CN103320372B - Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof - Google Patents
Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof Download PDFInfo
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- CN103320372B CN103320372B CN201310168980.1A CN201310168980A CN103320372B CN 103320372 B CN103320372 B CN 103320372B CN 201310168980 A CN201310168980 A CN 201310168980A CN 103320372 B CN103320372 B CN 103320372B
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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
Technical field
The present invention relates to improve toyokamycin output by strengthening 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
1connect the deazapurine ring of similar guanine, core texture is pyrroles's pyrimidine nucleoside analoys.The mechanism of action is mainly the growth that affects thalline of transcribing by suppressing microorganism, and its bioactivity research report mainly concentrates on clinical medicine domain.Studies have found that in the recent period toyokamycin has good prevention effect to various plants epidemic disease, life-time service can not cause environmental pollution, and plant-growth is also had to certain regulating effect.Therefore the application potential that, toyokamycin has in agricultural plants disease control field.With respect to chemical synthesis, the synthetic toyokamycin of biological process is taking renewable resources as raw material, has 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 current toyokamycin suitability for industrialized production.
Toyokamycin belongs to secondary metabolite, and route of synthesis complexity 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 addressing this problem is all generally the production bacterial strain as Main Means screening high yield and high quality by traditional selection by mutation, but screen, the uncertain factor of high efficient strain is many, the cycle is long.
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 taking the synthetic toyokamycin bacterial strain Streptomyces rimosus (ATCC14673) of a strain as initial research object, clone biosynthesizing toyokamycin gene cluster, building-up process and the regulatory mechanism of toyokamycin are illustrated, research finds that toyokamycin is precursor by GTP, through the synthetic toyokamycin of polystep reaction, be wherein one of key enzyme of pathways metabolism by the adenosine succsinic acid lyase of toyF genes encoding.Have no relevant report but utilize metabolic engineering technology further to improve toyokamycin output by increasing toyokamycin metabolic flux because relate to the challenge 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 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 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 build the recombinant vectors pIB139-that carries toyF gene
toyf, and utilize conjugal transfer method by its specific being incorporated on streptomyces diastatochromogenes 1628 karyomit(e)s.
One has been strengthened
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).
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 conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes karyomit(e), obtain described engineering bacteria.
Utilize promotor permE* on pIB139 carrier to start the expression of toyF gene, utilize conjugal transfer method by specific carrier pIB139-toyF be integrated into streptomyces diastatochromogenes (
streptomyces diastatochromogenes) 1628 karyomit(e), obtain the engineering bacteria of inheritance stability.
Described reinforcement
toythe purposes of the restructuring streptomyces diastatochromogenes that F expresses, compared with original strain, the work of recombinant bacterium adenosine succsinic acid lyase enzyme has improved 0.9 times, and toyokamycin output has at least improved 20.5%.
Beneficial effect of the present invention:
The present invention strengthens toyokamycin biosynthetic pathway key gene toyF in streptomyces diastatochromogenes after 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%.For further improving toyokamycin output, realize early suitability for industrialized production and lay a good foundation.
Brief description of the 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 toyF gene exists
e.colithe SDS-PAGE analysis chart of BL21.
1. pET28a-
toyf/
e.colibL21 induction; 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
Below in conjunction with the drawings and specific embodiments, the present invention is described further.
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 and contains
ndei and
notthe toyF gene of two restriction enzyme sites of I, is connected with pMD18-T Vector, builds cloning vector pMD18-T-toyF(
ndei+
noti), by cloning vector pMD18-T-toyF(
ndei+
noti) be converted in acceptor intestinal bacteria, coat on the LB agar plate containing ammonia benzyl resistance, after 37 DEG C of overnight incubation, random picking positive transformant enzyme is cut and is served that Hai Shenggong check order and carry out sequential analysis after qualification, use
ndei and
noti double digestion is contained
ndei and
notthe toyF gene of two restriction enzyme sites of I, connects with the streptomycete integrative shuttle expression vector pIB139 of same double digestion, obtains recombinating shuttle expression carrier pIB139-toyF after enzyme is cut checking, is proceeded to
e.colieT12567 (pUZ8002) screens positive transformant on the LB flat board of that resistance of card and apramycin resistance
e.colieT12567 (pUZ8002, pIB139-toyF), with
e.colieT12567 (pUZ8002, pIB139-toyF) is donor, and streptomyces diastatochromogenes is acceptor, utilizes conjugal transfer method that pIB139-toyF is incorporated into streptomyces diastatochromogenes
s. diastatochromogeneson 1628 karyomit(e)s, on apramycin resistance MS flat board, screen positive transformant, streptomyces diastatochromogenes 1628-TOYF 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 verifies as shown in Figure 2, and recombinant plasmid pMD18-T-toyF double digestion obtains the DNA fragmentation of about 1.4kb and 2.7kb, and in the same size with toyF gene fragment and plasmid pMD18-T respectively, illustrates that recombinant cloning vector connects correct.To recombinant plasmid, pMD18-T-toyF checks order, sequential analysis shows that Insert Fragment is the sequence of 1443 bp, 480 amino acid of encoding, relative molecular weight size is about respectively 53 kDa, there is higher homology with the adenosine succsinic acid lyase of many streptomyces of reporting, be wherein up to 89%.The toyF gene GenBank accession number that amplification obtains is: JQ267374.1.ToyF gene is connected into pET28a carrier, build pET28a-toyF recombinant vectors, and recombinant vectors is proceeded to e. coli bl21, and picking positive transformant is in the LB of 10mL substratum, and 37 DEG C of shaking culture are spent the night, switching next day, IPTG abduction delivering, as shown in Figure 3, after induction, recombinant bacterium has 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
notit is in the same size that I double digestion discharges fragment and the toyF gene of 1.4 kb, illustrates that plasmid pIB139-toyF builds correct, with conjugal transfer method be incorporated into streptomyces diastatochromogenes (
streptomyces diastatochromogenes) on 1628 karyomit(e), after in apramycin resistant panel, the random some single bacterium colonies of picking are cultivated repeatedly in CP substratum, extraction karyomit(e).PCR experiment all can amplify 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
Compared 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 250 mL shake flask fermentation experiments, and the enhancing of adenosine succsinic acid lyase enzyme streptomyces diastatochromogenes being lived from fermentation angle is verified the raising effect of toyokamycin output.Reciprocating shaking speed is 200 r/min, 28 DEG C, and fermentation 96h, control group is 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.Illustrate at toyokamycin and produce bacterial strain
s. diastatochromogenesthe expression that strengthens the key enzyme adenosine succsinic acid lyase-ToyF of biosynthetic pathway in 1628 contributes to improve the output of toyokamycin in 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 (3)
1. strengthened for one kind
toythe restructuring streptomyces diastatochromogenes that F expresses, is characterized in that: it has expressed adenosine succsinic acid lyase encoding gene
toyf, have than original bacterium streptomyces diastatochromogenes (
streptomyces diastatochromogenes) 1628 higher toyokamycin abilities to express;
Original bacterium is streptomyces diastatochromogenes 1628, and deposit number is CGMCC NO. 2060;
Described adenosine succsinic acid lyase encoding gene
toyf comes from original bacterium streptomyces diastatochromogenes 1628 to be reorganized, and GenBank accession number is: JQ267374.1;
Described adenosine succsinic acid lyase encoding gene
toyf is integrated into original bacterium streptomyces diastatochromogenes 1628 karyomit(e)s.
2. as claimed in claim 1 a 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;
2) utilize conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes 1628 karyomit(e)s, obtain described engineering bacteria;
Utilize the promotor permE* on pIB139 carrier to start
toythe expression of F gene, utilizes conjugal transfer method by the specific carrier pIB139-toyF karyomit(e) that is integrated into streptomyces diastatochromogenes 1628, has obtained the engineering bacteria of inheritance stability.
3. one kind is utilized as claimed in claim 1 reinforcement
toythe purposes of the restructuring streptomyces diastatochromogenes that F expresses, is characterized in that, compared 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%.
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| CN101283684A (en) * | 2007-11-19 | 2008-10-15 | 中国计量学院 | Application of a compound for preventing and controlling cucumber fusarium wilt and tomato gray mold |
| CN100560714C (en) * | 2007-11-19 | 2009-11-18 | 中国计量学院 | One biocontrol actinomyces-streptomyces diastatochromogenes D |
| CN101444216B (en) * | 2008-12-29 | 2012-02-08 | 中国热带农业科学院热带生物技术研究所 | Application of toyocamycin in preparing plant blight control medicines |
| CN101961013B (en) * | 2010-03-19 | 2013-01-16 | 中国计量学院 | Application of toyocamycin in controlling tomato gray mold |
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