 3 B it is found that at the 5th day, the per dry wt production element of M4018 Δs afs, which is higher by, to set out Bacterium 54% or so.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>East China University of Science

Datong District is the same as star antibiotic Co., Ltd

<120>The method for improving oxytetracycline yield based on afsQ1Q2-like bi-component regulating systems

<130> 180688

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 225

<212> PRT

<213>Streptomyces rimosus (S. rimosus)

<400> 1

Val Pro Phe Leu Leu Leu Ile Glu Asp Asp Asp Ala Ile Arg Thr Ala

1 5 10 15

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

20 25 30

Thr Gly Glu Asp Gly Leu Lys Leu Leu Arg Glu Gln Arg Pro Asp Leu

35 40 45

Ile Val Leu Asp Val Met Leu Pro Gly Ile Asp Gly Phe Glu Val Cys

50 55 60

Arg Arg Ile Arg Arg Thr Asp Gln Leu Pro Ile Ile Leu Leu Thr Ala

65 70 75 80

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

85 90 95

Asp Tyr Val Val Lys Pro Val Gln Gly Arg Val Leu Asp Ala Arg Ile

100 105 110

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

115 120 125

Phe Gly Ser Leu Val Ile Asp Arg Ser Ala Met Thr Val Thr Lys Asn

130 135 140

Gly Glu Asp Leu Gln Leu Thr Pro Thr Glu Leu Arg Leu Leu Leu Glu

145 150 155 160

Leu Ser Arg Arg Pro Gly Gln Ala Leu Ser Arg Gln Gln Leu Leu Arg

165 170 175

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

180 185 190

Cys Val Gln Arg Leu Arg Ala Lys Val Glu Asp Val Pro Ser Ser Pro

195 200 205

Thr Leu Ile Arg Thr Val Arg Gly Val Gly Tyr Arg Leu Asp Thr Pro

210 215 220

Gln

225

<210> 2

<211> 654

<212> PRT

<213>Streptomyces rimosus (S. rimosus)

<400> 2

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

1 5 10 15

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

20 25 30

Gly Arg Thr Gly Thr Asp Gly Ala Gly Ala Gly Arg Thr Asp Gly Arg

35 40 45

Ala Gly Gly Arg Thr Ala Asp Asp Thr Glu Arg Thr Ala Asp Gly Thr

50 55 60

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

65 70 75 80

Cys Val Arg Ala Leu Gly Leu Leu Arg Trp Thr Ser Leu Arg Leu Arg

85 90 95

Leu Val Phe Val Phe Ala Leu Val Ala Leu Thr Ala Ala Val Ser Ala

100 105 110

Ser Gly Ile Ala Tyr Trp Leu Asn Arg Asn Thr Val Leu Asp Arg Thr

115 120 125

Gln Asn Ser Ala Leu Asn Asp Phe His Lys Ser Leu Glu Asp Ser Thr

130 135 140

Arg Ser Leu Pro Leu Arg Pro His Cys Ala Glu Leu Gln Asp Ala Ala

145 150 155 160

Arg Gln Met Ala Gly Gly Pro Gln Asn Tyr Ala Val Leu Leu Val Met

165 170 175

Arg Gly Ala Asp Gly Arg Gln Cys Val Ala Thr Thr Asp Lys Asp Leu

180 185 190

Phe Thr Leu Lys Thr Val Pro Gln Ser Leu Gln Thr Ala Val Asn Glu

195 200 205

Lys Arg Ala Leu Asp Asp Gly Asn Arg Val Pro Tyr His Met Tyr Trp

210 215 220

Gln Arg Lys Glu Leu His Asp Thr Pro Tyr Leu Val Ala Gly Ala Arg

225 230 235 240

Met Asn Gly Gly Gly Pro Thr Gly Tyr Met Leu Lys Ser Leu Ala Thr

245 250 255

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

260 265 270

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

275 280 285

Thr Val Leu Arg Pro Val Gln Arg Leu Gly His Ala Ala Arg Gln Leu

290 295 300

Gly Glu Gly Arg Leu Asp Thr Arg Leu Arg Val Thr Gly Thr Asp Glu

305 310 315 320

Leu Ala Asp Leu Ser Arg Thr Phe Asn Arg Thr Ala Glu Arg Leu Glu

325 330 335

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

340 345 350

Val Ala Asp Met Ser His Glu Leu Arg Thr Pro Leu Thr Ala Ile Thr

355 360 365

Ala Val Thr Glu Val Leu Glu Glu Glu Glu Asp Ser Leu Asp Pro Met

370 375 380

Ile Ala Pro Ala Val Lys Leu Val Val Ser Glu Thr Arg Arg Leu Asn

385 390 395 400

Asp Leu Val Glu Asn Leu Met Glu Val Thr Arg Phe Asp Ala Gly Thr

405 410 415

Ala Arg Leu Val Gln Asp Asp Val Asp Val Ala Asp Gln Val Thr Ala

420 425 430

Cys Ile Asp Ala Arg Ala Trp Leu Asp Ala Val Glu Leu Asp Ala Glu

435 440 445

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

450 455 460

Ala Asn Leu Ile Gly Asn Ala Leu Lys His Gly Gly Ser Pro Val Arg

465 470 475 480

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

485 490 495

Arg Val Arg Asp His Gly Pro Gly Ile Pro Gln Glu Val Leu Pro His

500 505 510

Val Phe Asp Arg Phe Tyr Lys Ala Ser Ala Ser Arg Pro Arg Ser Glu

515 520 525

Gly Ser Gly Leu Gly Leu Ser Ile Ala Leu Glu Asn Ala His Ile His

530 535 540

Gly Gly Glu Ile Thr Ala Ala Asn Ser Pro Glu Gly Gly Ala Val Phe

545 550 555 560

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

565 570 575

Glu Asp Gly Gln Gly Ala Ala Gln Gly Val Ala Gln Gly Thr Ala Gln

580 585 590

Glu Ala Pro Lys Ala Pro Glu Thr Pro Glu Thr Pro Glu Thr Pro Asp

595 600 605

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

610 615 620

Ser Ala Pro Gly Pro Gly Ser Asp Pro Asp Ala Ala Pro Asp Ser Gly

625 630 635 640

Pro Asp Ser Gly Pro Arg Arg Lys Glu Gly Gly Asp Arg Arg

645 650

<210> 3

<211> 678

<212> DNA

<213>Streptomyces rimosus (S. rimosus)

<400> 3

gtgcctttcc tgttgctgat cgaggacgac gacgccatcc gcacggccct cgaactctcg 60

ctgtcccgcc agggtcaccg tgtggtgacc gcggcgacgg gcgaggacgg cctgaagctg 120

ctgcgcgagc agcgcccgga cctgatcgtg ctggacgtga tgctcccggg catcgacggt 180

ttcgaggtgt gccggcggat caggcggacg gaccagctgc ccatcatcct gctgaccgcg 240

cgcaacgacg acatcgacgt ggtggtcggg ctggagtcgg gcgcggacga ctacgtggtc 300

aagccggtgc agggccgggt gctggacgcc cgtatccggg ccgtgctgcg gcgcggggag 360

cgcgagtcca acgacgcggc gaccttcggc tcgctggtca tcgaccgctc ggcgatgacg 420

gtcaccaaga acggcgagga tctgcaactg acacccaccg agctgcggtt gctgctggag 480

ctcagccgcc gccccggcca ggcgctgtcc cggcagcagc tgctgcgcct ggtgtgggag 540

cacgactacc tgggtgactc gcggctggtg gacgcgtgcg tgcagcggct gcgcgcgaag 600

gtggaggacg taccgtcctc gccgaccttg atccgtacgg tccgcggtgt cggctaccgg 660

ctggacaccc cgcagtga 678

<210> 4

<211> 1965

<212> DNA

<213>Streptomyces rimosus (S. rimosus)

<400> 4

gtgaccgaac gctcgaccgg tccggacccg gaccgtacgg acggcccgcg ggacacggcc 60

cggccgggca gggacaggaa ggacagggac gtggggggcc gtacgggcac cgacggcgcg 120

ggtgccgggc ggacggacgg gcgtgcgggc ggccgtacgg cggacgacac ggagcggacc 180

gccgacggca cggccgacga gggcgcgccc ggacaggagc gctcctcgct gctcgtacgg 240

tgcgtacggg cgctcggcct gctgcgctgg accagcctgc ggctgcggct ggtcttcgtg 300

ttcgcgctgg tcgcgctgac cgcggcggtc tccgcctccg gcatcgcgta ctggctcaac 360

cgcaacacgg tgctggaccg cacccagaac tccgcgctca acgacttcca caagtcgctg 420

gaggacagca cccgctcgct gccgctgcgc ccgcactgcg cggagcttca ggacgccgcg 480

cggcagatgg cgggcgggcc gcagaactac gccgtgctgc tcgtcatgcg cggcgcggac 540

ggccggcagt gcgtcgcgac caccgacaag gacctcttca cgctcaagac ggtgccgcag 600

tcgctccaga ccgcggtcaa cgagaagcgc gcgctcgacg acggcaaccg ggtgccgtac 660

cacatgtact ggcagcgcaa ggagctgcac gacaccccgt acctcgtcgc cggtgcccgg 720

atgaacggcg gcggcccgac cggctacatg ctcaagtcgc tggccaccga gcgtcaggac 780

ctgaactcgc tggcctggtc gctgggcatc gccaccgcgc tggccctggt cggctcggcg 840

ctgctggcgc aggccgccgc gaccaccgtg ctgcggccgg tgcagcgcct gggccacgcg 900

gcccggcagc tgggcgaggg gcggctggac acccggctgc gggtgaccgg caccgacgag 960

ctcgccgacc tctcccgtac gttcaaccgc accgccgagc ggctggagca gcgggtggcc 1020

gagctgagcg ggcgggaggc ggcgtcccgc cgcttcgtcg ccgacatgtc gcacgagctg 1080

cgcacgccgc tgacggccat caccgcggtc accgaggtgc tggaggaaga ggaggactcc 1140

ctcgacccga tgatcgcgcc cgcggtgaag ctggtggtca gcgagacccg gcggctgaac 1200

gacctggtgg agaacctcat ggaggtcacc cgcttcgacg cggggacggc acggctggtg 1260

caggacgacg tggacgtggc ggaccaggtg accgcgtgca tcgacgcgcg ggcctggctg 1320

gacgcggtcg agctggacgc cgagcgcggc atcgtggccc gtctggaccc gcgccgcctc 1380

gacgtcatcc tcgccaacct catcggcaac gcgctcaagc acggcggctc gccggtccgg 1440

gtgtccgtac ggacccaggc cacgccggag ggcgaggacc tgctgatccg ggtacgcgac 1500

cacggccccg gcatcccgca ggaggtcctg ccgcacgtct tcgaccgctt ctacaaggcg 1560

agcgcgtccc ggccgcggtc ggagggcagc ggcctgggcc tgtcgatcgc cctggagaac 1620

gcgcacatcc acggtggcga gatcaccgcc gcgaactcgc cggagggcgg cgcggtcttc 1680

acgctgcggc tgccggtggg gaccggcggg gctgcggagg aggggcagga ggacgggcag 1740

ggggctgcgc agggcgtcgc gcaggggacc gcgcaggagg cgccgaaggc gccggagacg 1800

ccggagacgc cggagacgcc ggacgggacc gctgtatcgg agcgtgcgga ggacgcgacc 1860

ccgggtacgg gctccgcacc cggccccggc tccgaccccg acgccgcccc ggactccgga 1920

cccgactcag gcccccgccg caaggaaggc ggtgaccgcc gatga 1965

<210> 5

<211> 2639

<212> DNA

<213>Streptomyces rimosus (S. rimosus)

<400> 5

gtgcctttcc tgttgctgat cgaggacgac gacgccatcc gcacggccct cgaactctcg 60

ctgtcccgcc agggtcaccg tgtggtgacc gcggcgacgg gcgaggacgg cctgaagctg 120

ctgcgcgagc agcgcccgga cctgatcgtg ctggacgtga tgctcccggg catcgacggt 180

ttcgaggtgt gccggcggat caggcggacg gaccagctgc ccatcatcct gctgaccgcg 240

cgcaacgacg acatcgacgt ggtggtcggg ctggagtcgg gcgcggacga ctacgtggtc 300

aagccggtgc agggccgggt gctggacgcc cgtatccggg ccgtgctgcg gcgcggggag 360

cgcgagtcca acgacgcggc gaccttcggc tcgctggtca tcgaccgctc ggcgatgacg 420

gtcaccaaga acggcgagga tctgcaactg acacccaccg agctgcggtt gctgctggag 480

ctcagccgcc gccccggcca ggcgctgtcc cggcagcagc tgctgcgcct ggtgtgggag 540

cacgactacc tgggtgactc gcggctggtg gacgcgtgcg tgcagcggct gcgcgcgaag 600

gtggaggacg taccgtcctc gccgaccttg atccgtacgg tccgcggtgt cggctaccgg 660

ctggacaccc cgcagtgacc gaacgctcga ccggtccgga cccggaccgt acggacggcc 720

cgcgggacac ggcccggccg ggcagggaca ggaaggacag ggacgtgggg ggccgtacgg 780

gcaccgacgg cgcgggtgcc gggcggacgg acgggcgtgc gggcggccgt acggcggacg 840

acacggagcg gaccgccgac ggcacggccg acgagggcgc gcccggacag gagcgctcct 900

cgctgctcgt acggtgcgta cgggcgctcg gcctgctgcg ctggaccagc ctgcggctgc 960

ggctggtctt cgtgttcgcg ctggtcgcgc tgaccgcggc ggtctccgcc tccggcatcg 1020

cgtactggct caaccgcaac acggtgctgg accgcaccca gaactccgcg ctcaacgact 1080

tccacaagtc gctggaggac agcacccgct cgctgccgct gcgcccgcac tgcgcggagc 1140

ttcaggacgc cgcgcggcag atggcgggcg ggccgcagaa ctacgccgtg ctgctcgtca 1200

tgcgcggcgc ggacggccgg cagtgcgtcg cgaccaccga caaggacctc ttcacgctca 1260

agacggtgcc gcagtcgctc cagaccgcgg tcaacgagaa gcgcgcgctc gacgacggca 1320

accgggtgcc gtaccacatg tactggcagc gcaaggagct gcacgacacc ccgtacctcg 1380

tcgccggtgc ccggatgaac ggcggcggcc cgaccggcta catgctcaag tcgctggcca 1440

ccgagcgtca ggacctgaac tcgctggcct ggtcgctggg catcgccacc gcgctggccc 1500

tggtcggctc ggcgctgctg gcgcaggccg ccgcgaccac cgtgctgcgg ccggtgcagc 1560

gcctgggcca cgcggcccgg cagctgggcg aggggcggct ggacacccgg ctgcgggtga 1620

ccggcaccga cgagctcgcc gacctctccc gtacgttcaa ccgcaccgcc gagcggctgg 1680

agcagcgggt ggccgagctg agcgggcggg aggcggcgtc ccgccgcttc gtcgccgaca 1740

tgtcgcacga gctgcgcacg ccgctgacgg ccatcaccgc ggtcaccgag gtgctggagg 1800

aagaggagga ctccctcgac ccgatgatcg cgcccgcggt gaagctggtg gtcagcgaga 1860

cccggcggct gaacgacctg gtggagaacc tcatggaggt cacccgcttc gacgcgggga 1920

cggcacggct ggtgcaggac gacgtggacg tggcggacca ggtgaccgcg tgcatcgacg 1980

cgcgggcctg gctggacgcg gtcgagctgg acgccgagcg cggcatcgtg gcccgtctgg 2040

acccgcgccg cctcgacgtc atcctcgcca acctcatcgg caacgcgctc aagcacggcg 2100

gctcgccggt ccgggtgtcc gtacggaccc aggccacgcc ggagggcgag gacctgctga 2160

tccgggtacg cgaccacggc cccggcatcc cgcaggaggt cctgccgcac gtcttcgacc 2220

gcttctacaa ggcgagcgcg tcccggccgc ggtcggaggg cagcggcctg ggcctgtcga 2280

tcgccctgga gaacgcgcac atccacggtg gcgagatcac cgccgcgaac tcgccggagg 2340

gcggcgcggt cttcacgctg cggctgccgg tggggaccgg cggggctgcg gaggaggggc 2400

aggaggacgg gcagggggct gcgcagggcg tcgcgcaggg gaccgcgcag gaggcgccga 2460

aggcgccgga gacgccggag acgccggaga cgccggacgg gaccgctgta tcggagcgtg 2520

cggaggacgc gaccccgggt acgggctccg cacccggccc cggctccgac cccgacgccg 2580

ccccggactc cggacccgac tcaggccccc gccgcaagga aggcggtgac cgccgatga 2639

<210> 6

<211> 28

<212> DNA

<213>Primer (Primer)

<400> 6

cccaagctta gggtcaccgt gtggtgac 28

<210> 7

<211> 28

<212> DNA

<213>Primer (Primer)

<400> 7

tgctctagat ccaccagccg cgagtcac 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

ctggtccccc atgggccaga atg 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> 34

<212> DNA

<213>Primer (Primer)

<400> 14

ggaattccat atgcctttcc tgttgctgat cgag 34

<210> 15

<211> 29

<212> DNA

<213>Primer (Primer)

<400> 15

cggaattctc atcggcggtc accgccttc 29

<210> 16

<211> 27

<212> DNA

<213>Primer (Primer)

<400> 16

gtgcctttcc tgttgctgat cgaggac 27

<210> 17

<211> 25

<212> DNA

<213>Primer (Primer)

<400> 17

tcatcggcgg tcaccgcctt ccttg 25

<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 afsQ1Q2-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 afsQ1Q2-like is bis- in the downward terramycin production bacterium The activity of composition regulation system includes：

(a) gene of knockout or silence afsQ1Q2-like bi-component regulating systems in terramycin produces bacterium；

(b) the lower adjustment for lowering afsQ1Q2-like bi-component regulating systems is transferred in terramycin production bacterium；Or

(c) the stream signal access or upstream gene of afsQ1Q2-like bi-component regulating systems in terramycin production bacterium are adjusted, To lower afsQ1Q2-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 afsQ1Q2-like bi-component regulating systems in bacterium；Preferably, knocking out response regulator gene afsQ1 or induction Protein gene afsQ2.

4. method as claimed in claim 2, which is characterized in that in (b), the lower adjustment is specificity interference afsQ1Q2- The disturbing molecule of the expression of gene in like bi-component regulating systems；Preferably, the disturbing molecule is with afsQ1Q2- 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 afsQ1Q2-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 afsQ1 or induction protein gene afsQ2 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 afsQ1Q2-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.