BE1026767B1 - The method of construction and utility of the recombinant diastatochromogenic Streptomyces to enhance toyB expression - Google Patents

Abstract

The present invention discloses a genus of recombinant diastatochromogenic Streptomyces to enhance toyB expression and the method of its construction and utility. The genus Recombinant diastatochromogenic Streptomyces is capable of excessively expressing the toyB genes encoding the synthetic enzyme of 6-pyruvoyl-tetahydropterin, the key enzyme for the biological synthesis of toyocamycin and has the capacity for the synthesis of more powerful toyocamycin as Stastptomyces diastatochromogens 1628. The construction process is as follows: 1) construct the translation vector plB139-toyB; 2) take advantage of the welding and transfer method and insert the translation vector into the chromosome of diastatochromogenic Streptomyces, then obtain the engineering bacterium. Take advantage of the permE * promoter of the vector plB139 to start the translation of the toyB genes and of the probe and transfer method to introduce the peculiarity of the vector plB139-toyB to the chromosome of Streptomyces diastatochromogenic 1628 and obtain the engineered bacterium with stable inheritance . Compared to the original strain, the activity of the synthetic enzyme 6-pyruvoyl-tetahydropterin of the recombinant bacterium amounts to at least 1.3 times and the production of toyocamycin rises to at least 1.3 times. 23.4%.

Description

The method of construction and the utility of the recombinant diastatochromogenic Streptomyces to enhance toyB expression

TECHNICAL AREA

The present invention relates to increasing the production of toyocamycin by enhancing the expression of toyB genes in diastatochromogenic Streptomyces and belongs to the field of genetic engineering technology.

TECHNICAL BACKGROUND

Toyocamycin is a new nucleoside antibiotic, the molecular formula being C12H13N5O4, ribose Ci connecting the purine denitrogenation ring similar to guanine, the key structure being Pyrryl-pyrimidine, a nucleoside analogue. Its functioning mechanism mainly lies in the influence on the growth of bacteria by inhibiting the transcription of the microbe and research reports on its bioactivity focus considerably on the field of clinical medicine. The most recent research shows that toyocamycin can have a good effect on the prevention and cure of epidemic diseases of several plants. In addition, its long use does not cause environmental pollution and to some extent plays a role in adjusting plant growth. As a result, toyocamycin has the potential to be useful in the prevention and cure of agricultural plant diseases. In comparison with the synthesis method

- 2 BE2019 / 5493 chemical, the biological synthesis of toyocamycin uses renewable resources as a raw material and in addition it has the advantages of mild condition reaction, low pollution and low cost, etc. So the synthesis method organic is an economical and efficient method of industrial production of toyocamycin at the moment.

Toyocamycin belongs to the secondary metabolite and its synthesis pathway is complex due to the restriction and control of several factors, all of which give rise to a low level of synthesis and the difficulty of industrial production. The present way of solving this problem lies in the selection of productive bacteria with higher quality and high yield, obtaining seeds by mutagenesis in the traditional way being considered as the important means of selection. But many uncertain factors and the too long period always accompany the selection of good productive bacteria.

How to take advantage of molecular biology and other advanced technical means improving the microbe and increasing the production of toyocamycin is a practicable way. McCarty's group considers a strain of Streptomyces rimosus (ATCC14673) which can synthesize toyocamycin as an object of research, clones the genetic cluster of biological synthetic toyocamycin, elucidates the process of the synthesis of toyocamycin and the mechanism of adjustment and control. In addition, studies discover that it is the prosome of GTP which undergoes several reactions and synthesizes toyocamycin and the synthetic enzyme of 6-pyruvoyl-tetahydropterin coded by the toyB genes is one of the key enzymes of the metabolic pathway. Because taking advantage of metabolic engineering technology, further raising the production of toyocamycin by increasing the metabolic flow of toyocamycin affects the difficulty of establishing the

- 3 BE2019 / 5493 translation system and other complicated problems, we have not yet seen the related report.

CONTENT OF THE INVENTION

In order to overcome the drawbacks of current technology, the present invention provides the method of construction and the utility of an engineering bacterium of the diastatochromogenic Streptomyces producing toyocamycin.

Diastatochromogenic streptomyces 1628 is an antagonistic actinomycete, and its fermented drink has a strong inhibiting effect on several plant pathogenic fungus. After separation and extraction, it is proven that the main active component of the fermented drink is toyocamycin. And we have not yet seen the reports of studies on the molecular modification of the metabolic engineering of diastatochromogenic Streptomyces.

First, this invention clones the toyB genes encoding the 6-pyruvoyl-tetahydropterin synthetic enzyme for the first time using Streptomyces diastatochromogens 1628 (the library number is CGMCC NO. 2060), connects the genes and the integrated plasmid. expressive type pIB139 of Streptomyces, then succeeds in constructing the recombination vector pIB139-toyB carrying the toyB genes, and finally introduces the peculiarity of the vector pIB139-toyB in the chromosome of Streptomyces diastatochromogenic 1628 by taking advantage of the probe and transfer method .

For the toyB genes of a diastatochromogenic Streptomyces genus, its sequence is as shown in SEQ ID NO: 1

For a recombinant diastatochromogenic Streptomyces genus to reinforce toyB expression, it translates the toyB coding genes for the synthetic enzyme of 6-pyruvoyl-tetahydropterin, which is the key enzyme for the biological synthesis of toyocamycin and in addition has the

- 4 BE2019 / 5493 more powerful ability to translate toyocamycin than Streptomyces diastatochromogens 1628

The original bacterium is Streptomyces diastatochromogens 1628.

The toyB genes encoding the synthetic enzyme 6-pyruvoyltetahydropterin comes from the original bacterium of Streptomyces diastatochromogen 1628 ready to recombine.

The toyB genes coding for the synthetic enzyme 6-pyruvoyltetahydropterin are inserted into the chromosome of the original diastatochromogenic Streptomyces bacteria.

The method of constructing recombinant diastatochromogenic Streptomyces to enhance toyB expression is characterized in that the method is as follows:

1) Construct the translation vector pIB139-toyB; .

2) Take advantage of the welding and transfer method and insert the translation vector into the chromosome of diastatochromogenic Streptomyces, then obtain the engineered bacteria.

Take advantage of the permE * promoter of the vector pIB139 to start the translation of the toyB genes and take advantage of the probe and transfer method to introduce the peculiarity of the vector pIB139-toyB to the chromosome of Streptomyces diastatochromogens 1628 and obtain the engineering bacterium with a stable heredity.

The usefulness of recombinant diastatochromogenic Streptomyces for enhancing toyB expression shows that after the fermentation of the recombinant bacterium 1628-TOYB, the enzymatic activity of the synthetic enzyme 6-pyruvoyl-tetahydropterin of the recombinant bacterium s breeds at least 1.3 times and the production of toyocamycin is at least 23.4%, compared to the original strain.

The good effect of the present invention:

The present invention enhances the excessive translation of the toyB genes of the key enzyme of the synthetic synthetic pathway of toyocamycin in

- 5 BE2019 / 5493

Diastatochromogenic streptomyces and the enzymatic activity of the synthetic enzyme 6-pyruvoyl-tetahydropterin of the recombinant bacterium amounts to at least 1.3 times and the production of toyocamycin amounts to at least 23.4%, compared to the original strain. And that lays the foundation for further increasing the production of toyocamycin and achieving industrial production in the shortest possible time.

ADDITIONAL DRAWINGS:

Drawing 1 is the diagram of the construction of the recombination plasmid pIB139-toyB.

Drawing 2 is the demonstration of the enzymatic section of recombinant plasmid pMD18-T-toyB.

1.pMD18-T-toyB / Nde I + Not I; 2. AND DL2000 marker;

Figure 3 is the SDS-PAGE diagram of the toyB genes in E.coli BL21.

1. pET28a-toyB / E.coli BL21 provocation; 2. Marker Protein; 3. pET28a-toyB /E.coli BL21 unprovoked.

Drawing 4 is the electrophoresis demonstration of the enzymatic section of shuttle expression plasmid and of recombination pIB139-toyB. ÀAND / Hin dIII Marker; 2. pIB139-toyB / Nde I + Not I; 3. toyB gene; 4. DL2000 Marker.

Drawing 5 is the demonstration of PCR electrophoresis of the recombination bacterium 1628-TOYB.

1-3 the strain of genetic engineering; 4 the original strain; 5 DL2000 marker.

CONCRETE IMPLEMENTATION METHOD

We present to you a more detailed description combining the annexed drawings and concrete methods of execution.

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Practical example 1: amplification of the target gene and construction of recombinant diastatochromogenic Streptomyces

Genomes of the chromosome of S. diastatochromogens 1628 as a model, PtoyB F Nde I and PtoyB R Not I as a genetic primer, obtaining the toyB genes containing Nde I and Not I, two enzyme sites by amplification of PCR technology, conjugating vector pMD18-T, construct the cloning vector pMD18-T-toyB (Nde I + Not I) and then transform the vector pMD18-T-toyB (Nde I + Not I) in the Escherichia coli receptor (abbreviated as E. coli) , coat the E.coli on the LB agar tray containing the resistant ampicillin, cultivate them overnight at a temperature of 37 T, randomly choose positive transformants, identify them by enzymic section and then bring them to the Limited Engineering Company biological laboratory to test the sequence and then analyze the sequence, use Nde I and Not I two enzymes to cut positive transformants, acquire the toyB genes containing Nde I and Not I, two enzyme sites, connect integrated expression vector of streptomyces shuttle pIB139 cut by the same two enzymes, acquire shuttle expression vector and recombination pIB139-toyB, after the demonstration of enzymic cut transform them in E.coli ET12567 (pUZ8002), select the positive transformant E.coli ET12567 (pUZ8002, pIB139-toyB ) on the LB tray containing resistant kanamycin and resistant apramycin, and finally E. coli ET12567 (pUZ8002, pIB139-toyB) as a donor, S. diastatochromogens as receptor, introduce the peculiarity of the vector pIB139-toyB to the chromosome of S. diastatochromogens 1628 by the probing and transfer method, select the positive transformants on an MS tray containing resistant apramycin, and thus acquire Streptomyces. 1628-TOYB recombinant diastatochromogens.

The chromosome of S. diastatochromogens 1628 as a model, design two primers, amplify toyB with PCR technology, and the design of the primers is as follows:

- 7 BE2019 / 5493 toyB F Nde I: 5 '-CGCCATATGTTGTTCAGCATCACCGTC -3'; toyB R Not I: 5 '-CGCGCGGCCGCTCACAGCGCACGCTCGTAG -3' The demonstration of enzymatic cutting of the two restriction enzymes Nde I and Not I for the recombination plasmid pMD18-T-toyB is as shown in drawing 2. After the enzymatic cutting of two enzymes of the recombination plasmid pMD18-T-toyB, approximately 0.4 kb and 2.7 kb of DNA fragment are obtained. If the lengths were respectively identical to the fragment of the toyB genes and to plasmid pMD18-T, it will be proved that the conjugation of recombinant cloning vector is correct. The plasmid sequence test pMD18-T-toyB and the sequence analysis indicates the insertion fragment is the sequence of 399 bp which codes 132 amino acids with a relative molecular mass of approximately 14 kDa and has a high homology to the synthetic enzyme 6pyruvoyl-tetahydropterin from many Streptomyces reported, the largest homology reaching 90%. The toyB nucleic acid sequence is as shown in SEQ ID NO: 1. Insert the toyB genes into the pET28a vector, construct the pET28a-toyB recombination vector, then introduce the recombination vector into Escherichia coli BL21, select the positive transformants and put them in 10 ml of LB culture medium, cultivate them by oscillation at temperature 37 ° C overnight and the following day provoke expression using IPTG. As shown in Figure 3, the recombination bacterium shows the obvious specificity band after the challenge. The activity of the synthetic enzyme of 6pyruvoyl-tetahydropterin of the test is 39 U / mg total protein and this demonstrates the translation of the toyB genes with success in Escherichia coli.

The result of the enzymatic section demonstration of the shuttle expression and recombination plasmid of the ingested type pIB139-toyB is as shown in drawing 4: the recombination plasmid pIB139toyB cut by two enzymes Nde I and Not I delivers 0.4 kb fragment with a length identical to the toyaB genes, it demonstrates the success

- 8 BE2019 / 5493 constructing plasmid pIB139-toyB. Introduce the plasmid pIB139toyB to the chromosome of S. diastatochromogens 1628 by the probing and transfer method, randomly choose a few unique microbial colonies on a resistant apramycin plate and place them in the CP culture medium, cultivate them several times, extract the chromosome. If in all the chromosome amplifications by the PCR test appeared the apramycin resistant genes apr (desiccator 5), it will be proven that the Streptomyces diastatochromogenic recombination 1628-TOYB was successfully constructed with a stable inheritance.

Practical example 2: demonstration of the fermentation performance of the original strain and of the recombinant strain for diastatochromogenic Streptomyces

In comparison with the original strain, the activity of the synthetic enzyme of 6-pyruvoyl-tetahydropterin of the recombinant strain increases by 1.3 times; fermentation tests are carried out in 250 ml of oscillation flask for the recombination strain 1628-TOYB and the original strain S. diastatochromogenes 1628 in order to demonstrate that the increase in the activity of the synthetic enzyme of 6-pyruvoyltetahydropterin from S. diastatochromogens induces an increase in the production of toyocamycin from the point of view of fermentation. With a revolution speed of 200 r / min for an alternating oscillator, at a temperature of 28 ° C. ferment 96 hours and the control group is the starting strain of diastatochromogenic Streptomyces. As Table 1 shows: the final production of toyocamycin from the recombinant strain is higher than that of the original strain and reaches 166.55 mg / L, increasing by approximately 23.4% compared to the strain of origin, with good repeatability. As a result, it is attested that reinforcing the expression toyB coding for the synthetic enzyme of 6-pyruvoyltetahydropterin, that is the key enzyme of the biological synthesis pathway in the production strain of toyocamycin S. diastatochromogenes

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1628 promotes the increase in the production of toyocamycin during fermentation.

Table 1 is the comparison of the final production of toyocamycin between the recombinant strain and the original strain.

Table 1

Strain Toyocamycin production 1628 134.97 1628-TOYB 166.55

Claims (2)

1. The toyB genes of a diastatochromogenic Streptomyces genus are characterized by the fact that its sequence is as shown in SEQ ID NO: 1. 2. A genus Streptomyces diastatochromogens of recombination to reinforce the expression toyB is characterized by the fact that Streptomyces diastatochromogens 1628 whose stock number is CGMCC NO. 2060 is recombined and then translated the toyB genes coding for the synthetic enzyme of 6-pyruvoyl-tetahydropterin, i.e. the key enzyme for the biological synthesis of toyocamycin and the sequence of the toyB genes is as shown in SEQ ID NO: 1 as well as has the more powerful capacity to translate toyocamycin than Streptomyces diastatochromogens 1628 with the stock number of CGMCC NO. 2060; The toyB genes encoding the synthetic enzyme 6-pyruvoyltetahydropterin are introduced into the chromosome of Streptomyces diastatochromogenic 1628 described above. 3. The method of constructing a recombinant diastatochromogenic Streptomyces genus of reinforcing the toyB expression according to claim 2 is characterized in that the method is as follows: 1) Construct the translation vector pIB139-toyB; 2) Take advantage of the welding and transfer method and insert the above translation vector into the chromosome of diastatochromogenic Streptomyces and obtain the engineered bacteria. The translation vector pIB139-toyB takes advantage of the permE * promoter of the vector pIB139 to start the translation of the toyB genes and the step 2) described above takes advantage of the method - 11 BE2019 / 5493 probing and transfer to introduce the peculiarity of the vector pIB139-toyB to the chromosome of Streptomyces diastatochromogens 1628 and obtain the engineered bacterium with stable inheritance. 4. The usefulness of a recombinant diastatochromogenic Streptomyces genus for enhancing the toyB expression according to claim 2 is characterized in that after the fermentation of recombinant diastatochromogenic Streptomyces, the activity of the synthetic enzyme of 6- pyruvoyl-tetahydropterin of the recombinant bacterium increased at least 1.3 times and the production of toyocamycin increased at least 23.4% compared to the original strain.