CN103320372A

CN103320372A - Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof

Info

Publication number: CN103320372A
Application number: CN2013101689801A
Authority: CN
Inventors: 马正; 俞晓平; 陶立彬; 申屠旭萍; 边亚琳; 郝培应; 许益鹏
Original assignee: China Jiliang University
Current assignee: China Jiliang University
Priority date: 2013-05-08
Filing date: 2013-05-08
Publication date: 2013-09-25
Anticipated expiration: 2033-05-08
Also published as: CN103320372B

Abstract

The invention discloses recombinant streptomyces diastatochromogenes with reinforced toyF expression, a construction method and uses thereof. The recombinant streptomyces diastatochromogenes over-expresses coding gene toyF of toyocamycin biosynthesis key enzyme adenylosuccinate lyase, and has higher toyocamycin synthesis capacity compared with streptomyces diastatochromogenes 1628. The construction process comprises: 1) constructing an expression vector pIB139-toyF; and 2) adopting a conjugal transfer method to integrate the expression vector into streptomyces diastatochromogenes chromosome to obtain engineering bacteria. According to the present invention, a promoter permE* on the pIB139 vector is adopted to start toyF gene expression, and the conjugal transfer method is adopted to specially integrate the vector pIB139-toyF into the streptomyces diastatochromogenes 1628 chromosome to obtain the genetically stable engineering bacteria, wherein activity of the recombinant adenylosuccinate lyase is increased by 0.9 time and a toyocamycin yield is increased by at least 20.5% compared with the original bacterial.

Description

Restructuring streptomyces diastatochromogenes and construction process and purposes that toyF expresses have been strengthened

Technical field

The present invention relates to improve toyokamycin output by strengthening the toyF gene in the expression of streptomyces diastatochromogenes, belong to gene engineering technology field.

Background technology

Toyokamycin is a kind of novel nucleoside microbiotic, and molecular formula is C ₁₂H ₁₃N ₅O ₄, ribose C ₁The deazapurine ring that connects similar guanine, core texture are pyrroles's pyrimidine nucleoside analoys.The mechanism of action mainly is the growth that affects thalline of transcribing by the inhibition microorganism, and its bioactivity research report mainly concentrates on clinical medicine domain.Have in the recent period research to find that toyokamycin has good prevention effect to the various plants epidemic disease, life-time service can not cause environmental pollution, and plant-growth is also had certain regulating effect.Therefore, the application potential that has in agricultural plants disease control field of toyokamycin.With respect to chemical synthesis, the synthetic toyokamycin of biological process is take renewable resources as raw material, has the reaction conditions gentleness, pollutes less and the advantage such as with low cost, and therefore, biological synthesis process is the both economical effective means of present toyokamycin suitability for industrialized production.

Toyokamycin belongs to secondary metabolite, and route of synthesis is complicated, is subject to restriction and the regulation and control of many factors, causes the synthetic level of toyokamycin lower, is difficult to large-scale production.The existing way that addresses this problem generally all is by the production bacterial strain of traditional selection by mutation as Main Means screening high yield and high quality, and the uncertain factor of high efficient strain is many, the cycle is long but screen.

Output how to utilize the advanced persons' such as molecular biology technique means improvement microorganism further to improve toyokamycin becomes a feasible thinking.The people such as McCarty are take the synthetic toyokamycin bacterial strain Streptomyces rimosus (ATCC14673) of a strain as initial research object, cloned biosynthesizing toyokamycin gene cluster, building-up process and the regulatory mechanism of toyokamycin have been illustrated, research finds that toyokamycin is precursor by GTP, through the synthetic toyokamycin of polystep reaction, wherein the adenosine succsinic acid lyase by the toyF genes encoding is one of key enzyme of pathways metabolism.But utilize the metabolic engineering technology further to improve toyokamycin output by increasing the toyokamycin metabolic flux and have no relevant report because relate to the challenges such as foundation of expression system.

Summary of the invention

In order to overcome the deficiencies in the prior art, the invention provides a kind of strengthened restructuring streptomyces diastatochromogenes and construction process and purposes that toyF expresses.

Streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628 are strain Antagonistic Actinomycetes, its fermented liquid has stronger restraining effect to the various plants pathogenic fungi, through separation and Extraction, determine that its main effective constituent is toyokamycin, there is not yet streptomyces diastatochromogenes metabolic engineering molecular modification aspect research report.

The present invention at first from streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) clone the toyF gene of coding adenosine succsinic acid lyase in 1628 (deposit number of bacterial strain is CGMCC NO. 2060), and this gene is connected with streptomycete integrative plasmid pIB139, successfully made up the recombinant vectors pIB139-that carries the toyF gene ToyF, and utilize the conjugal transfer method with its specific being incorporated on streptomyces diastatochromogenes 1628 karyomit(e)s.

A kind of reinforcement ToyThe restructuring streptomyces diastatochromogenes that F expresses, it has expressed adenosine succsinic acid lyase encoding gene ToyF, have than streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628 higher toyokamycin abilities to express.

Described original bacterium be streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628.

Described adenosine succsinic acid lyase encoding gene ToyF come from original bacterium streptomyces diastatochromogenes to be reorganized ( Streptomyces diastatochromogenes).

Described adenosine succsinic acid lyase encoding gene ToyF is integrated into original bacterium streptomyces diastatochromogenes karyomit(e).

The described reinforcement ToyThe construction process of the restructuring streptomyces diastatochromogenes that F expresses, process is as follows:

1) construction of expression vector pIB139-toyF;

2) utilize the conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes karyomit(e), obtain described engineering bacteria.

Utilize promotor permE* on the pIB139 carrier to start the expression of toyF gene, utilize the conjugal transfer method with carrier pIB139-toyF specific be integrated into streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628 karyomit(e), obtained the engineering bacteria of inheritance stability.

The described reinforcement ToyThe purposes of the restructuring streptomyces diastatochromogenes that F expresses is compared with original strain, and the work of recombinant bacterium adenosine succsinic acid lyase enzyme has improved 0.9 times, and toyokamycin output has improved 20.5% at least.

Beneficial effect of the present invention:

The present invention strengthens toyokamycin biosynthetic pathway key gene toyF in streptomyces diastatochromogenes behind the overexpression, and the work of recombinant bacterium adenosine succsinic acid lyase enzyme has improved 0.9 times, and the original bacterium of energy force rate of synthetic toyokamycin has improved 20.5%.Be further to improve toyokamycin output, realize that early suitability for industrialized production lays a good foundation.

Description of drawings

Fig. 1 is recombinant plasmid pIB139 -The structure schematic diagram of toyF.

Fig. 2 is that the enzyme of recombinant plasmid pMD18-T-toyF is cut checking.

1.?pMD18-T-toyF/ Nde?I+ Not?I；2.?toyF?gene；3.?DNA?Marker?DL2000。

Fig. 3 is that the toyF gene exists E.coliThe SDS-PAGE analysis chart of BL21.

1. pET28a- ToyF/ E.coliBL21 induces; 2. Protein Marker; 3. pET28a- ToyF/ E.coliBL21 does not induce.

Fig. 4 is the restriction enzyme digestion and electrophoresis proof diagram of restructuring shuttle expression plasmid pIB139-toyF.

1.?toyF?gene；2.?DL2000?Marker；3.?pIB139-toyF/ Nde?I+ Not?I；4.?pIB139/ Nde?I+ Not?I；5.?λ?DNA/ Hind?III?Marker。

Fig. 5 is the PCR electrophoresis proof diagram of recombinant bacterium 1628-TOYF.

1. original strain; 2-4. engineering strain; 5. DL2000 Marker.

Embodiment

The present invention is described further below in conjunction with the drawings and specific embodiments.

Embodiment 1:The structure of the amplification of goal gene and restructuring streptomyces diastatochromogenes

With S. diastatochromogenes1628 chromogene groups are template, with PtoyF F NdeI and PtoyF R NotI is primer, and pcr amplification obtains to contain NdeI and NotThe toyF gene of two restriction enzyme sites of I is connected with pMD18-T Vector, makes up cloning vector pMD18-T-toyF( NdeI+ NotI), with cloning vector pMD18-T-toyF( NdeI+ NotI) be converted in the acceptor intestinal bacteria, coat on the LB agar plate that contains ammonia benzyl resistance, after 37 ℃ of overnight incubation, random picking positive transformant enzyme is served the Hai Shenggong order-checking and is carried out sequential analysis after cutting evaluation, uses NdeI and NotThe I double digestion is contained NdeI and NotThe toyF gene of two restriction enzyme sites of I is connected with the streptomycete integrative shuttle expression vector pIB139 of same double digestion, obtains recombinating shuttle expression carrier pIB139-toyF after enzyme is cut checking, and it is changed over to E.coliET12567 (pUZ8002) screens positive transformant at the LB flat board that blocks that resistance and apramycin resistance E.coliET12567 (pUZ8002, pIB139-toyF), with E.coliET12567 (pUZ8002, pIB139-toyF) is donor, and streptomyces diastatochromogenes is acceptor, utilizes the conjugal transfer method that pIB139-toyF is incorporated into streptomyces diastatochromogenes S. diastatochromogenesOn 1628 karyomit(e)s, screen positive transformant at apramycin resistance MS flat board, streptomyces diastatochromogenes 1628-TOYF namely obtains recombinating.

With S. diastatochromogenes1628 karyomit(e)s are template, design two primers, pcr amplification toyF, and design of primers is as follows:

toyF?F? Nde?I：5’-CGC CATATGGTGACTGCTG?CGTCTGC-3’

toyF?R? Not?I：5’-CGC GCGGCCGCTCAGAGAATCGCTCCGGG-3’

Recombinant plasmid pMD18-T-toyF is with restriction enzyme NdeI and NotI double digestion checking as shown in Figure 2, recombinant plasmid pMD18-T-toyF double digestion obtains the approximately dna fragmentation of 1.4kb and 2.7kb, with the in the same size of toyF gene fragment and plasmid pMD18-T, illustrate that recombinant cloning vector connects correctly respectively.PMD18-T-toyF checks order to recombinant plasmid, sequential analysis shows that Insert Fragment is the sequence of 1443 bp, 480 amino acid of encoding, the relative molecular weight size is about respectively 53 kDa, have higher homology with the adenosine succsinic acid lyase of many streptomyces of having reported, wherein be up to 89%.The toyF gene GenBank accession number that amplification obtains is: JQ267374.1.The toyF gene is connected into the pET28a carrier, make up the pET28a-toyF recombinant vectors, and change recombinant vectors over to e. coli bl21, and the picking positive transformant is in the LB of 10mL substratum, and 37 ℃ of shaking culture are spent the night, switching next day, the IPTG abduction delivering as shown in Figure 3, induces rear recombinant bacterium to have obvious signature band to occur, recording adenosine succsinic acid lyase enzyme activity is 24 U/mg total proteins, shows toyF gene successful expression in intestinal bacteria.

The enzyme of integrated restructuring shuttle expression plasmid pIB139-toyF is cut the result as shown in Figure 4, recombinant plasmid pIB139-toyF warp NdeI and NotThe I double digestion discharges fragment and the toyF gene in the same size of 1.4 kb, illustrates that plasmid pIB139-toyF makes up correct, with the conjugal transfer method with its be incorporated into streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) on 1628 the karyomit(e), after the random some single bacterium colonies of picking are cultivated repeatedly in the CP substratum on the apramycin resistant panel, extraction karyomit(e).The PCR experiment all can amplify the apramycin resistant gene Apr(Fig. 5), prove that restructuring streptomyces diastatochromogenes 1628-TOYF successfully constructs, and inheritance stability.

Embodiment 2:The leavening property checking of the original bacterium of streptomyces diastatochromogenes and recombinant bacterium

Compare with original strain, the adenosine succsinic acid lyase enzyme work of recombinant bacterium has improved 0.9 times, to recombinant bacterium 1628-TOYF and original bacterium S. diastatochromogenes1628 carry out the experiment of 250 mL shake flask fermentations, from the enhancing that the fermentation angle is lived to adenosine succsinic acid lyase enzyme the streptomyces diastatochromogenes raising effect of toyokamycin output are verified.Reciprocating type shaking speed is 200 r/min, and 28 ℃, fermentation 96h, control group are the streptomyces diastatochromogenes strain of setting out.As shown in table 1, the output of recombinant bacterium toyokamycin is higher than original bacterium, and recombinant bacterium toyokamycin ultimate capacity reaches 162.64 mg/L, and more original bacterium has improved approximately 20.5%, and repeatability is good.Explanation is produced bacterial strain at toyokamycin S. diastatochromogenesThe expression that strengthens the key enzyme adenosine succsinic acid lyase-ToyF of biosynthetic pathway in 1628 helps to improve the output of toyokamycin in the fermenting process.

The comparison of table 1 recombinant bacterium and the final toyokamycin output of original bacterium

Thalline	Toyokamycin output (mg/L)
		1628	134.97
1628-TOYF	162.64

Claims

1. strengthened for one kind ToyThe restructuring streptomyces diastatochromogenes that F expresses, it is characterized in that: it has expressed adenosine succsinic acid lyase encoding gene ToyF, have than streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628 higher toyokamycin abilities to express.

2. as claimed in claim 1 the reinforcement ToyThe restructuring streptomyces diastatochromogenes that F expresses is characterized in that: described original bacterium be streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628.

3. as claimed in claim 1 the reinforcement ToyThe restructuring streptomyces diastatochromogenes of F genetic expression is characterized in that: described adenosine succsinic acid lyase encoding gene ToyF come from original bacterium streptomyces diastatochromogenes to be reorganized ( Streptomyces diastatochromogenes).

4. the restructuring streptomyces diastatochromogenes of having strengthened the toyF expression as claimed in claim 1 is characterized in that: described adenosine succsinic acid lyase encoding gene ToyF is integrated into original bacterium streptomyces diastatochromogenes karyomit(e).

5. as claimed in claim 1 reinforcement ToyThe construction process of the restructuring streptomyces diastatochromogenes that F expresses is characterized in that, process is as follows:

1) construction of expression vector pIB139-toyF;

6. method as claimed in claim 5 is characterized in that, utilizes promotor permE* on the pIB139 carrier to start the expression of toyF gene, utilize the conjugal transfer method with carrier pIB139-toyF specific be integrated into streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628 karyomit(e), obtained the engineering bacteria of inheritance stability.

7. one kind is utilized as claimed in claim 1 the reinforcement ToyThe purposes of the restructuring streptomyces diastatochromogenes that F expresses is characterized in that compare with original strain, the work of recombinant bacterium adenosine succsinic acid lyase enzyme has improved 0.9 times, and toyokamycin output has improved 20.5% at least.