CN104531598A - Recombined streptomycete, construction method thereof and method for increasing antibiotic yield - Google Patents

Abstract

The invention discloses a recombinant streptomyces, a construction method thereof and a method for increasing antibiotic production. The recombinant Streptomyces overexpresses the small subunit ilvH of acetolactate synthase. By overexpressing the regulatory subunit gene of acetolactate synthase through the recombinant bacteria, the products of secondary metabolism with L-isoleucine and/or L-valine and/or L-leucine as precursors can be significantly increased (such as antibiotics) production.

Description

A kind of recombinant streptomyces, its construction method and the method for improving antibiotic production

technical field

The invention belongs to the field of genetic engineering, relates to a recombinant streptomyces and its application, in particular to improving the output of antibiotics by transforming key genes in the synthetic pathway of antibiotic precursors.

Background technique

Streptomyces is a Gram-positive actinomycete, which belongs to the genus Streptomyces (Strptomyces) in systems biology, and has a complex life cycle and secondary metabolic pathways. Many members of this genus produce a variety of biologically active secondary metabolites. Among the tens of thousands of known antibiotics, more than 70% are produced by Streptomyces. In addition, it can also produce immunosuppressants, antineoplastic agents, insecticides and a variety of extracellular hydrolytic enzymes, such as cellulase, amylase, pectinase and protease.

In the process of Streptomyces producing secondary metabolites, amino acids can be used as carbon sources and nitrogen sources, and many amino acids are precursors of some secondary metabolites, such as valine, isoleucine, leucine amino acids, and their metabolites include acetyl-CoA, propionyl-CoA, butyryl-CoA, and isovaleryl-CoA, etc., which are precursors for the biosynthesis of macrolide antibiotics. Such as the biosynthesis of avermectin (Hafner E W, Holley B W, Holdom K S, et al.Branched-chain fatty acid requirement for avermectin production by a mutant of Streptomyce avermitilis lacking branched-chain 2-oxo acid dehydrogenase activity[ J]. J Antibiot (Tokyo), 1991, 44 (3): 349 ~ 356), spiramycin Streptomyces producing Bite spiramycin (Li Zhenlin, Jiang Wei, Wang Yonghong, etc. Val, Ile and Leu on Bite spiramycin Influence of Isoleucine upon quinomycin biosynthesis by Streptomyces sp. 732 [J]. J Bacteriol, 1967, 93 (4): 1327-1331), etc.

Avermectin is a class of macrolide antibiotics produced by the fermentation of Streptomyces avermitilis, which has a wide range of anthelmintic and insecticidal activities. Abamectin Streptomyces fermentation broth usually contains 8 kinds of abamectin components A1a, A1b, A2a, A2b, B1a, B1b, B2a, B2b, among which, the insecticidal effect is best with B1 component, especially B1a components. Abamectin has a unique mechanism of action, which is different from conventional chemical pesticides. The insecticidal effect is dozens of times that of general chemical pesticides, and it is not easy to make pests resistant. The target of abamectin is the neurotransmitter γ-aminobutyric acid of nematodes and arthropod parasites. It has low toxicity to mammals, high selectivity, and a short residual time in plants, and microorganisms in the soil can quickly decompose it into non-toxic substances. Due to its outstanding advantages such as broad spectrum, high efficiency, no residue, and high safety to animals and plants, abamectin has been widely used in agriculture, animal husbandry, medicine and other fields, and has broad market prospects and application value.

In addition, with Abamectin as the parent, a series of derivative products with higher activity, stronger selectivity, and safety for animals and plants have been developed. The commercialized products include doramectin, ivermectin, Emamectin etc.

Abamectin has been industrialized in China and has achieved huge economic and social benefits. But the production strain of abamectin also has low fermentation unit, problems such as high production cost. How to improve the output of abamectin and reduce the production cost is an important topic in the research of abamectin.

Contents of the invention

Aiming at the demand in the prior art, the present invention provides a kind of recombinant streptomyces, which promotes acetolactate synthase to the L-isoleucine in the recombinant streptomyces by overexpressing the small subunit of acetolactate synthase. , L-valine and/or L-leucine biosynthesis, thereby improving Streptomyces with L-isoleucine, L-valine and/or L-leucine as precursor The production of metabolic products. The present invention also provides a kind of product (such as avermectin, biterspiramycin) that carries out secondary metabolism with L-isoleucine, L-valine, L-leucine as precursor in a kind of improving streptomyces , quinomycin, etc.) yield method.

According to the first aspect of the present invention, a recombinant Streptomyces is provided, wherein the recombinant Streptomyces overexpresses the small acetolactate synthase subunit ilvH shown in the amino acid sequence 2 of the Sequence Listing.

According to one embodiment, the recombinant Streptomyces is obtained by cloning the ilvH gene encoding the small subunit of the acetolactate synthase into an expression vector, and then introducing the obtained recombinant expression vector into Streptomyces cerevisiae.

The Streptomyces initiatus is a Streptomyces producing antibiotics with L-isoleucine and/or L-valine and/or L-leucine as precursors. For example, it can be wild-type Streptomyces, or Streptomyces obtained by modification, mutation, mutagenesis or gene recombination.

Preferably, the aforementioned streptomyces is selected from a strain of Streptomyces avermitilis, Streptomyces spiramyceticus and Streptomyces sp.732.

Most preferably, the Streptomyces avermitilis is Streptomyces avermitilis MA4680 (ATCC31267).

The expression vector is preferably an Escherichia coli-Streptomyces shuttle vector.

According to one embodiment, the Escherichia coli-Streptomyces shuttle vector is pSET152, and the promoter and ilvH gene fragments are inserted into the multiple cloning site of pSET152 to obtain a recombinant expression vector.

The ilvH gene is most preferably the ilvH gene encoding the small subunit of acetolactate synthase in Streptomyces abamectin, i.e. the nucleotide sequence 1 in the sequence listing; the promoter is the nucleotide sequence in the sequence listing The strong promoter ermE*p of the erythromycin resistance gene shown in 3.

Most preferably, the recombinant Streptomyces is Streptomyces avermitilis AV-2320-8, which was deposited on December 08, 2014 at No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing. General Microbiology Center of the Culture Collection Management Committee, the preservation number is CGMCC 10146.

According to yet another aspect of the present invention, there is provided a method for preparing the above-mentioned recombinant Streptomyces, comprising overexpressing the acetolactate synthase small subunit ilvH shown in amino acid sequence 2 of the Sequence Listing in Streptomyces cerevisiae.

According to one embodiment, the recombinant Streptomyces is obtained by cloning the ilvH gene encoding the small subunit of the acetolactate synthase into an expression vector, and then introducing the obtained recombinant expression vector into Streptomyces cerevisiae.

The method for introducing the recombinant expression vector into Streptomyces spp. can adopt common methods in the field of bioengineering, such as combined transfer method, electroporation method, protoplast transformation method and the like.

Preferred is the conjoint transfer method. Preferably, the expression vector is first transformed into Escherichia coli ET12567 (pUZ8002) deficient in restriction modification, and the resistant colonies are collected and combined with the Streptomyces initiating for transfer.

The Streptomyces initiatus is a Streptomyces producing antibiotics with L-isoleucine and/or L-valine and/or L-leucine as precursors. For example, it can be wild-type Streptomyces, or Streptomyces obtained by modification, mutation, mutagenesis or gene recombination.

Preferably, the above-mentioned starting Streptomyces is selected from a strain in Streptomyces avermitilis, Streptomyces spiramyceticus and Streptomyces sp.732.

Most preferably, the Streptomyces avermitilis is Streptomyces avermitilis MA4680 (ATCC31267).

The expression vector is preferably the Escherichia coli-Streptomyces shuttle vector pSET152.

The ilvH gene is most preferably the ilvH gene encoding the small subunit of acetolactate synthase in Streptomyces abamectin, i.e. the nucleotide sequence 1 in the sequence listing; the promoter is the nucleotide sequence in the sequence listing The strong promoter ermE*p of the erythromycin resistance gene shown in 3.

According to another aspect of the present invention, there is provided a method for improving the yield of products of secondary metabolism with L-isoleucine, L-valine, and L-leucine as precursors in Streptomyces, the method It includes the step of using the above-mentioned recombinant Streptomyces to ferment.

According to the present invention, take Streptomyces avermitilis (Streptomyces avermitilis MA4680 (ATCC31267))) as starting bacterial strain, the recombinant Streptomyces avermitilis that obtains, after shake flask culture 240 hours, avermectin output can reach 3486.32 ±497.97μg/mL, which is 5.13 times of the abamectin production of the starting strain.

The recombinant Streptomyces abamectin genetically engineered bacterium constructed in the specific example of the present invention can be directly used in the fermentation production of Streptomyces abamectin, so that the output of Abamectin can be improved, thereby reducing the production cost. Those skilled in the art should understand that the method of the present invention, by overexpressing the small subunit of acetyl synthase, increases the acetolactate in the synthetic pathway of L-isoleucine, L-valine, and L-leucine in Streptomyces. Synthase enzyme amount, thereby increasing the production of L-isoleucine, L-valine, L-leucine, and further improving the production of products of secondary metabolism with these amino acids as precursors, this method is suitable for Streptomyces that need to go through this synthetic pathway when producing secondary metabolites (such as antibiotics), thereby obtaining high-yielding recombinant bacteria of the target product, and thus obtaining the effect of improving the target product.

Description of drawings

The invention will be better understood with reference to the following figures.

Figures 1A and 1B are plasmid maps of recombinant plasmids pH-19 and pH-152 containing ilvH gene and strong promoter of erythromycin resistance gene, respectively.

Fig. 2 is the abamectin output that abamectin streptomyces strain Streptomyces avermitilis MA4680 (ATCC31267) and its recombinant avermectin streptomyces (CGMCC10146) prepared according to the present invention obtain through fermentation.

Fig. 3 A is abamectin B1a standard product HPLC collection of illustrative plates, Fig. 3B and 3C are respectively the avermectin bacteria Streptomyces avermitilis MA4680 (ATCC31267) and the recombinant avermectin streptomyces prepared according to the present invention produce Avermectin Streptomyces HPLC profile of protein B1a.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the present invention is not limited to the following examples.

Based on the excellent characteristics and wide application of abamectin, the present invention mainly takes abamectin Streptomyces as an example to study, by overexpressing acetolactate synthase small subunit in abamectin Streptomyces, obtained A strain with significantly improved abamectin production.

Similarly, in other Streptomyces that use L-isoleucine and/or L-valine and/or L-leucine as precursors to produce secondary metabolites, it is also believed that the corresponding secondary metabolites can be effectively improved. output. Examples of such Streptomyces include spiramycin-producing Streptomyces spirulina, quinomycin-producing Streptomyces sp.732, and the like.

Abamectin is a group of glycosides formed by a sixteen-membered ring lactone and a disaccharide (oleanose), and there is a spiroketal system containing two six-membered rings around the sixteen-membered lactone and Hexahydrobenzofuran ring system. The backbone of the glycosyl ligand is a branched-chain fatty acid S(+)-methylbutyryl fatty acid or isobutyryl fatty acid as the starting point, and 7 acetates and 5 propionates are polymerized head-to-tail. The different substituents at the C-25 position are divided into components a and b. The dimethyl butyryl group of component a is derived from S(+)-methylbutyryl CoA, and the isobutyryl group of component b is derived from isobutyl Acyl-CoA. S(+)-methylbutyryl-CoA and isobutyryl-CoA are formed from L-isoleucine and L-valine through deamination, transamination and decarboxylation, respectively.

Acetolactate synthase is an enzyme that biocatalyzes the first step in the biosynthesis of L-isoleucine, L-valine, and L-leucine. In the process of biosynthesis of secondary metabolites with leucine as the precursor, increasing the activity or enzyme amount of acetolactate synthase will increase the output of the corresponding secondary metabolites.

In Streptomyces abamectin, acetolactate synthase contains 3 large subunits and 1 small subunit. The large subunit plays a catalytic role, and the small subunit plays an activation and feedback regulation role. The present invention finds through research that overexpressing the small subunit of the acetolactate synthase can significantly increase the production of abamectin with L-isoleucine and L-valine as precursors.

In the following examples, unless otherwise specified, all are conventional methods.

Embodiment 1, the construction of ilvH gene expression vector

1. Amplification of ilvH structural gene

Primers were designed for amplifying the ilvH structural gene [NC_003155.4 (3353885..3354412)] located on the chromosome of Streptomyces avermitilis MA4680 (ATCC31267), that is, sequence 1 in the sequence listing.

Upstream primer Primer F1: ATGAGCAAGCACACCCTCTCCGTCCT,

Downstream primer Primer R1: TTGTAAAACGACGGCCAGT GAATTC TTACGCGGATCGGTCCAGCGCGCGC,

The underlined base is the recognition site of the restriction endonuclease EcoRI, and the amplified product should be 557bp.

Using the total DNA of Streptomyces avermitilis MA4680 (ATCC31267) as a template, Primer F1 and Primer R1 as primers, the Q5Hot Start High-Fidelity DNA Polymerase from New England Biolabs was used to perform PCR amplification. The amplification conditions were: 98°C, 3min; (98°C, 10s; 72°C, 40s)×30 cycles; 72°C, 2min. The amplified product was detected by agarose gel electrophoresis, and there was a specific amplified band at about 500 bp.

2. Amplification of the strong promoter (ermE*p) of the erythromycin resistance gene

Design primers for amplifying the plasmid located in pZW221 (Li Jia, Xiang Sihai, Yang Xiushan, Yang Keqian, comparing the activities of two actinomycete promoters by reporter gene method, Acta Microbiology, 2009, 49(11): 1454- 1458.) on the ermE*p gene (ie, sequence listing sequence 3).

Upstream primer Primer F2: GCATGCCTGCAGGTCGAC TCTAGA AGAGCGAGTGTCCGTTCGAGTGGCGGCTT,

Downstream primer Primer R2: GGACGGAGAGGGTGTGCTTGCTCATATGTGGATCCTACCAACCGGCACGGTT.

The underlined bases are restriction endonuclease XbaI restriction sites, and the amplified product should be 235bp.

Using the pZY125 plasmid as a template, Primer F2 and Primer R2 as primers, the Q5Hot Start High-Fidelity DNA Polymerase from New England Biolabs was used for PCR amplification. The amplification conditions were 98°C, 3min; (98°C, 10s; 72°C , 30s)×30 cycles; 72°C, 2min. The amplified product was detected by agarose gel electrophoresis, and there was a specific amplified band at about 250 bp.

3. Construction of recombinant plasmid pH-152

A DNA recovery kit (OMEGA, D2500-02) was used to recover the PCR amplified fragments of the ilvH gene and ermE*p, and the pUC19 vector was digested with XbaI and EcoRI to recover a fragment of about 2660 bp. The three fragments were ligated using Gibson Assembly kit (New England Biolabs), named pH-19 (see Figure 1A). Sequencing and comparison showed that the amplified fragments were indeed ermE*p and ilvH genes. The pH-19 vector containing ermE*p and ilvH genes was digested with XbaI and EcoRI, and the junction fragment of the ermE*p and ilvH genes of about 710bp was reclaimed, and this fragment was combined with the vector pSET152 (Bierman M. , Logan R, O'Brien K, et al.Plasmid cloning vector for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.Gene, 1992,116:43-49) connection, the connection products were respectively transformed into the competent state of Escherichia coli DH5α Cells (TransGen Biotech Company), the plasmid was extracted from the transformant for PCR and enzyme digestion verification, and the correct recombinant plasmid was named pH-152 (see Figure 1B). pH-152 is an integrative plasmid, and the ilvH gene in this plasmid is placed under the strong promoter ermE*p of the erythromycin resistance gene.

Embodiment 2, transformation of recombinant plasmid

Due to the strong restriction modification in Streptomyces abamectin, E.coli DH5α is directly combined with Streptomyces abamectin for transfer, the transformation efficiency is extremely low, and sometimes even no transformants can be obtained. However, using E.coli ET12567 (PUZ8002) without restriction modification for binding transfer, the transformation efficiency was significantly improved. Therefore, the constructed recombinant plasmid was transformed into E.coli ET12567 (PUZ8002) (Kieser T, Bibb M J, Buttner M J, et al. Practical Streptomyces Genetics, 2000, Norwich: The John Innes Foundation.) to obtain non- Methylated DNA is then transferred by binding.

In this example, Streptomyces avermitilis MA4680 (ATCC31267) is selected as the starting strain, which produces off-white spores on the plate.

The E.coli ET12567 (PUZ8002) containing the ilvH gene expression plasmid pH-152 constructed in Example 1 was combined with the above-mentioned Streptomyces abamectin for transfer, spread on an MS plate containing 10mM MgCl ₂ , and cultivated at 28°C After 16-20 hours, cover evenly with 1 mL of sterile water containing 1000 μg nalidixic acid and 1000 μg apramycin, and incubate at 28°C for 5-7 days. After the PCR verification is correct, the next step of fermentation research is carried out.

The obtained transformant is the recombinant Streptomyces obtained according to the construction method of the present invention. The recombinant streptomyces is named as Streptomyces avermitilis AV-2320-8, and it is preserved in the General Microorganism Center of China Microbiology Culture Collection Management Committee, and the preservation number is CGMCC10146. The optimum culture condition of this strain is 28 ℃, and the pH is close to neutral. When cultured on a solid medium (such as MS) for 5-7 days, it will produce off-white spores with few air filaments and base filaments.

Embodiment 3, the fermentation research of recombinant bacterial strain

1. Shake flask fermentation of abamectin streptomyces

Abamectin Streptomyces starting bacterial strain Abamectin Streptomyces avermitilis MA4680 (ATCC31267) and the recombinant bacterium that embodiment 2 obtains grow abundant spores on the slant culture medium, to the avermectin Streptomyces transformant Abamectin production levels were tested. Dig 1 cm ² of the lawn from the inclined surface, put it into a seed bottle containing 40 mL of sterilized seed medium, and incubate on a shaker at 28°C for 44-48 hours with a rotation speed of 200 rpm and a rotation radius of 50 mm to obtain a seed culture solution. Get above-mentioned seed culture solution and inoculate in the Erlenmeyer flask that 30mL sterilized fermentation medium is housed by the inoculation amount of 5% (percentage by volume), 28 ℃ of shaker cultures 10 days, put bottle, measure Avermella with HPLC method The fermentation unit of element, specific HPLC analysis is as described in the following step 2.

The slant medium in this experiment (glucose 1.5%, beef extract 0.3%, asparagine 0.05%, KH ₂ PO ₄ 0.05%, agar 1.8%), seed medium (cornstarch with a final concentration of 30g/L, final Concentration is the soybean cake powder of g/L, the peanut cake powder that the final concentration is 10g/L, the yeast powder that the final concentration is 4g/L, the CoCl ₂ that the final concentration is 0.03g/L) and fermentation medium (final concentration is 150g/L cornstarch, 28g/L soybean meal powder, 9g/L yeast powder, 0.25g/L (NH ₄ ) ₂ SO ₄ , 0.02g/L final concentration CoCl ₂ , Na ₂ MoO ₄ at a final concentration of 0.022g/L, MnSO ₄ at a final concentration of 0.0023g/L, amylase at a final concentration of 4000U/L, and CaCO ₃ at a final concentration of 0.8g/L). (See Chinese Patent Application No. 200810227639.8, a method for preparing avermectin and its special strain, the full text of which is incorporated herein by reference.)

2. HPLC analysis of fermentation products

1) Sample treatment: take 1.0mL of fermentation broth, add 9.0mL of methanol, ultrasonically shake for more than 30 minutes, and let stand for 10-20 minutes;

2) Take 1 mL of the supernatant, filter it with a 0.2 μm microporous membrane, and obtain the filtrate for HPLC (Agilent 1200 high performance liquid chromatograph) analysis.

3) HPLC analysis conditions: C18 reversed column (Agilent), column length 150mm, column inner diameter 4.6mm, mobile phase methanol: water (90:10), flow rate 1.0mL/min, autosampler injection, injection The sample volume is 10 μL, and the detection wavelength is 245 nm. Under these conditions, the retention time of the avermectin B1a standard substance (DR, Germany) is about 6 minutes, and the peak area in the fermentation filtrate with a retention time of about 6 minutes is calculated to calculate the abamectin B1a yield. The experiment was repeated 3 times, and the results were averaged.

The fermentation result in Fig. 2 shows that the abamectin fermentation unit of the transformant containing the ilvH expression vector is significantly improved compared with the starting strain, which is increased by about 4 times.

It should be understood that, without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps and conditions of the present invention fall within the scope of the present invention.

Claims (10)

1. a restructuring streptomycete, the acetolactate synthase small subunit ilvH shown in aminoacid sequence 2 of wherein said restructuring streptomycete process LAN sequence table.

2. restructuring streptomycete according to claim 1, wherein pass through the ilvH gene clone of the described acetolactate synthase small subunit of coding to expression vector, again the recombinant expression vector obtained is incorporated into the streptomycete that sets out and obtains described restructuring streptomycete, the wherein said streptomycete that sets out be with ILE and/or Valine and/or L-Leu for precursor produces antibiotic streptomycete; Preferably, the streptomycete that sets out described in is selected from a kind of bacterial strain in Avrmectin streptomycete Streptomyces avermitilis, spiramycin streptomyces Streptomyces spiramyceticus and streptomycete Streptomyces sp.732; More preferably, the streptomycete that sets out described in is Avrmectin streptomycete Streptomyces avermitilis MA4680.

3. restructuring streptomycete according to claim 2, wherein said expression vector is intestinal bacteria-streptomycete shuttle vectors;

Preferred shuttle vectors is pSET152, promotor and described ilvH gene fragment is inserted the multiple clone site of pSET152, obtains recombinant expression vector;

More preferably, described ilvH gene is the ilvH gene of encoding acetolactate synthase small subunit in the Avrmectin streptomycete shown in nucleotide sequence 1 in sequence table, and described promotor is the erythromycin resistance gene strong promoter ermE*p shown in the nucleotide sequence 3 in sequence table;

Most preferably, described restructuring streptomycete is Avrmectin streptomycete Streptomyces avermitilis AV-2320-8, and it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC 10146.

4. build a method for the restructuring streptomycete in claims 1 to 3 described in any one, be included in the acetolactate synthase small subunit ilvH shown in aminoacid sequence 2 of process LAN sequence table in the streptomycete that sets out.

5. method according to claim 4, the wherein said streptomycete that sets out be with ILE and/or Valine and/or L-Leu for precursor produces antibiotic streptomycete; Be preferably selected from a kind of bacterial strain in Avrmectin streptomycete Streptomyces avermitilis, spiramycin streptomyces Streptomyces spiramyceticus and streptomycete Streptomyces sp.732; Be more preferably Avrmectin streptomycete Streptomyces avermitilis MA4680.

6. method according to claim 5, wherein pass through the ilvH gene clone of the described acetolactate synthase small subunit of coding to expression vector, again the recombinant expression vector obtained is incorporated into the streptomycete that sets out and obtains described restructuring streptomycete, preferably, first expression vector is transformed in the intestinal bacteria ET12567 (pUZ8002) of restriction modification effect defect, collects resistant clones and do Conjugative tiansfer with the described streptomycete that sets out again.

7. method according to claim 6, wherein said expression vector is intestinal bacteria-streptomycete shuttle vectors pSET152.

8. method according to claim 6, wherein said ilvH gene is the ilvH gene of encoding acetolactate synthase small subunit in the Avrmectin streptomycete shown in nucleotide sequence 1 in sequence table, and described promotor is the erythromycin resistance gene strong promoter ermE*p shown in the nucleotide sequence 3 in sequence table.

9. improve a method for the Product yields carrying out secondary metabolism in streptomycete with ILE, Valine, L-Leu for precursor, described method comprises and utilizes the restructuring streptomycete in claim 1 ~ 3 described in any one to carry out the step of fermenting.

10. method according to claim 9, the wherein said product carrying out secondary metabolism for precursor with ILE, Valine, L-Leu is microbiotic, preferably Avrmectin.