CN116323959A - Method for improving spinosyn yield - Google Patents
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- CN116323959A CN116323959A CN202280005732.2A CN202280005732A CN116323959A CN 116323959 A CN116323959 A CN 116323959A CN 202280005732 A CN202280005732 A CN 202280005732A CN 116323959 A CN116323959 A CN 116323959A
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
Abstract
The invention discloses a method for improving spinosyn yield, belonging to the field of genetic engineering. The method of the invention is to over-express the spnF gene and/or the spnP gene in the spinosyns, and the spinosyns genetic engineering bacteria with high spinosyns can be obtained by over-expressing the spnF gene and/or the spnP gene in the spinosyns. The invention discovers that the spinF gene and/or the spnP gene can be over-expressed to obviously improve the spinosyn yield of the spinosyns.
Description
The invention belongs to the field of genetic engineering, and particularly relates to a method for improving spinosyn yield.
Spinosyns (spinosyns) is a secondary metabolite obtained by aerobic fermentation of spinosyns (Saccharopolyspora spinosa), is a novel green broad-spectrum biological pesticide, and belongs to macrolide antibiotics. The unique insecticidal mechanism of spinosad represents the safety of biopesticides and the rapidity of chemical pesticides, and is harmless to the environment, crops and mammals due to the readily degradable nature of spinosad, without carcinogenic, teratogenic, mutagenic or neurotoxic, thus three times have been achieved in the united states "general green chemical challenge prize". Currently, spinosad is still produced by aerobic fermentation from spinosyns. Although there are several patents reporting that the yield of spinosad is improved by improving the control method of the culture medium or the fermenter, the mass production is difficult to realize by fermentation optimization because the fermentation yield of the strain is poor, so that industrialization of the strain has not been realized yet in China.
Disclosure of Invention
The invention aims to solve the problem of low spinosyn yield in the existing spinosyn fermentation production, and provides a method for improving spinosyn yield.
The aim of the invention is achieved by the following technical scheme:
a method for improving spinosyn yield by over-expressing spnF gene and/or spnP gene in spinosyn.
The spnF gene catalyzes intermolecular crosslinking reaction of spinosad macrolide.
The spnP gene catalyzes the loading of the foglibose in spinosad.
A spinosyn genetic engineering bacterium for high-yield spinosyn is spinosyn over-expressing spnF gene and/or spnP gene.
In some embodiments, the method of increasing spinosyn production is over-expressing the spnF gene and/or the spnP gene in the spinosyn WHU1107 strain. The spinosyn genetically engineered bacterium for high-yield spinosyn is obtained by over-expressing the spnF gene and/or the spnP gene in spinosyn WHO 1107 strain.
The spinosyn WHO 1107 strain is preserved in China center for type culture collection (CCTCC for short, address: university of Wuhan, china) for 3 months and 31 days in 2021, and is classified and named as Saccharopolyspora spinosa WHU1107, and the preservation number is CCTCC NO: m2021307. The spinosyn WHO 1107 strain is far higher than the existing reported strain in the yield of spinosyn produced by fermentation, and the spinF gene and/or the spnP gene are overexpressed in the WHO 1107 strain, so that the yield of spinosyn can be further improved.
The spinosyn genetically engineered bacteria with high spinosyn yield can be applied to spinosyn production.
A method of producing spinosad comprising the steps of: inoculating the spinosyn genetic engineering bacteria with high spinosyn yield into a fermentation medium, and fermenting to obtain a fermentation product containing spinosyn.
In some embodiments, the fermentation medium is formulated as follows: 8g of glucose, 2g of cottonseed meal, 1g of protein powder, 0.5g of yeast powder, 0.4g of trisodium citrate, 0.2g of dipotassium hydrogen phosphate, 0.3g of calcium carbonate, 0.2g of ammonium sulfate, 5g of rapeseed oil and pH 7.0 are added into 100mL of water.
In some embodiments, the fermentation conditions are: 250rpm, 28 ℃ and 60% humidity.
The invention has the advantages and beneficial effects that: the invention discovers that the spinF gene and/or the spnP gene can be over-expressed to obviously improve the spinosyn yield of the spinosyns. The invention provides a new transformation method and a new material for industrialized production of spinosad.
FIG. 1 is a graph showing the results of LC-MS detection of the composition of fermentation broth of a strain WHO 1107 of Saccharopolyspora spinosa.
FIG. 2 is a graph showing the results of HPLC determination of spinosyn content in fermentation broth of a strain WHO 1107 of Saccharopolyspora spinosa.
FIG. 3 is a construction diagram of pIB139-spnF plasmid. A: schematic diagram of pIB139-spnF plasmid; b: pIB139-spnF plasmid restriction enzyme analysis.
FIG. 4 is a construction diagram of pSET152-spnP plasmid. A: schematic diagram of pSET152-spnP plasmid; b: pSET152-spnP plasmid restriction enzyme analysis map.
FIG. 5 is a schematic diagram of pIB139-spnF-spnP plasmid.
FIG. 6 is a graph showing the results of spinosyn content in various spinosyn fermentation broths.
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
The Saccharopolyspora spinosa WHO 1107 strain used in the following examples was preserved in China center for type culture collection (CCTCC, address: university of Wuhan, china), with a classification designation of Saccharopolyspora spinosa WHU1107, and a preservation number of CCTCC NO: m2021307. The strain is cultured in a culture bottle filled with 25mL of fermentation medium, and the shake flask yield of spinosad is up to more than 4g/L after fermentation culture.
EXAMPLE 1 screening of the Saccharopolyspora spinosa WHU1107 Strain
1. Spinosyn strain isolation
Soil samples were collected from multiple regions at 7 months 2017 and were subjected to actinomycete isolation by the following method: the soil is split into 0.1g/mL with sterile water, 10 glass beads are added after the soil is suspended with sterile water, and then the soil is diluted step by step according to decreasing 10 times, and 10 pieces of separation culture medium are coated at each concentration. Wherein the basic formula of the separation medium is as follows: adding 5g of glucose, 3g of yeast extract and 10g of enzymatic casein (N-Z amine type A) into 1L of distilled water, mixing, fully dissolving, and regulating the pH to 7.0 by using NaOH; subpackaging according to 200mL, adding 4g of agar into each bottle; sterilizing at 115 deg.C for 30min. After sterilization is completed, the temperature is cooled to about 65 ℃, nalidixic acid is added to a final concentration of 25 mug/mL to inhibit the growth of gram-negative bacteria, and nystatin is added to a final concentration of 50 mug/mL to inhibit the growth of fungi. And selecting Streptomyces sample with bacterial colony morphology generated by spores from the bacterial colonies which can grow finally, preserving the seeds, and collecting 78 strains.
Every 5 strains of bacteria are subjected to mixed fermentation, and the fermentation method comprises the following steps:
(1) Related culture medium
Seed culture medium: adding 1g of glucose, 1g of yeast extract, 0.2g of N-Z amine type A, 2.5g of cotton seed cake powder, 2g of corn starch, 0.2g of magnesium sulfate heptahydrate and 0.1g of ammonium sulfate into 100mL of distilled water, mixing, fully dissolving, and regulating the pH to 7.0 by using NaOH; sub-packaging 25mL of seed culture medium per bottle of seed culture; sterilizing at 121deg.C for 30min.
Fermentation medium: adding 8g of glucose, 2g of cotton seed cake powder, 1g of protein powder (dolphin board), 0.5g of yeast powder, 0.4g of trisodium citrate, 0.2g of dipotassium hydrogen phosphate, 0.3g of calcium carbonate, 0.2g of ammonium sulfate and 5g of rapeseed oil into 100mL of distilled water; after fully dissolving, naOH is used for adjusting the pH value to 7.0; sterilizing at 121deg.C for 30min; split charging 25mL of fermentation medium per bottle for fermentation culture; and (3) injection: the calcium carbonate is packaged into each bottle.
(2) Fermentation process
Each isolated strain is picked up, single bacterial colony is scratched on another new flat plate to grow, bacterial blocks with the size of about 1cm are scratched in a seed culture medium, 5 isolated strains are inoculated in a culture bottle (16 bottles in total), primary seeds are cultivated for 96 hours at the temperature of 28 ℃ and the humidity of 60% at the speed of 250rpm, whether the primary seeds are infected with bacteria or not and the growth state of the seeds are observed by microscopic examination, and secondary seeds are transferred according to the transfer quantity of 1% when the bacterial infection is not infected and the growth state is good; the second-level seed culture condition is the same-level seed, the second-level seed microscopic examination is carried out when the second-level seed culture condition is cultured for 60 hours, whether the seed is infected with bacteria or not is observed, if the second-level seed culture condition is normal, the second-level seed culture condition is carried out fermentation transfer, the second-level seed culture condition is transferred to a culture bottle filled with a fermentation culture medium according to the transfer quantity of 5%, and three bacteria in each bottle are parallel; the fermentation culture conditions are as follows: culturing at 28deg.C and 60% humidity for 12 days at 250 rpm.
The fermentation broth was analyzed for spinosad by the detection method described in the reference (GaoYi Tan, et al Heterologous Biosynthesis of Spinosad: an graphics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces [ J ]. Acs Synth Biol, 2017.). The production of spinosad was found in the 1 st and 7 th bottles, 10 strains in total of the 1 st and 7 th bottles were fermented one by one according to the above method, and finally 3 strains capable of producing spinosad were found, the production amounts of which were 51.2mg/L, 253mg/L and 336mg/L, respectively, designated WHO 1100, WHO 1101 and WHO 1102.
2. Strain mutagenesis
Preparing strain inclined plane of WHU1102 into spore suspension with physiological saline, filtering with absorbent cotton, and adjusting spore concentration to 10 -6 ～10 -7 10mL of the monospore suspension was taken at a concentration of 30s in a sterile 9cm diameter plate, irradiated at a distance of 20cm from a 15W UV lamp for 30s, and then serially diluted by 10-fold decrease.
Every 20 strains are divided into a group, fermentation screening is carried out according to the method, about 3000 strains are screened, 1 group with highest yield is selected, and 20 strains are selected as primary screening strains. These 20 strains were mixed and subjected to a second round of mutagenesis as described above, and about 3000 strains were similarly selected, and a group of 20 strains with the highest spinosad yield was selected as strains to be selected (designated as WHU1103-WHU1122, respectively).
The 20 strains were fermented one by one according to the method described above, wherein the strain with the highest yield was WHO 1107, detection method in reference (GaoYi Tan, et al Heterologous Biosynthesis of Spinosad: an graphics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces [ J ]. Acs Synthh Biol, 2017.), detection of the components of the fermented product by LC-MS, and the results (FIG. 1) showed that spinosad and spinosad could be detected in the fermented liquid; the sum of the yields of spinosad a and spinosad was about 4.1g/L, with about 95% of the a component and about 5% of the D component, as analyzed by HPLC (fig. 2).
3. Identification of Strain WHO 1107
The strain WHU1107 is identified, the 16s rRNA sequence of the strain WHU1107 is shown as SEQ ID NO.1, and the sequence similarity with GENBANK ACCESSION NR _024839.1 is the highest, and the similarity is 99%. According to the above results, strain WHU1107 belongs to the genus spinosyns (Saccharopolyspora spinosa).
4. Preservation of Strain WHO 1107
The strain WHO 1107 belongs to spinosyns (Saccharopolyspora spinosa) and is preserved in China center for type culture collection (CCTCC, address: university of Chinese, wuhan, and Wuhan) in 2021, wherein the preservation number is CCTCC NO: m2021307.
5. Yield comparison of strain WHO 1107 and other Saccharopolyspora spinosa strains
The shake flask yield of the multi-killing high-yield strain obtained by mutagenesis and other methods reported in the previous literature or patent is about 1-2g/L, which is far lower than the 4g/L yield of the WHO 1107 strain of the invention. The commercially available wild-type strain NRRL18395 of Saccharopolyspora spinosa was tested for spinosad production at 78.7mg/L according to the above fermentation method, wherein the A component was about 84% and the D component was about 16% and was also much lower than the 4g/L production of WHO 1107 strain. These results demonstrate that WHO 1107 yields as high as 4g/L are a manifestation of its own high spinosad, which can be developed as an industrial strain of spinosad with high yields.
EXAMPLE 2 construction of spinosyn Gene engineering bacteria overexpressing the spnF Gene and/or the spnP Gene
(1) Construction of plasmids pIB139-spnF, pSET152-spnP and pIB139-spnF-spnP
In this example, attB-attP sites were used for integration, the promoter ermEp was used for overexpression of spnF and the original promoter PspnP was used for overexpression of spnP.
1) Construction of plasmid pIB139-spnF
The genome DNA of extracted spinosyn WHO 1107 is used as a PCR template, a cloning fragment spnF primer (an upstream primer: 5'-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3' and a downstream primer: 5'-tatgacatgattacgaattcgatatctcagccgaccggcttccgcgccgtc-3') is used for amplifying a target band spnF with the size of 880bp, the target band is assembled with a pIB139 vector which is digested by NdeI/EcoRV to obtain a vector which is subjected to Gibson assembly and overexpresses the spnF by an ermEp strong promoter, an assembled product is transformed into escherichia coli DH10B to be competent, after the escherichia coli is randomly picked up and subjected to monoclonal culture, plasmids are extracted, and then the plasmids are subjected to double digestion identification by NcoI and EcoRI (FIG. 3B), so that 4462bp target fragments with the sizes of 2256bp2 are obtained, and plasmids with correct digestion identification are selected. Subsequently, a first generation sequencing method was used to verify that the plasmid that was successful was pIB139-spnF (FIG. 3A).
2) Construction of plasmid pSET152-spnP
The genomic DNA of the extracted spinosyn WHU1107 is used as a PCR template, a cloning fragment Pspnp-spnP primer (an upstream primer: 5'-gactctagagcaggcgacgatcagtcttcgcgc-3' and a downstream primer: 5'-cgcgcggccgctcacggatggccatcagactgcccag-3') is used for amplifying a target fragment Pspnp-spnP with the size of 1719bp, the target band and a pSET152 vector are simultaneously digested and recovered by XbaI and NotI, then enzyme ligation is carried out by using T4 ligase to obtain a vector for over-expressing the spnP by using the spnP original promoter PspnP, and EcoRV is used for enzyme digestion identification (FIG. 4B) to obtain target fragments with the sizes of 6012bp and 1396bp 2, thus the plasmid pSET152-spnP is successfully constructed. Subsequently, a first generation sequencing method was used to verify that the plasmid was pSET152-spnP (FIG. 4A).
3) Construction of plasmid pIB139-spnF-spnP
The genomic DNA of the extracted spinosyn WHU1107 is used as a PCR template, a cloning fragment spnF primer (upstream primer: 5'-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3', downstream primer: 5'-cgtcgcctgctcagccgaccggcttccgcgccgtc-3') is used for amplifying a target band spnF with the size of 864bp, a cloning fragment Pspnp-spnP primer (upstream primer: 5'-ggtcggctgagcaggcgacgatcagtcttc-3', downstream primer: 5'-aacagctatgacatgattacgaattctcacggatggccatcagactg-3') is used for amplifying a target band spnP with the size of 1735bp, gibson assembly is carried out on the target band spnF and Pspnp-spnP and a pIB139 vector cut by NdeI/EcoRI, the assembled product is transformed into E.coli DH10B competent, after a plasmid is extracted by random E.coli monoclonal culture, plasmid pIB 139-spnF-nP is successfully constructed after enzyme cutting to identify that the correct one plasmid is sequenced correctly (FIG. 5).
(2) Screening of strains overexpressing spnF and spnP
The plasmid constructed above and pSET152 were transformed into competent E.cooli ET12567/pUZ8002, respectively, and the monoclone was picked up and expanded with LB medium as donor strain for conjugation transfer, and subjected to intergeneric transfer between two parents with Saccharopolyspora spinosa WHO 1107, and the conjugator was picked up in ABB13 (5 g/L soluble starch, 5g/L soybean peptone, 2.1g/L MOPS, 3g/L CaCO) 3 Thiamine hydrochloride 0.01g/L, feSO 0.046g/L 4 ·7H 2 O, 20g/L agar) plate transfer expansion culture, then picking part of colony of each zygote to TSB-M liquid culture medium (30 g/L soybean trypsin broth, 50g/L mannitol) for 3 days, extracting genome DNA as template, using upstream primer: 5'-cagcggtggagtgcaatgtcgt-3', downstream primer: 5'-cagaggcgggatgcgaagaatg-3' strains capable of amplifying the arabinomycin resistance gene with 750bp are strains which are correctly overexpressed spnF or spnP or spnF and spnP, are named WHU1107/pIB139-spnF, WHU1107/pSET152-spnP and WHU1107/pIB139-spnF-spnP respectively, and take the WHU1107/pSET152 strain as a control group.
(3) Fermentation of spnF and spnP overexpressing strains
The selected spnF and spp overexpressing strains were fermented and tested as in example 1. The results (FIG. 6) show that when fermentation culture is carried out in a culture flask filled with 25mL of fermentation medium, compared with the spinosad yield of control strain WHO 1107/pSET1524105mg/L, the spinF gene spinosad yield is up to 4410mg/L by over-expression of the strong promoter ermEp, and the yield is improved by 7.4%; the yield of spinosyn of the spnP gene is 4499mg/L by using the spnP original promoter, and the yield is improved by 9.6%. Meanwhile, the yield of spinosyns of the over-expressed spnF and spnP genes reaches 4725mg/L, and the yield is improved by 15.1 percent.
Both SpnF and spnP belong to modified genes after synthesis of the polyketide skeleton in spinosyn biosynthesis, and among spinosyn biosynthesis genes, 7 genes, in addition to SpnF and spnP, also belong to modified genes after synthesis of the polyketide skeleton, including spnJ, spnM, spnG, spnL, spnI, spnK and spnH, wherein it was confirmed in literature (GaoYi Tan, et al Heterologous Biosynthesis of Spinosad: an graphics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces [ J ]. Acs Synth Biol,2017 ])thatoverexpression of spnI in streptomyces albus heterologously expressed spinosyn increases spinosyn yield. Therefore, we tried to construct plasmids overexpressing spnI, spnH and spnJ in WHU1107 using ermEp as a vector and transferring them into WHU1107 according to the above described conjugation transfer method, and examined that spinosad production was 2317mg/L in the strain overexpressing spnI, 2259mg/L in the strain overexpressing spnJ, 2301mg/L in the strain overexpressing spnH, and lower than the original strain production in all three strains. These results indicate that there is an unreasonable expectation based on the prior art that there is a link between overexpression of spinosyn biosynthesis-related genes and an increase in spinosyn production.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
- A method for increasing spinosyn production, characterized by: for over-expression of the spnF gene and/or the spnP gene in Saccharopolyspora spinosa.
- The method for increasing spinosyn production according to claim 1, characterized in that: the spinosyns are CCTCC NO: m2021307 Saccharopolyspora spinosa (Saccharopolyspora spinosa) WHO 1107.
- A spinosyn genetically engineered bacterium for high-yield spinosyn is characterized in that: is a spinosyn that overexpresses the spnF gene and/or the spnP gene.
- The spinosyn genetically engineered bacterium of high spinosyn of claim 3, characterized in that: by keeping the number CCTCC NO: the spinosyns WHU1107 of M2021307 is obtained by over-expressing the spnF gene and/or the spnP gene.
- Use of a spinosyn genetically engineered bacterium according to claim 3 or 4 for the production of spinosad.
- A method of producing spinosad, characterized by: the method comprises the following steps: inoculating the spinosyn genetically engineered bacterium of claim 3 or 4 into a fermentation medium, and fermenting to obtain a spinosyn-containing fermentation product.
- The method for producing spinosyn according to claim 6, characterized in that: the formula of the fermentation medium is as follows: 8g of glucose, 2g of cottonseed meal, 1g of protein powder, 0.5g of yeast powder, 0.4g of trisodium citrate, 0.2g of dipotassium hydrogen phosphate, 0.3g of calcium carbonate, 0.2g of ammonium sulfate, 5g of rapeseed oil and pH 7.0 are added into 100mL of water.
- The method for producing spinosyn according to claim 6, characterized in that: the fermentation conditions are as follows: 250rpm, 28 ℃ and 60% humidity.
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| PCT/CN2022/085682 WO2022262384A1 (en) | 2021-06-17 | 2022-04-08 | Method for increasing yield of spinosyns of saccharopolyspora spinosa |
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| CN115927033A (en) * | 2022-06-07 | 2023-04-07 | 合肥学院 | Saccharopolyspora spinosa strain and method for high yield of spinosad by same |
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