65 results about "Saccharopolyspora spinosa" patented technology

Spinosyn biosynthetic genes from Saccharopolyspora spinosa, spinosyn producing microorganisms transformed with the biosynthetic genes, methods using the biosynthetic genes to increase production of spinosyn insecticidal macrolides, and methods using the genes or fragments thereof to change the products produced by spinosyn-producing microorganisms are provided.

The invention discloses a process for preparing spinosad, which comprises two parts, namely an actinomyces saccharopolyspora spinosa S078-8 fermentation process and a process for extracting a spinosad crystal product by using fermentation liquor which is obtained by fermenting the actinomyces saccharopolyspora spinosa S078-8 and contains the spinosad. The process has the advantages that firstly, the yield is high, the yield of the spinosad finally achieves more than 2,000 to 4,000 milligrams per liter through HPLC measurement, and in particular, the content of the n-butyl spinosad component with highest insecticidal potency in the fermentation liquor reaches 1,500 to 2,800 milligrams per liter; and secondly, the spinosad crystal product has high purity which reaches 95 to 98 percent.

The invention discloses gene engineering bacteria capable of highly producing pleocidin and a preparation method thereof. In the gene engineering bacteria, an expression box of NDP-glucose synthesized enzyme gene gtt of NDP-4-ketone-6-deoxidation-Dglucose and an expression box of genes of NDP-glucose reductase gene gdh are integrated in genomes of Saccharopolyspora spinosa wild bacterial strains. According to fermentation validation, the fermenting units of the pleocidin of the gene engineering bacteria is increased by more than three times compared with that of the wild bacterial strains.

The invention provides a method for producing pleocidin by adopting saccharopolyspora spinosa through fermentation. According to the method, saccharopolyspora spinosa Z68 is adopted as a fermentation strain, an improved fermentation medium is adopted, fermentation cultivation is carried out under the conditions that the temperature is 26-30 DEG C, the rotation speed is 150-220 rpm, the compression-ventilation ratio is 1 to (0.3-0.7), the pot pressure is 0.03-0.04 MPa, and the dissolved oxygen is controlled to be 40% or above, and pleocidin is extracted from the fermentation liquid. According to the method provided by the invention, staring from the physicochemical properties of the fermentation medium of saccharopolyspora spinosa, the fermentation medium with stable yield of pleocidin and little foaming amount, and a material supplementation method are provided. The soybean meal / soybean cake powder in the fermentation medium are replaced by high-temperature soybean cake powder, and the experiment proves that in the fermentation process, the high-temperature soybean cake powder has small possibility of foaming compared with the soybean meal and low-temperature soybean cake powder, while part of fat not available in the high-temperature soybean cake powder but available in the soybean meal and the low-temperature soybean cake powder can be added from the outside in the middle and later periods of fermentation, and the adding at the time can achieve the effect of eliminating foams and solve the problem that the carbon source is insufficient in the later period.

The invention discloses a process for extracting pleocidin from fermentation liquor of saccharopolyspora spinosa, which comprises the following steps of: (1) preprocessing fermentation liquor; (2) adding a high dielectric constant polar organic solvent into the fermentation liquor for soaking and extracting the pleocidin, performing solid-liquid separation and collecting clear leach liquor; (3) removing the high dielectric constant polar organic solvent by vacuum concentration and volatilization to obtain the concentrated solution of the pleocidin; (4) adding a low dielectric constant or highalcohol extraction solvent for extraction to obtain a loaded organic phase; (5) adding acid water into the loaded organic phase to perform reextraction, and collecting a reextraction phase; and (6) removing the residual extraction solvent in the reextraction phase by volatilization, adjusting the pH value to 8.5 to 11.5 by using solution of sodium hydroxide (NaOH), allowing the pleocidin to precipitate, filtering, washing the pleocidin precipitate for 1 to 3 times by using diluted alkali liquor, and drying the washed product under vacuum to obtain pleocidin powder. The process for extracting pleocidin from fermentation liquor of saccharopolyspora spinosa has a total yield of over 80 percent and has the advantages of high efficiency, high quality, low cost, easy industrialization and the like.

The invention provides, biologically active spinosyns, hybrid spinosyn polyketide synthases capable of functioning in Saccharopolyspora spinosa to produce the spinosyns, and methods of controlling insects using the spinosyns.

The invention relates to a Saccharopolyspora spinosa high-pleocidin-yield engineering strain and application thereof. The Saccharopolyspora spinosa high-pleocidin-yield engineering strain is Saccharopolyspora spinosa S.sp-DELTApepA which is collected by China Center for Type Culture Collection on June 8th, 2015, and the culture collection number is CCTCC NO:M2015362. The pleocidin yield of the Saccharopolyspora spinosa S.sp-DELTApepA is enhanced to 137% of the initial strain.

The invention discloses a method of increasing the yield of spinosad by improving the fermentation condition of saccharopolyspora spinosa based on metabonomics. The method comprises the following steps: (1) determining intracellular metabolite of the saccharopolyspora spinosa; (2) analyzing the capability of the saccharopolyspora spinosa of producing the spinosad; (3) carrying out PLS analysis; and (4) carrying out process analysis. According to the method disclosed by the invention, the metabolite change law in the process of producing the spinosad by virtue of fermentation of the saccharopolyspora spinosa is revealed by utilizing the metabonomics means and combining multivariate statistics, and a metabolic change mechanism related to production of the spinosad is further explained; by analyzing the spinosad production capability and the metabolic level of the saccharopolyspora spinosa in different growth environments, the metabolin related to the production of the spinosad is found, thus providing a direction for optimizing the culture process of the saccharopolyspora spinosa and increasing the fermentation yield of the spinosad. The invention also provides new thinking and method for study in other culture technological processes of producing macrolides microorganisms.

The invention discloses a genetically engineered bacterium capable of increasing the yield of spinosads as well as a construction method and an application. The construction method of the genetically engineered bacterium capable of increasing the yield of spinosads comprises the following steps: (1) inserting an spnk expression cassette into an escherichia coli-saccharopolyspora spinosa shuttle plasmid pOJ260 to construct a recombinant plasmid pLU101; (2) guiding the recombinant plasmid pLU101 into saccharopolyspora spinosa ATCC49460, and integrating the recombinant plasmid with a genome through homologous recombination, thus obtaining the genetically engineered bacterium capable of increasing the yield of spinosads and naming the genetically engineered bacterium LU101. The genetically engineered bacterium constructed by the method can increase the yield of spinosads.

The invention relates to saccharopolyspora spinosa strain, application of the saccharopolyspora spinosa strain and a method for preparing spinosad. The strain related to the invention has the preservation number of CGMCC No.3460. The invention also relates to a mutant strain obtained by the strain. The invention also relates to the method for preparing the spinosad, which comprises the following steps of: a, fermenting the saccharopolyspora spinosa strain or the mutant strain thereof disclosed by the invention to obtain fermentation liquor containing the spinosad; and b, separating the spinosad from the fermentation liquor. The strain disclosed by the invention has strong capability of producing the spinosad and has high industrial application value.

The invention provides a two-stage dissolved oxygen control method for increasing pleocidin output. The method is characterized in that the volumetric mass transfer coefficient kLa of oxygen is adjusted and controlled by adjusting throughput and stirring speed at the beginning of fermentation of saccharopolyspora spinosa in the process of preparing saccharopolyspora spinosa fermentation liquor, thus the initial oxygen volumetric mass transfer coefficient kLa can be controlled to be 100-130h<-1> and the accumulation of biomass of saccharopolyspora spinosa is promoted; then the volumetric mass transfer coefficient kLa of oxygen is adjusted and controlled by adjusting throughput and stirring speed when the fermentation enters the final cell growth exponential phase, and thus the initial oxygen volumetric mass transfer coefficient kLa can be controlled to be 60-92h<-1> and the synthesis and accumulation of pleocidin are promoted. According to the method, the two-stage dissolved oxygen control is performed, which obviously increases the pleocidin output and shortened the fermentation cycle. The two-stage dissolved oxygen control and fermentation method is easily realized and has a high application value.

The invention provides a fermentation method for improving yield of spinosad. Macroporous resin is added into a fermentation medium of Saccharopolyspora spinosa, whole milk powder is added during a fermentation process, so that the yield of the spinosad is improved. The macroporous resin added in the fermentation medium can effectively and rapidly reduce concentration of the spinosad, and decrease repression of metabolite; the whole milk powder added as supplementary food in the fermentation process is good for continue growth and product synthesis of bacteria, and can further improve yield of the spinosad.

The invention discloses saccharopolyspora spinosa for producing spinosad and belongs to the field of microorganisms. The strain number of the saccharopolyspora spinosa BCSsBFSg<5>-006 is CGMCC (China general microbiological culture collection center) No.5546, and a fermentation medium for the saccharopolyspora spinosa of the spinosad comprises glucose 40g / L, soluble starch 30g / L, corn steep liquor 15g / L, cottonseed concentration powder 20g / L, yeast powder 20g / L and calcium carbonate 1g / L, and is prepared by distilled water with the pH (potential of hydrogen) of 7.0-7.2 regulated by NaOH 1mol / L. Compared with conventional spinosad production strains, the mutant strain BCSsBFSg<5>-006 is higher in spinosad production capacity, and accordingly yield of the spinosad can be effectively increased, and production cost is reduced.

The invention relates to a culture medium and a culture method for producing spinosad through fermentation of saccharopolyspora spinosa. A carbon source, a nitrogen source and optimal compatibility ratio in a primary seed medium, a secondary seed medium and a fermentation medium for production of spinosad through fermentation of the saccharopolyspora spinosa in a three-stage fermentation mode areconfirmed. By utilizing the culture medium formula and the fermentation control process provided by the invention, the fermentation unit of rice spinosad can be improved, the fermentation cost is reduced, the sources of raw materials and auxiliary materials are furthest reduced and are not influenced by the environment, sufficient supply of the raw materials and the auxiliary materials is ensured,and stable and efficient production of the spinosad is realized.

The invention discloses a spinosad high-yield gene engineering bacterium. The preservation number of the gene engineering bacterium is CGMCC No. 14193. The invention further discloses a method by enhancing a fatty acid metabolism pathway to construct the spinosad high-yield gene engineering bacterium and application of the engineering bacterium to produce spinosad. According to the construction method, by conducting expression enhancement on the oxidation pathway of fatty acid Beta of saccharopolyspora spinosa, supply for the spinosad to biosynthesize the important precursor of acetyl coenzyme A is improved, and compared with original strains, the yield of the spinosad of obtained gene engineering strains is improved obviously.

The invention provides a culture medium for fermenting pleocidin producing bacteria by using Saccharopolyspora spinosa NRRL18395 as strains, which comprises the following components: 5.0-9.0 percent of carbon source, 3.0-7.0 percent of nitrogen source, 0.2-2.5 percent of seed oil, 0.1-0.5 percent of amino acid, 0.1-1.0 percent of CaCO3 and 81.0-91.6 percent of water. The fermentation level of the pleocidin producing bacteria can be improved by 19 times to the maximum based on the original fermentation level, and thereby the culture medium for fermenting pleocidin producing bacteria is beneficial to the industrialization of pleocidin in China.

The invention discloses a saccharopolyspora spinosa genome scale metabolic network model and a construction method and application of the saccharopolyspora spinosa genome scale metabolic network model. The construction method includes the following steps that according to annotation information of saccharopolyspora spinosa genome sequences in KEGG and NCBI databases, a characteristic reaction for spinosad biosynthesis and a thallus sythesis reaction are added, a network reaction is manually refined, and then the saccharopolyspora spinosa genome scale metabolic network model is acquired. Through the saccharopolyspora spinosa genome scale metabolic network model, influences on improvement of the output of spinosad on a gene target spot can be predicted, the modification direction is finally determined, and the bacterial strain path molecule modification method is achieved. Experiments show that through genes predicted by the saccharopolyspora spinosa genome scale metabolic network model, genetically engineered bacteria obtained through modification can make the output of spinosad improved by 86.5% compared with wild bacterial strains. An instruction platform is provided for construction, research and analysis of the bacterial strains for efficiently producing and synthesizing spinosad.

The invention relates to a Saccharopolyspora spinosa rhamnose biosynthesis gene duplication engineering strain (S. sp-RM, Saccharopolyspora spinosa) which is collected by China Center for Type Culture Collection on June 8th, 2015, and the culture collection number is CCTCC NO:M2015363. The PCR (polymerase chain reaction) identification result indicates that the plasmid is successfully integrated to the Saccharopolyspora pogona genome. The real-time fluorescent quantitative PCR result indicates that the transcription levels of genes gdh, kre, epi and gtt in the engineering strain are respectively enhanced by 80.3 times, 30.8 times, 23.8 times and 18.3 times as compared with the initial strain. The HPLC (high performance liquid chromatography) detection result indicates that the pleocidin yield of the engineering bacterium S. sp-RM is enhanced by 73% as compared with the initial strain.

The invention discloses a saccharopolyspora spinosa recombinant strain with double bldD genes. The provided saccharopolyspora spinosa recombinant strain with double bldD genes is prepared by integrating bldD gene into saccharopolyspora spinosa genome. The provided saccharopolyspora spinosa recombinant strain with double bldD genes is named as S.spinosa-BldD, and is preserved in the China Center for Type Culture Collection (CCTCC) in May 31st, 2015, and the preservation number is CCTCC No. M2015347. The PCR detection results show that bldD gene has been successfully integrated into a target bacterial strain chromosome. In the plate culture, people find that in BHI and TSB plates, the spores of recombinant strains are prominently inhibited. The results of shaking flask fermentation show that the spinosad yield of recombinant strain is increased by 1.35 times compared with that of a referential strain.

The invention provides a novel integrase and application thereof to genetically modifying saccharopolyspora spinosa. The integrase particularly comes from saccharopolyspora endophytica CCTCC AA208003, and exogenous DNA (deoxyribonucleic acid) can be specifically locally integrated on saccharopolyspora endophytica chromosome by the integrase via specific cis-form components. In addition, independent free replication regions of different strain sources are discovered in the saccharopolyspora spinosa by an inventor via research on genetic factors of different strains of the saccharopolyspora spinosa. The novel integrase and the application have the advantages that vectors constructed on the basis of the integrase and sequences of the replication regions can be used for genetically modifying the saccharopolyspora spinosa, accordingly, novel spinosad analogues can be possibly discovered, and the spinosad fermentation yields can be possibly increased; different resistance molecular markers can be carried by plasmids, accordingly, the novel integrase can be used for inter-species or intergenetic genome shuffling operation on the saccharopolyspora spinosa, and efficient genome shuffling can be implemented.

The invention relates to a fermenting method for increasing pleocidin output. The method comprises the following steps: putting saccharopolyspora spinosa in a fermenting culture medium to ferment; after fermentation in 72-120hr, supplementing a compound organic nitrogen source for the first time, wherein the adding amount is the amount, by which the final concentration of the compound organic nitrogen source in a fermenting liquid is 5-20g / L; and after fermentation in 120-160hr, supplementing the compound organic nitrogen source for the first time, wherein the adding amount is the amount, by which the final concentration of the compound organic nitrogen source in the fermenting liquid is 5-20g / L. Continuous production of thalli and product synthesis are facilitated, so that the pleocidin output can be further increased.

The invention relates to a method for improving a saccharopolyspora spinosa spinosad fermentation yield. The method utilizes mutagenesis treatment to screen a saccharopolyspora spinosa spinosad high-yield mutant strain with a good starch utilization capability. The mutagenesis treatment comprises at least two of UV-streptomycin combined mutagenesis, 5-fluorouracil mutagenesis, microwave-streptomycin combined mutagenesis and nitrosoguanidine-streptomycin combined mutagenesis. Preferably, the mutagenesis treatment comprises the above four mutagenesis methods. Preferably, the UV-streptomycin combined mutagenesis, 5-fluorouracil mutagenesis, microwave-streptomycin combined mutagenesis and nitrosoguanidine-streptomycin combined mutagenesis processes are orderly carried out. The broth of the obtained mutant strain has spinosad titer substantially higher than that of an original strain screened through mutagenesis and thus the method can improve a saccharopolyspora spinosa spinosad fermentation yield.

The invention relates to a method for extracting spinosad from a Saccharopolyspora spinosa fermentation solution. The method comprises the following steps: pretreatment of the fermentation solution, active carbon column treatment for removing a lot of pigments and gummy impurities, extraction of spinosad, back-extraction, fine filtration, crystallization, separation and drying. A compound enzyme is adopted during the pretreatment to treat mycelia of the Saccharopolyspora spinosa, so the spinosad generated in the mycelia is released into the fermentation solution; and an active carbon column is used to remove a lot of the pigments, colloids and other impurities from the fermentation solution, so the improvement of the purity of the spinosad product is greatly benefited. The method has the advantages of simplicity in operation, short period, safety in process, low cost, high yield and high purity.

The invention relates to a method for extracting pleocidin from saccharopolyspora spinosa fermentation liquor. The method comprises the following steps: (1), pre-treating saccharopolyspora spinosa fermentation liquor and carrying out solid-liquid separation; (2), adsorbing centrifuged organic substances in a water phase by an adsorbent; (3), combining solids which are centrifuged and separated for the first time and the second time, and drying in vacuum; (4), extracting pleocidin from solids; and (5), recrystallizing by using a solvent to obtain a pleocidin product, decompressing and desolventizing. According to the method disclosed by the invention, a polar solvent is not needed to be added for extracting during pre-treatment, so that usage amount of an organic solvent is greatly reduced, pollution is reduced, cost is lowered, a centrifugal method can be adopted, operation is safe and separating effect is good; moreover, the adsorbent is added into the water phase for adsorbing organic substances in water, so that COD (chemical oxygen demand) in wastewater is greatly lowered, wastewater treating difficulty is reduced, the yield of the obtained product pleocidin is higher, and a total yield is 70%-95%; after the product is purified, the pleocidin content with mass content of 75%-93.2% is higher.

The invention discloses a vegetable planting method. The vegetable planting method comprises the steps of 1 preparing before planting, wherein green environment-friendly base fertilizer is applied to soil, wherein a making method of the green environment-friendly base fertilizer comprises the steps that swill is filtered, pulverized and sterilized to obtain nutrient powder and a nutrient solution, straw, cottonseed meal, bran, dry braches and fallen leaves are mixed and pulverized to obtain plant powder, the plant powder, the nutrient solution and saccharopolyspora spinosa are mixed and fermented to obtain fermented fertilizer, and the fermented fertilizer, the nutrient powder and nitrogen-fixing bacteria are mixed and then fermented to obtain the green environment-friendly base fertilizer; 2 seedling raising, wherein seeds are soaked with a copper sulfate solution and processed with ultrasonic waves under infrared ray irradiation and then mixed with a urea solution and the nutrient solution; 3 planting and managing, wherein the nutrient powder is applied, and a cleaning agent is sprayed before picking. The vegetable planting method has the advantages that the vegetable yield is increased, the vegetable quality is improved, waste is comprehensively utilized, the germination condition is created, residual fertilizer and pesticides on the outer surfaces of the vegetables are removed, and therefore the vegetables are cleaner and safer.

The invention discloses an insecticide for sugarcane planting and a preparation method of the insecticide. The insecticide for sugarcane planting is formed by fermenting compound bacteria and a Chinese herbal culture medium, wherein the compound bacteria are selected from one or two of saccharopolyspora spinosa and aspergillus niger; the Chinese herbal culture medium is prepared from, by weight, 20-40 parts of fructus forsythiae, 5-15 parts of garlic, 3-12 parts of celastrus angulatus rhizomes, 1-8 parts of fructus crotonis, 10-30 parts of stems and leaves of chrysanthemum coronarium, 30-50 parts of nutrients and 30-50 parts of deionized water. The insecticide for sugarcane planting and the preparation method of the insecticide have the advantages that effective components in Chinese herbal medicine are extracted through melanomyces and the saccharopolyspora spinosa, and secondary metabolic components generated in the fermentation process of the melanomyces and saccharopolyspora spinosa synergize with the active components contained in the Chinese herbal medicine so that the insecticide can reduce diseases and insect pests of crops to the maximum extent.

This disclosure concerns methods for producing a spinosyn producing strain that comprises modifying a nucleic acid molecule encoding for spinactin by introducing, mutating, deleting, replacing or inactivating a nucleic acid sequence encoding one or more activities encoded by said nucleic acid molecule. Methods for producing a modified Saccharopolyspora spinosa organism are also disclosed.