WO2019141272A1 - Erythromycin producing strain having improved fermentation malodor and use thereof - Google Patents

Erythromycin producing strain having improved fermentation malodor and use thereof Download PDF

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WO2019141272A1
WO2019141272A1 PCT/CN2019/072500 CN2019072500W WO2019141272A1 WO 2019141272 A1 WO2019141272 A1 WO 2019141272A1 CN 2019072500 W CN2019072500 W CN 2019072500W WO 2019141272 A1 WO2019141272 A1 WO 2019141272A1
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klcndx
erythromycin
strain
fermentation
culture
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PCT/CN2019/072500
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French (fr)
Chinese (zh)
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邓旭衡
周路
张婷
胡晓非
张云辉
刘思川
吴杰群
刘�文
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伊犁川宁生物技术有限公司
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Publication of WO2019141272A1 publication Critical patent/WO2019141272A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
    • B01D53/84—Biological processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2257/00—Components to be removed
    • B01D2257/90—Odorous compounds not provided for in groups B01D2257/00 - B01D2257/708
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

Definitions

  • the invention belongs to the field of microbial fermentation engineering, and relates to a novel erythromycin producing strain capable of effectively reducing malodor odor in fermentation and application thereof.
  • Erythromycin is a secondary metabolite produced by the genus Saccharopolyspora erythraea and is a broad-spectrum fourteen-membered macrolide antibiotic against Gram-positive bacteria. According to the difference of side chain groups, erythromycin is divided into six kinds, namely erythromycin A, erythromycin B, erythromycin C, erythromycin D, erythromycin E and erythromycin F. Among them, erythromycin A is the most commonly used pharmaceutical ingredient in the clinic, and the components B, C and D are intermediate products in the synthesis of erythromycin A. Erythromycin B has lower biological activity than erythromycin A, but its toxicity is twice that of erythromycin A.
  • Erythromycins E and F are two natural products with little content and low activity, and few studies [Mironov V et al, Appl. Biochem. Microbiol. 2004; And Zou X et al, J Ind Microbiol Biotechnol, 2008] .
  • Elililly Company of the United States isolated erythromycin A from the fermentation broth of Saccharopolyspora erythraea and developed the first generation of macrolide antibiotic erythromycin. After the market, erythromycin became the gram-positive treatment at that time. An important drug for bacterial infection. So far, erythromycin is still a commonly used antibiotic in clinical practice, and it has a very effective therapeutic effect on some common bacterial infection diseases.
  • Second and third generation erythromycin derivatives synthesized with erythromycin A as a precursor have also been widely used in clinical practice.
  • erythromycin The widespread use of erythromycin in the clinic has greatly increased the supply of erythromycin raw materials, thus promoting the development of the entire erythromycin industry.
  • China is the world's largest producer and exporter of erythromycin, with an annual output of more than 9,000 tons.
  • the erythromycin bulk drug is mainly produced by fermentation of the strain. Due to the development of genomics and proteomics in recent years, combined with the microbial breeding screening of domestic researchers for many years, the domestic erythromycin fermentation production capacity has been improved, but there is still room for improvement compared with the higher production levels abroad. .
  • Fan Xiaoyu et al (Fan Xiaoyu, Medical Engineering Design, 34(4), 2013) used ozone to produce tail gas with erythromycin thiocyanate for mixed oxidation reaction, and then entered the UV irradiation chamber.
  • the ozone was decomposed by ultraviolet irradiation to generate hydroxyl radicals.
  • the volatile organic compounds VOCs in the oxidation tower of the wet oxidation tower are degraded into CO 2 , H 2 O and trace organic acids, and then enter the spray washing tower, washed and purified, absorbed by the trace organic acid and then discharged.
  • the post-fermentation tail gas is free from pollution.
  • the above method has obvious effect of eliminating the bitter odor of the erythromycin fermentation tail gas.
  • Zhang Zhihong et al (Zhang Zhihong et al., Journal of Henan Normal University, 37(5), 2009) prepared a composite microbial agent with Geotrichum candidum, Bacillus licheniformis, Candida, Lactobacillus and Photosynthetic bacteria to carry out solid-state fermentation of erythromycin slag. The special odor in the erythromycin slag is removed.
  • An object of the present invention is to provide an erythromycin producing strain capable of effectively reducing malodorous odor in fermentation and use thereof.
  • One aspect of the present invention provides a Saccharopolyspora erythraea having the name KLCNDX-01, the accession number is CCTCC NO: M 2017647; or the name is KLCNDX-02, and the accession number is CCTCC NO: M 2017648.
  • Another aspect of the present invention provides a Saccharopolyspora erythraea cultured from the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647; or a red sugar obtained by culturing the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 Spores.
  • Another aspect of the present invention provides a S. erythraea having the identification characteristics of the strain KLCNDX-01 with the accession number CCTCC NO: M2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  • Another aspect of the present invention provides a S. erythraea culture comprising a strain KLCNDX-01 having a deposit number of CCTCC NO: M2017647 or a strain KLCNDX-02 having a deposit number of CCTCC NO: M 2017648, or containing KLCNDX therein -01 or KLCNDX-02 identifies strains of the characteristics.
  • the S. erythraea culture of the present invention is a biologically pure culture obtained by culturing strain KLCNDX-01 or KLCNDX-02; or the biologically pure culture having KLCNDX- Identification characteristics of 01 or KLCNDX-02 cultures.
  • Saccharopolyspora erythraea or a culture thereof the Saccharomyces cerevisiae strain S strain KLCNDX-01 deposited under the accession number CCTCC NO: M 2017647 or the strain deposited under the accession number CCTCC NO: M 2017648 KLCNDX-02 is derived.
  • the S. erythraea of the present invention has an equal or higher erythromycin production capacity prior to derivatization, and/or does not produce or produce less malodor odor during culture or fermentation.
  • Another aspect of the present invention provides a Saccharopolyspora erythraea having the same or substantially the same genomic sequence as KLCNDX-01 or KLCNDX-02, wherein said substantially identical genomic sequence refers to proliferation only in the genome of the strain in the presence of natural conditions Spontaneous mutations that occur during passage.
  • the S. erythraea has substantially the same erythromycin production capacity as KLCNDX-01 or KLCNDX-02 and/or does not produce or produce malodorous odor in culture or fermentation.
  • the S. erythraea genomic sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and KLCNDX-01 or KLCNDX-02, 98%, 99% or higher homology.
  • Another aspect of the present invention provides a fermentation method which is carried out by the strain KLCNDX-01 having the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 having the accession number CCTCC NO: M 2017648.
  • the fermentation method of the present invention comprises incubating the spores of the cryopreserved strains, and then culturing the spore suspension obtained by the slant culture through a multi-stage seed tank, and finally fermenting into the fermenter.
  • the bevel culture comprises a mother bevel culture and a sub-slope culture;
  • the multi-stage seed tank culture is a first stage seed tank culture, or a two-stage, three-stage or more seed tank continuous culture.
  • the seed tank is cultured in a tertiary culture.
  • the culture temperature of the strain in the fermentation process of the invention is 28-37 ° C, preferably 30-35 ° C.
  • the fermentation method of the present invention comprises the following steps: (1) erythromycin seed culture: 1 mother slant spore preparation: erythromycin Streptomyces spores frozen in a sand tube, inoculated The culture medium is cultured on a slant medium at a temperature of 32-34 ° C for 7-9 days; 2 slant spore preparation: the mother slant spores are inoculated on a sterilized slant medium at a temperature of 32-34 ° C.
  • 3 seed culture seed slant spores, inoculated in sterilized liquid seed culture medium, through three-stage seed expansion culture to obtain erythromycin seed liquid
  • (2) erythromycin fermentation The erythromycin seed solution is inoculated into the sterilized medium at a volume ratio of 10-20%, and the sterile air is introduced and stirred, the nutrient is added, and the culture is carried out for 7-10 days at a temperature of 32-34 ° C. Erythromycin fermentation broth.
  • the bevel culture medium of the present invention comprises the following components: 0.1-1.0 parts by weight of starch, 0.05-0.2 parts by weight of corn syrup, 0.1-1.0 parts by weight of agar, 0.1-1.0 parts by weight of glucose, sodium chloride. 0.5-2.0 parts by weight, 0.1-1.0 parts by weight of calcium chloride, and made up to 100 parts by volume with tap water.
  • the primary seed expanded culture condition of the present invention is: (1) medium composition: soybean powder 2-4 parts by weight, starch 1-2 parts by weight, CaCO 3 0.6-0.8 parts by weight, dextrin 1 - 2 parts by weight, ammonium sulfate 0.2-0.4 parts by weight, corn syrup 1-4 parts by weight, vegetable oil 0.5-1 parts by weight, made up to 100 parts by volume with tap water; (2) temperature 32-34 ° C; (3) The culture time is 48-96 hours.
  • the second and third stage seed expansion culture conditions of the present invention are: (1) medium composition: soybean powder 2.0-4.5 parts by weight, starch 2.5-4.0 parts by weight, CaCO 3 0.5-0.7 weight Parts, sodium chloride 0.4-0.6 parts by weight, dextrin 2.5-4.0 parts by weight, ammonium sulfate 0.1-0.3 parts by weight, corn syrup 1-2 parts by weight, vegetable oil 0.8-1.5 parts by weight, defoaming agent 0-0.02 parts by weight , dilute to 100 parts by volume with tap water; (2) temperature is 32-34 ° C; (3) culture time 24-48 hours.
  • the fermentation medium of the present invention comprises: 3.5-5.5 parts by weight of soy flour, 2.0-4.0 parts by weight of starch, 0.08-0.15 parts by weight of ammonium sulfate, 0.6-1.0 parts by weight of CaCO 3 , 0.5-1.0 vegetable oil. Parts by weight, defoaming agent 0.005-0.02 parts by weight, made up to 100 parts by volume with tap water.
  • the nutritional supplement composition of the present invention comprises: 10-50 parts by weight of 20-70% glucose solution, 1-10 parts by weight of vegetable oil, and 0.5-5 parts by weight of n-propanol solution.
  • the vegetable oil of the present invention comprises peanut oil, rapeseed oil, soybean oil, cottonseed oil, sesame oil, sunflower oil, olive oil, zedoary oil, garlic oil, sea buckthorn oil, angelica oil, ginger oil, Any of forsythia oil, perilla oil, tangerine oil, castor oil, peppermint oil, and nutmeg oil; preferably any of soybean oil and rapeseed oil.
  • the n-propanol solution of the present invention is a 20-70% n-propanol solution;
  • the antifoaming agent of the present invention is a polyether antifoaming agent; preferably, the present invention
  • the polyether antifoaming agent is any one of polyoxypropylene glycerin and polyoxyethylene oxypropylene glycerin.
  • Another aspect of the present invention provides a method for producing erythromycin or erythromycin thiocyanate comprising a strain KLCNDX-01 having a deposit number of CCTCC NO: M 2017647 or a strain KLCNDX having a deposit number of CCTCC NO: M 2017648- 02 Fermentation was carried out to obtain an erythromycin fermentation broth.
  • the method of producing erythromycin of the present invention comprises: 1) cultivating the microorganism of the present invention to obtain a cell culture; and 2) collecting erythromycin fermentation broth from a cell culture or microorganism, by passing on a red mold The fermentation broth is processed to obtain a finished erythromycin or erythromycin thiocyanate.
  • the method for producing erythromycin of the present invention comprises: filtering the fermentation broth of the erythromycin producing strain through a ceramic membrane, the slag is subjected to environmental protection, and the filtrate is further subjected to nanofiltration, crystallization, and drying to obtain erythromycin. product.
  • the method for producing erythromycin thiocyanate according to the present invention comprises: filtering the erythromycin fermentation broth through a ceramic membrane, the slag is subjected to environmental protection, and the filtrate is concentrated through a nanofiltration membrane to obtain an erythromycin concentrate. Adding an appropriate amount of sodium thiocyanate solution to the concentrated solution to obtain a crude erythromycin thiocyanate, and obtaining a finished erythromycin thiocyanate by secondary crystallization and drying.
  • the method of producing erythromycin thiocyanate of the present invention comprises the steps of:
  • Concentration of fermentation broth The erythromycin fermentation liquid is adjusted to a pH of 7.5-9.0, filtered through a ceramic membrane having a pore size of 50 nm to 100 nm, and the filtrate is concentrated with a 200 molecular weight nanofiltration membrane to obtain an erythromycin concentrate;
  • Another aspect of the present invention provides the use of the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 for the production of erythromycin.
  • Another aspect of the present invention provides a method for reducing malodor odor in erythromycin fermentation production, the method comprising the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX with the accession number CCTCC NO: M2017648 02 Fermentation to produce erythromycin.
  • the method of the present invention for reducing malodors in erythromycin fermentation production further comprises treating exhaust gas, wastewater, and/or waste generated by fermentation to reduce malodors therein.
  • the method of the present invention for treating tail gas, wastewater, and/or waste generated by fermentation includes treating the tail gas, waste water, and/or waste residue by physical and/or chemical means to reduce the malodor odor therein.
  • the physical methods include adsorption methods (eg, adsorption using hydrophobic activated carbon, silica gel, activated alumina, zeolite molecular sieves, etc.), plasma methods, and the like.
  • adsorption methods eg, adsorption using hydrophobic activated carbon, silica gel, activated alumina, zeolite molecular sieves, etc.
  • plasma methods and the like.
  • the chemical method includes an absorption method (e.g., alkali water absorption), a combustion method (e.g., direct combustion method, catalytic combustion method, thermal combustion method), photocatalytic oxidation, and the like.
  • absorption method e.g., alkali water absorption
  • combustion method e.g., direct combustion method, catalytic combustion method, thermal combustion method
  • photocatalytic oxidation e.g., photocatalytic oxidation, and the like.
  • the red sugar multi-saturated bacteria will form a characteristic tail gas during the fermentation process, but since the fermentation gas is mostly a mixture of various odor gases, it is not easy to identify, so a standard fermentation tail gas of red sugar multi-saturated bacteria is needed as a reference to identify Whether there is a characteristic tail gas produced by fermentation of red sugar polysaturated bacteria in the fermentation gas.
  • Another aspect of the present invention provides an application of the fermentation tail gas of the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 as standard fermentation tail gas, applied to red Identification of characteristic tail gas from Saccharopolysaccharide fermentation.
  • the invention provides the use of the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 in the characteristic tail gas recognition of S. erythraea fermentation.
  • Another aspect of the present invention provides a method for identifying characteristic tail gas of Saccharopolyspora erythraea, the strain KLCNDX-01 with the accession number CCTCC NO:M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO:M 2017648
  • the fermentation tail gas is used as a standard fermentation tail gas to identify the characteristic tail gas of the fermentation of S. cerevisiae.
  • Another aspect of the present invention provides a standard fermentation tail gas of Saccharopolyspora erythraea which is obtained by fermentation collection of strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or strain KLCNDX-02 with the accession number CCTCC NO: M 2017648. .
  • the identification of tail gas by red sugar multi-fermentation fermentation can be carried out according to GB/T 14675-93 "Air quality - malodor measurement - three-point comparative odor bag method" method.
  • the standard fermentation tail gas of the genus Saccharomyces cerevisiae of the invention can be used for the technical training of the olfactory discriminator or the olfactory identification of the olfactory discriminator, so that the scent finder can understand the odor characteristic of the characteristic tail gas of the fermentation of S. cerevisiae and improve the corresponding exhaust gas.
  • the ability to sniff can be used for the technical training of the olfactory discriminator or the olfactory identification of the olfactory discriminator, so that the scent finder can understand the odor characteristic of the characteristic tail gas of the fermentation of S. cerevisiae and improve the corresponding exhaust gas.
  • the ability to sniff can be used for the technical training of the olfactory discriminator or the olfactory identification of the
  • the method for identifying the standard fermentation tail gas and characteristic tail gas of the Saccharopolyspora erythraea of the present invention can also be used for identifying non-professional olfactory personnel to identify the characteristic tail gas of S. erythraea fermentation or to distinguish the fermentation tail gas of S. cerevisiae.
  • the S. erythraea KLCNDX-01 and KLCNDX-02 of the present invention are obtained by genetic modification and domestication breeding of Saccharopolyspora erythraea used in the industrial production of erythromycin, and the colonies of the strain are chrysanthemum-type, and the bacteriophre pigment is The mouse is gray, and the mycelium pigment is reddish brown.
  • the strain effectively reduced the generation of malodorous odors in the fermentation production of the red mold relative to the original strain before transformation and acclimation.
  • the domesticated breeding after transformation further improved the production capacity of erythromycin.
  • KLCNDX-01 was obtained after genetic modification of the strain after two generations of domestication, and KLCNDX-02 was obtained for further passage and domestication to the fourth generation.
  • KLCNDX-01 increased the titer of shake flask by 64% compared with the strain before acclimation, and the potency of KLCNDX-02 was 67.8% higher than that of the strain before acclimation.
  • the production of erythromycin by the two strains was higher than that of the strain before the genetic modification. 14-16.7%.
  • the tail gas discharge of the seed tank and the fermenter can reach the straight discharge standard, and the treatment cost of the fermentation tail gas is reduced.
  • the pressure of the fermentation tail gas pipeline system can be reduced and the risk of contamination can be reduced.
  • the strain of the present invention solves the problem of malodorous odor in fermentation production and improves the production capacity of erythromycin, and has very important industrial production value and environmental protection value.
  • Saccharopolyspora erythraea KLCNDX-01 of the present invention has been deposited with the China Center for Type Culture Collection (CCTCC, China, Wuhan, Wuhan University, China) on November 1, 2017, under the accession number CCTCC NO: M 2017647.
  • the Saccharopolyspora erythraea KLCNDX-02 of the present invention has been deposited with the China Center for Type Culture Collection (CCTCC, China, Wuhan, Wuhan University, China) on November 1, 2017, under the accession number C CCTCC NO :M 2017648.
  • the term "cultivating” means allowing the microorganism to grow under artificially controlled conditions.
  • a variety of methods well known in the art can be used to culture the strain of the present invention, and can be cultured by a batch method or a continuous method such as a fed-batch method or a repeated fed-batch method, but the present invention is not limited thereto.
  • the medium used for the culture must meet the requirements of the species used.
  • Culture media suitable for the cultivation of actinomycetes such as Gaussian No. 1 medium and flour agar medium are well known in the art.
  • the carbon source of the medium may be: soluble starch, dextrin, liquefied sugar, glucose, and the like.
  • the nitrogen source for cultivating the strain of the present invention may be: soybean cake powder, peptone, corn syrup, ammonium sulfate, potassium nitrate or the like.
  • Other components of the medium may include sodium chloride, magnesium sulfate, dipotassium hydrogen phosphate, and the like. These materials may be used singly or in combination.
  • a strain having an identification characteristic of [prescribed strain] or a "culture having an identification characteristic of [specified strain]", including a homologue or a mutant of a prescribed strain, is closely related to a prescribed strain (ie, It has a common ancestor or is derived from a prescribed strain, but usually differs from the prescribed strain in one or more genotypes or phenotypic characteristics. Mutants can usually be identified by assessment of genetic differences.
  • a strain having an identifying characteristic includes a homologous strain or a mutant strain having all the identifying characteristics of a prescribed strain (for example, a DNA analysis-based DNA fingerprint corresponding to a DNA fingerprint of a prescribed strain).
  • the term "culture” refers to a product obtained by growing and/or amplifying a microorganism, which may be a biologically pure culture of microorganisms, or may contain a certain amount of medium, metabolite or culture process. Other ingredients produced as long as they do not substantially affect the activity of the product produced by the culture.
  • the term "culture” also includes subcultures obtained by passage of microorganisms, which may be a generation of cultures, or a mixture of several generations. The ability of the (passage) culture to produce erythromycin is substantially the same as KLCNDX-01 or KLCNDX-02 of the present invention.
  • biologically pure culture refers to a culture (eg, cultured) that is substantially free of biological contamination and has genetic consistency such that different subcultures derived therefrom will exhibit substantially the same genotype and phenotype (eg, culture) At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99%, up to 100% pure purity).
  • phenotype eg, culture
  • a strain obtained by "derived” refers to a strain which is transformed by any means, for example, by one or more crosses and/or by mutation and/or by transgene.
  • a strain derived by hybridization can be obtained by hybridizing a strain according to the present invention with the same strain or another strain according to the present invention, or any other strain.
  • the strain derived by the variation may be a strain that undergoes at least one spontaneous mutation in its genome or undergoes at least one variation induced by, for example, mutagenesis.
  • the variation of the derived strain is or is not silent.
  • mutagenic means a standard mutagen obtained by irradiation (for example, ultraviolet light) or a mutagenic chemical, and insertional mutagenesis by integration of a transposition or a foreign DNA fragment. Mutagenicity by radiation involves the use of ultraviolet light, X-rays or gamma rays.
  • Mutagenic chemicals such as EMS (ethyl-methylsulfonate), EES (ethyl-ethylsulfonate), nitrosoguanidine, nitrous acid, aflatoxin B1, hydroxylamine, 5-bromo-urine Pyrimidine, 2-amino-indole, pre-flavin, acridine orange.
  • the strain derived by the transgene is a strain into which foreign DNA is introduced.
  • the foreign DNA is preferably introduced by a plasmid or directly integrated into the genome.
  • the term "homology” refers to a certain percentage of "sequence identity" of a polynucleotide or polypeptide to another sequence. When comparing two sequences, these percentage bases or amino acids are identical when conjugated.
  • Such mating and homology percentages or sequence identity can be determined using software programs known in the art, as described, for example, in CURRENT PROTOCOLS INMOLECULAR BIOLOGY (FMAusubel et al., eds., 1987) Supplement 30, Section 7.7.18. Those programs.
  • Another sequence of software programs that can be applied is the TFastA Data Searching Program, available from the Sequence Analysis Software Package version 6.0 (Genetic Computer Group, University of Wisconsin, Madison, WI).
  • the equivalent erythromycin production capacity in the term "equivalent or higher erythromycin production capacity” means that when cultured under the same conditions, the changed payout is compared with the actinomycete host cell before derivatization.
  • the amount of erythromycin produced in the bacterial cells or the shake flask titer is the same as before the change, or the amount of change is not more than ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1% or less;
  • the erythromycin production capacity means that the above change is an increase of at least 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60% or more.
  • the term "no odor is produced or produced in a culture or fermentation” does not produce malodorous odors, meaning that no malodorous gas is generated in culture or fermentation, or the amount of malodorous gas generated is lower than the olfactory threshold.
  • the olfactory threshold includes a sensory threshold at which an olfactory odor can exist or an identification threshold capable of determining odor characteristics (see National Standard GB/T 14675-93).
  • the production of less malodorous odor means that the concentration of malodorous gas produced in the culture or fermentation is lower.
  • the malodorous gas concentration refers to the mass of the malodorous gas per unit volume.
  • the malodorous gas concentration can also be measured by the method in "Air Quality - Measurement of malodor - three-point comparative odor bag method" described in the national standard GB/T 14675-93.
  • the malodorous gas concentration refers to an index indicating the magnitude of the odor odor according to the olfactory organ test method, and the dilution factor when the odorous sample is continuously diluted to the sniffer threshold by the odorless clean air.
  • the comparison of the erythromycin production capacity and the level or ability of malodorous odor generation may be carried out by culturing two or more microbial host cells to be compared under the same conditions, and using the same conditions, preferably using the same measurement method. The amount of erythromycin produced or the magnitude of the malodor odor was measured.
  • malodor means that the stimulating olfactory organ produced during the erythromycin fermentation production process causes an unpleasant gas odor, and may be a collection of various gas odors or a single gas odor.
  • the odor can be analyzed and judged by people's perceptual thinking, or can be compared and identified according to the standard fermentation tail gas of Saccharopolyspora erythraea, or a three-point comparison formula prescribed by the professional olfactory discriminator according to the national standard GB/T14675-93.
  • the odor bag method is used for identification.
  • the fermentation method of the fermentation strain KLCNDX-01 or KLCNDX-02 of the present invention can be carried out by referring to the invention patent CN102409071A disclosed in the company, which is incorporated herein by reference.
  • the culture and fermentation method may include the following steps:
  • Erythromycin seed culture 1 preparation of mother slant spores: Spores of Streptomyces erythromycin stored in a sand tube, inoculated on sterilized slant medium, cultured at a temperature of 34 ° C 9 days; preparation of 2 slanted spores: inoculation of mother slant spores on sterilized slant medium, cultured for 9 days at 34 °C; 3 seed culture: inoculation of slanted spores, sterilized Seed culture medium, after three-stage seed expansion culture, to obtain erythromycin seed liquid; (2) Fermentation: inoculate erythromycin seed liquid in a sterilized medium at a ratio of 10% by volume, and pass through sterile air. The mixture was stirred for 20 hours, nutrients were added, and after 8 days of culture, an erythromycin fermentation broth was obtained.
  • the composition of the mother slant medium is: starch 0.5g, corn syrup 0.1g, agar 0.5g, glucose 0.5g, sodium chloride 1g, calcium chloride 0.5g, toluene to 100ml with tap water; : starch 0.4g, corn syrup 0.2g, agar 0.4g, glucose 0.4g, sodium chloride 1.5g, calcium chloride 1.0g, with tap water to 100ml; fermentation medium composition: soybean powder 4.5g, starch 3.0 g, ammonium sulfate 0.12g, CaCO 3 0.8g, soybean oil 0.7g, polyoxypropylene glycerol 0.01g, dilute to 100ml with tap water; nutrient is 40% glucose solution 20g, soybean oil 4g, 60% n-propanol solution 2g; the first seed medium is: soybean powder 3g, starch 1.5g, CaCO30.7g, dextrin 1.5g, ammonium sulfate 0.3g, corn syrup 2g, soybean oil 0.7
  • the method for producing erythromycin thiocyanate from the fermentation broth of the strain KLCNDX-01 or KLCNDX-02 of the present invention can be carried out by referring to the invention patent CN102408462A disclosed in the company, the disclosure of which is incorporated herein by reference.
  • the preparation method may include the following steps:
  • Adjust the pH to 9.6-9.8 with a 15% sodium hydroxide solution convert erythromycin thiocyanate into erythromycin base, let stand, take acetone solution, add 15% sodium thiocyanate solution (per ten 100 million units of erythromycin added 0.15kg of 15% sodium thiocyanate solution), adjusted to pH 6.5 with 40% glacial acetic acid solution, added 50-55 ° C purified water 1.0 times the volume of the solution, control crystallization temperature 25-30 °C, stop stirring, let stand for 2 hours into the centrifuge to obtain erythromycin thiocyanate wet product, erythromycin thiocyanate wet product with acetone, 50-55 ° C purified water rinse, dry, that is, thiocyanate Finished acid erythromycin.
  • the method for measuring the foul odor is to use the "three-point comparative odor bag method", which is determined according to the method of the People's Republic of China national standard GB/T14675-93 "Air quality - measurement of malodor - three-point comparative odor bag method". The entire content of this standard is incorporated into the present invention as part of the present invention.
  • the three-point comparative odor bag method for measuring the concentration of malodorous gas is: firstly, two of the three odorless bags are filled with odorless air, and the other is charged with odorless air and the measured malodorous gas according to a certain dilution ratio.
  • the sample is sniffed by the sniffing discriminator. When the sniffing staff correctly identifies the odorous bag, it is diluted and smeared step by step until the odor level of the diluted sample is lower than the olfactory threshold of the sniffer.
  • Each sample is simultaneously determined by a number of sniffers (usually no less than 6), and finally the odor concentration is determined based on the individual threshold of the sniffer and the average threshold of the members of the sniffing panel.
  • the tail gas of different fermentation time of the erythromycin producing strains KLCNDX-01 and KLCNDX-02 of the present invention is collected, and the sampling method is referred to the "bad smell training material" (prepared by the national key laboratory for environmental odor pollution control). Chapter 2 malodorous samples The collection was carried out by the "three-point comparison odor bag method".
  • the strain KLCNDX-01 of the present invention when the fermentation time is 136 hours, the average odor concentration is 220, and before the genetic engineering transformation, the original production strain is fermented for 136 hours, and the odor concentration is 549, and the odor of the KLCNDX-01 strain is measured at the same time. The odor concentration was reduced by 60% compared to the original production strain before genetic engineering.
  • the productivity of the strain was determined by chemical colorimetry and high performance liquid chromatography as follows:
  • Reagent, mixed coloring solution Phosphoric acid and purified water are prepared in a ratio of 7:3, mixed and shaken, and used.
  • the absorbance was measured at a wavelength of 483 nm using a spectrophotometer with purified water as a blank.
  • a standard curve was prepared by adding the erythromycin titer content (U) to the absorbance value, and the regression line equation was obtained.
  • Sample preparation 1 standard solution preparation: Take erythromycin standard 0.04 g, accurately weighed, placed in a 10 ml volumetric flask, dissolved with mobile phase and diluted to the mark.
  • Preparation of test solution Take 5 grams of erythromycin fermentation broth filtrate into a 25ml volumetric flask, add appropriate amount of methanol, dissolve in ultrasound, let stand, and dilute with methanol. The solution was centrifuged (3500 r/min, 20 min), and the supernatant was taken. The organic membrane was passed through 0.22 um and used.
  • Erythromycin A content erythromycin A peak area * standard product A component concentration * dilution factor / erythromycin standard product A component peak area
  • Erythromycin B content erythromycin B peak area * standard product A component concentration * dilution factor / erythromycin standard product B component peak area
  • Erythromycin C content erythromycin C peak area * standard product A component concentration * dilution factor / erythromycin standard product C component peak area
  • Erythromycin E content erythromycin E peak area * standard product A component concentration * dilution factor / erythromycin standard product E component peak area
  • the titer is the result of chemical coloration.
  • the total components, erythromycin A, erythromycin B, erythromycin C, and erythromycin E are the results of high performance liquid chromatography
  • the total component titer of erythromycin produced by KLCNDX-02 was increased by 16.7% compared with the strain before genetic engineering.
  • the main active ingredient erythromycin A was increased by 9.66%, and the erythromycin B component was reduced by 44%.
  • the total component titer of KLCNDX-02 fermented to produce erythromycin increased by 67.8%, and the main active ingredient erythromycin A increased by 70.34%.
  • the mycin B component was reduced by 36.8%.

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Abstract

The present invention relates to an erythromycin producing strain having improved fermentation malodor and use thereof. The Saccharopolyspora erythraea of the present invention is obtained by means of genetic engineering, domestication and breeding of a strain used in industrial production of erythromycin. The strain effectively reduces the production of malodor in the fermentation production of erythromycin relative to the original strain before engineering and domestication while further improving the production capacity of erythromycin, having important industrial production value and environmental protection value.

Description

一种发酵恶臭味改善的红霉素生产菌株及其应用Erythromycin producing strain with improved malodor of fermentation and application thereof 技术领域Technical field
本发明属于微生物发酵工程领域，涉及一种能够有效降低发酵中恶臭气味的新型红霉素生产菌株及其应用。The invention belongs to the field of microbial fermentation engineering, and relates to a novel erythromycin producing strain capable of effectively reducing malodor odor in fermentation and application thereof.
背景技术Background technique
红霉素是由链霉属红色糖多孢菌(Saccharopolyspora erythraea)产生的次级代谢产物，是一类抗革兰氏阳性菌的广谱十四元大环内酯类抗生素。根据侧链基团的不同，将红霉素分为六种，分别为红霉素A、红霉素B、红霉素C、红霉素D、红霉素E和红霉素F。其中红霉素A是临床上最常用的药用成分，而B、C、D组分是红霉素A合成过程中的中间产物。红霉素B的生物活性比红霉素A低，但毒性却是红霉素A的两倍。红霉素E和F是两种天然产物，含量很少，活性很低，研究较少[Mironov V et al,Appl.Biochem.Microbiol.2004；And Zou X et al,J Ind Microbiol Biotechnol,2008]。美国Elililly公司在1952年从红色糖多孢菌发酵液中分离得到红霉素A，开发出第一代大环内酯类抗生素红霉素，上市之后，红霉素成为当时治疗***感染的重要药物。目前为止，红霉素仍然是一种临床常用的抗生素，对于一些常见的细菌感染类疾病具有非常有效的治疗效果。以红霉素A为前体合成的第二、三代红霉素衍生物也已经被广泛应用于临床。Erythromycin is a secondary metabolite produced by the genus Saccharopolyspora erythraea and is a broad-spectrum fourteen-membered macrolide antibiotic against Gram-positive bacteria. According to the difference of side chain groups, erythromycin is divided into six kinds, namely erythromycin A, erythromycin B, erythromycin C, erythromycin D, erythromycin E and erythromycin F. Among them, erythromycin A is the most commonly used pharmaceutical ingredient in the clinic, and the components B, C and D are intermediate products in the synthesis of erythromycin A. Erythromycin B has lower biological activity than erythromycin A, but its toxicity is twice that of erythromycin A. Erythromycins E and F are two natural products with little content and low activity, and few studies [Mironov V et al, Appl. Biochem. Microbiol. 2004; And Zou X et al, J Ind Microbiol Biotechnol, 2008] . In 1952, Elililly Company of the United States isolated erythromycin A from the fermentation broth of Saccharopolyspora erythraea and developed the first generation of macrolide antibiotic erythromycin. After the market, erythromycin became the gram-positive treatment at that time. An important drug for bacterial infection. So far, erythromycin is still a commonly used antibiotic in clinical practice, and it has a very effective therapeutic effect on some common bacterial infection diseases. Second and third generation erythromycin derivatives synthesized with erythromycin A as a precursor have also been widely used in clinical practice.
红霉素在临床上的广泛使用，大大增加了对红霉素原料的供应需求，从而推动了整个红霉素产业的发展。目前，我国是世界最大的红霉素生产和出口国，年产量超过9000吨。红霉素原料药主要依靠菌株发酵生产。由于近年来基因组学与蛋白组学的发展，结合国内研究者多年的微生物育种筛选，国内红霉素的发酵生产能力有了一定的提高，但与国外较高的生产水平相比仍然存在提高空间。The widespread use of erythromycin in the clinic has greatly increased the supply of erythromycin raw materials, thus promoting the development of the entire erythromycin industry. At present, China is the world's largest producer and exporter of erythromycin, with an annual output of more than 9,000 tons. The erythromycin bulk drug is mainly produced by fermentation of the strain. Due to the development of genomics and proteomics in recent years, combined with the microbial breeding screening of domestic researchers for many years, the domestic erythromycin fermentation production capacity has been improved, but there is still room for improvement compared with the higher production levels abroad. .
同时，随着近年来企业和民众环保意识的提高，红霉素生产中所伴随的发酵尾气恶臭气味问题也越来越受到人们的重视。目前去除恶臭气味的主要方法包括采用生化或物理的方法对尾气进行处理和降解。At the same time, with the improvement of environmental awareness among enterprises and the public in recent years, the problem of malodorous odor of fermentation tail gas accompanying erythromycin production has also received more and more attention. The current main methods of removing malodors include the treatment and degradation of exhaust gases by biochemical or physical methods.
樊晓宇等(樊晓宇，医药工程设计，34(4)，2013年)使用臭氧与硫氰酸红霉素生产尾气进行混合氧化反应，随后进入UV照射室，臭氧经紫外照射分解产生羟基自由基，在湿式氧化塔内羟基自由基氧化发酵尾气挥发性有机物VOCs，降解成CO 2、H 2O和微量的有机酸，再进入喷淋洗浆塔，洗涤净化，对微量有机酸用水吸收后排放，洗涤后发酵尾气无污染排空。上述方法消除红霉素发酵尾气的苦涩气味效果明显。 Fan Xiaoyu et al (Fan Xiaoyu, Medical Engineering Design, 34(4), 2013) used ozone to produce tail gas with erythromycin thiocyanate for mixed oxidation reaction, and then entered the UV irradiation chamber. The ozone was decomposed by ultraviolet irradiation to generate hydroxyl radicals. The volatile organic compounds VOCs in the oxidation tower of the wet oxidation tower are degraded into CO 2 , H 2 O and trace organic acids, and then enter the spray washing tower, washed and purified, absorbed by the trace organic acid and then discharged. The post-fermentation tail gas is free from pollution. The above method has obvious effect of eliminating the bitter odor of the erythromycin fermentation tail gas.
伊犁川宁生物技术有限公司(经济观察者网，2016-4-25)采用分子筛、疏水性活性炭、高温热氧化燃烧等集成技术，对尾气进行***性的处理；同时通过“生化处理+MVR深度处理及回用”工艺技术净化污水，并循环利用，减少排放；对固体废弃物进行无害化处理和资源化利用，解决了发酵生产过程带来的尾气异味等问题。Yili Chuan Ning Biotechnology Co., Ltd. (Economic Observer Network, 2016-4-25) uses molecular sieves, hydrophobic activated carbon, high temperature thermal oxidation combustion and other integrated technologies to systematically treat the exhaust gas. At the same time, it passes “biochemical treatment + MVR depth”. Treatment and reuse of “process technology to purify wastewater, recycling, reduce emissions; harmless treatment and resource utilization of solid waste, to solve the problem of tail gas odor caused by fermentation production process.
张志宏等(张志宏等，河南师范大学学报，37(5)，2009)以白地霉、地衣芽胞杆菌、假丝酵母、乳酸菌、光合菌制备复合微生物菌剂,对红霉素菌渣进行固态发酵，去除了红霉素菌渣中特殊异味。Zhang Zhihong et al (Zhang Zhihong et al., Journal of Henan Normal University, 37(5), 2009) prepared a composite microbial agent with Geotrichum candidum, Bacillus licheniformis, Candida, Lactobacillus and Photosynthetic bacteria to carry out solid-state fermentation of erythromycin slag. The special odor in the erythromycin slag is removed.
上述针对红霉素发酵尾气、废料、污水进行理化处理的方式虽然去除了恶臭气味，但大大增加了红霉素的生产成本。而通过菌株改造和驯化，减弱和消除红霉素生产菌株自身产生恶臭气味的能力，无疑是解决上述问题的根本方法。目前，国内外尚未见能够解决红霉素菌株自身发酵恶臭气味问题且适宜工业化生产的优良菌株和发酵技术。The above physical and chemical treatment method for erythromycin fermentation tail gas, waste water and sewage has greatly reduced the production cost of erythromycin although the malodor odor is removed. The ability to attenuate and eliminate the odor of the erythromycin producing strain itself by strain modification and domestication is undoubtedly the fundamental method to solve the above problems. At present, there are no excellent strains and fermentation techniques that can solve the problem of odor of erythromycin strain self-fermentation and suitable for industrial production at home and abroad.
发明内容Summary of the invention
本发明目的是提供一种能够有效降低发酵中恶臭气味的红霉素生产菌株及其应用。An object of the present invention is to provide an erythromycin producing strain capable of effectively reducing malodorous odor in fermentation and use thereof.
本发明一个方面提供一种红色糖多孢菌(Saccharopolyspora erythraea)，其名称为KLCNDX-01，保藏编号为CCTCC NO:M 2017647；或名称为KLCNDX-02，保藏编号为CCTCC NO:M 2017648。One aspect of the present invention provides a Saccharopolyspora erythraea having the name KLCNDX-01, the accession number is CCTCC NO: M 2017647; or the name is KLCNDX-02, and the accession number is CCTCC NO: M 2017648.
本发明另一个方面提供由保藏编号为CCTCC NO:M 2017647的菌株KLCNDX-01培养得到的红色糖多孢菌；或由保藏编号为CCTCC NO:M 2017648的菌株KLCNDX-02培养得到的红色糖多孢菌。Another aspect of the present invention provides a Saccharopolyspora erythraea cultured from the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647; or a red sugar obtained by culturing the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 Spores.
本发明另一个方面提供一种红色糖多孢菌，其具有保藏编号为CCTCC NO:M2017647的菌株KLCNDX-01或保藏编号为CCTCC NO:M 2017648的菌株KLCNDX-02的鉴定特征。Another aspect of the present invention provides a S. erythraea having the identification characteristics of the strain KLCNDX-01 with the accession number CCTCC NO: M2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
本发明另一个方面提供一种红色糖多孢菌培养物，其中含有保藏号为CCTCC NO:M2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02，或其中含有具有KLCNDX-01或KLCNDX-02鉴定特征的菌株。Another aspect of the present invention provides a S. erythraea culture comprising a strain KLCNDX-01 having a deposit number of CCTCC NO: M2017647 or a strain KLCNDX-02 having a deposit number of CCTCC NO: M 2017648, or containing KLCNDX therein -01 or KLCNDX-02 identifies strains of the characteristics.
在某些优选的实施方案中，本发明红色糖多孢菌培养物为生物学纯培养物，其由菌株KLCNDX-01或KLCNDX-02培养获得；或所述生物学纯的培养物具有KLCNDX-01或KLCNDX-02培养物的鉴定特征。In certain preferred embodiments, the S. erythraea culture of the present invention is a biologically pure culture obtained by culturing strain KLCNDX-01 or KLCNDX-02; or the biologically pure culture having KLCNDX- Identification characteristics of 01 or KLCNDX-02 cultures.
本发明另一个方面提供一种红色糖多孢菌或其培养物，所述红色糖多孢菌由保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02衍生获得。Another aspect of the present invention provides a Saccharopolyspora erythraea or a culture thereof, the Saccharomyces cerevisiae strain S strain KLCNDX-01 deposited under the accession number CCTCC NO: M 2017647 or the strain deposited under the accession number CCTCC NO: M 2017648 KLCNDX-02 is derived.
在优选的实施例中，本发明红色糖多孢菌相对于衍生前具有同等或更高的红霉素生产能力，和/或在培养或发酵中不产生或产生更少的恶臭气味。In a preferred embodiment, the S. erythraea of the present invention has an equal or higher erythromycin production capacity prior to derivatization, and/or does not produce or produce less malodor odor during culture or fermentation.
本发明另一个方面提供一种红色糖多孢菌，其与KLCNDX-01或KLCNDX-02具有相同的或基本相同的基因组序列，所述基本相同的基因组序列指菌株基因组内仅存在自然条件下增殖传代中发生的自发突变。在优选的实施方案中，所述红色糖多孢菌与KLCNDX-01或KLCNDX-02具有基本相同的红霉素生产能力和/或在培养或发酵中不产生或少产生恶臭气味能力。在优选的实施方案中，所述红色糖多孢菌基因组序列与KLCNDX-01或KLCNDX-02具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。Another aspect of the present invention provides a Saccharopolyspora erythraea having the same or substantially the same genomic sequence as KLCNDX-01 or KLCNDX-02, wherein said substantially identical genomic sequence refers to proliferation only in the genome of the strain in the presence of natural conditions Spontaneous mutations that occur during passage. In a preferred embodiment, the S. erythraea has substantially the same erythromycin production capacity as KLCNDX-01 or KLCNDX-02 and/or does not produce or produce malodorous odor in culture or fermentation. In a preferred embodiment, the S. erythraea genomic sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and KLCNDX-01 or KLCNDX-02, 98%, 99% or higher homology.
本发明另一方面提供一种发酵方法，其以保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02进行发酵。Another aspect of the present invention provides a fermentation method which is carried out by the strain KLCNDX-01 having the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 having the accession number CCTCC NO: M 2017648.
在某些实施方式中，本发明的发酵方法包括将低温保存的菌株孢子进行斜面培养，再将斜面培养获得的孢子悬浮液经多级种子罐培养，最终接入发酵罐中发酵。In certain embodiments, the fermentation method of the present invention comprises incubating the spores of the cryopreserved strains, and then culturing the spore suspension obtained by the slant culture through a multi-stage seed tank, and finally fermenting into the fermenter.
在某些实施方式中，所述斜面培养包括母斜面培养和子斜面培养；所述多级种子罐培养为一级种子罐培养，或两级、三级或更多级种子罐连续培养。优选的，所述种子罐培养为三级培养。In certain embodiments, the bevel culture comprises a mother bevel culture and a sub-slope culture; the multi-stage seed tank culture is a first stage seed tank culture, or a two-stage, three-stage or more seed tank continuous culture. Preferably, the seed tank is cultured in a tertiary culture.
在某些实施方式中，本发明的发酵方法中菌株的培养温度为28-37℃，优选30-35℃。In certain embodiments, the culture temperature of the strain in the fermentation process of the invention is 28-37 ° C, preferably 30-35 ° C.
在某些实施方式中，本发明的发酵方法包括如下步骤：(1)红霉素种子液培养：①母斜面孢子制备：将冷冻保存在砂土管中的红霉素链霉菌孢子，接种于灭菌的斜面培养基上，在温度32-34℃条件下，培养7-9天；②子斜面孢子制备：将母斜面孢子，接种于灭菌的斜面培养基上，在温度32-34℃条件下，培养7-9天；③种子培养：将子斜面孢子，接种于灭菌的液体种子培养基中，经过三级种子扩大培养，得到红霉素种子液；(2)红霉素发酵：将红霉素种子液按10-20％体积比接种于灭菌的培养基中，通入无菌空气并开启搅拌，添加营养物质，在温度32-34℃条件下，培养7-10天，得红霉素发酵液。In certain embodiments, the fermentation method of the present invention comprises the following steps: (1) erythromycin seed culture: 1 mother slant spore preparation: erythromycin Streptomyces spores frozen in a sand tube, inoculated The culture medium is cultured on a slant medium at a temperature of 32-34 ° C for 7-9 days; 2 slant spore preparation: the mother slant spores are inoculated on a sterilized slant medium at a temperature of 32-34 ° C. Next, culture for 7-9 days; 3 seed culture: seed slant spores, inoculated in sterilized liquid seed culture medium, through three-stage seed expansion culture to obtain erythromycin seed liquid; (2) erythromycin fermentation: The erythromycin seed solution is inoculated into the sterilized medium at a volume ratio of 10-20%, and the sterile air is introduced and stirred, the nutrient is added, and the culture is carried out for 7-10 days at a temperature of 32-34 ° C. Erythromycin fermentation broth.
在某些实施方式中，本发明的斜面培养基包括如下组分：淀粉0.1-1.0重量份，玉米浆0.05-0.2重量份，琼脂0.1-1.0重量份，葡萄糖0.1-1.0重量份，氯化钠0.5-2.0重量份，氯化钙0.1-1.0重量份，用自来水定容到100体积份。In certain embodiments, the bevel culture medium of the present invention comprises the following components: 0.1-1.0 parts by weight of starch, 0.05-0.2 parts by weight of corn syrup, 0.1-1.0 parts by weight of agar, 0.1-1.0 parts by weight of glucose, sodium chloride. 0.5-2.0 parts by weight, 0.1-1.0 parts by weight of calcium chloride, and made up to 100 parts by volume with tap water.
在某些实施方式中，本发明一级种子扩大培养条件为：(1)培养基组成：黄豆粉2-4重量份，淀粉1-2重量份，CaCO 3 0.6-0.8重量份，糊精1-2重量份，硫酸铵0.2-0.4重量份，玉米浆1-4重量份，植物油0.5-1重量份，用自来水定容到100体积份；(2)温度为32-34℃；(3)培养时间48-96小时。 In certain embodiments, the primary seed expanded culture condition of the present invention is: (1) medium composition: soybean powder 2-4 parts by weight, starch 1-2 parts by weight, CaCO 3 0.6-0.8 parts by weight, dextrin 1 - 2 parts by weight, ammonium sulfate 0.2-0.4 parts by weight, corn syrup 1-4 parts by weight, vegetable oil 0.5-1 parts by weight, made up to 100 parts by volume with tap water; (2) temperature 32-34 ° C; (3) The culture time is 48-96 hours.
在某些实施方式中，本发明所述的二、三级种子扩大培养条件为：(1)培养基组成：黄豆粉2.0-4.5重量份，淀粉2.5-4.0重量份，CaCO 3 0.5-0.7重量份，氯化钠0.4-0.6重量份，糊精2.5-4.0重量份，硫酸铵0.1-0.3重量份，玉米浆1-2重量份，植物油0.8-1.5重 量份，消沫剂0-0.02重量份，用自来水定容到100体积份；(2)温度为32-34℃；(3)培养时间24-48小时。 In certain embodiments, the second and third stage seed expansion culture conditions of the present invention are: (1) medium composition: soybean powder 2.0-4.5 parts by weight, starch 2.5-4.0 parts by weight, CaCO 3 0.5-0.7 weight Parts, sodium chloride 0.4-0.6 parts by weight, dextrin 2.5-4.0 parts by weight, ammonium sulfate 0.1-0.3 parts by weight, corn syrup 1-2 parts by weight, vegetable oil 0.8-1.5 parts by weight, defoaming agent 0-0.02 parts by weight , dilute to 100 parts by volume with tap water; (2) temperature is 32-34 ° C; (3) culture time 24-48 hours.
在某些实施方式中，本发明的发酵培养基组成：黄豆粉3.5-5.5重量份，淀粉2.0-4.0重量份，硫酸铵0.08-0.15重量份，CaCO 3 0.6-1.0重量份，植物油0.5-1.0重量份，消沫剂0.005-0.02重量份，用自来水定容到100体积份。 In certain embodiments, the fermentation medium of the present invention comprises: 3.5-5.5 parts by weight of soy flour, 2.0-4.0 parts by weight of starch, 0.08-0.15 parts by weight of ammonium sulfate, 0.6-1.0 parts by weight of CaCO 3 , 0.5-1.0 vegetable oil. Parts by weight, defoaming agent 0.005-0.02 parts by weight, made up to 100 parts by volume with tap water.
在某些实施方式中，本发明所述的添加营养物质组成为：20-70％葡萄糖溶液10-50重量份，植物油1-10重量份，正丙醇溶液0.5-5重量份。In certain embodiments, the nutritional supplement composition of the present invention comprises: 10-50 parts by weight of 20-70% glucose solution, 1-10 parts by weight of vegetable oil, and 0.5-5 parts by weight of n-propanol solution.
在某些实施方式中，本发明所述的植物油包括花生油、菜籽油、大豆油、棉籽油、香油、葵花籽油、橄榄油、莪术油、大蒜油、沙棘油、当归油、生姜油、连翘油、紫苏油、陈皮油、蓖麻油、薄荷油、肉豆蔻油中的任一种；优选为大豆油，菜籽油中的任一种。In certain embodiments, the vegetable oil of the present invention comprises peanut oil, rapeseed oil, soybean oil, cottonseed oil, sesame oil, sunflower oil, olive oil, zedoary oil, garlic oil, sea buckthorn oil, angelica oil, ginger oil, Any of forsythia oil, perilla oil, tangerine oil, castor oil, peppermint oil, and nutmeg oil; preferably any of soybean oil and rapeseed oil.
在某些实施方式中，本发明所述的正丙醇溶液为20-70％正丙醇溶液；本发明所述的消沫剂为聚醚类消沫剂；优选的，本发明所述的聚醚类消沫剂为聚氧丙烯甘油和聚氧乙烯氧丙烯甘油中的任一种。In some embodiments, the n-propanol solution of the present invention is a 20-70% n-propanol solution; the antifoaming agent of the present invention is a polyether antifoaming agent; preferably, the present invention The polyether antifoaming agent is any one of polyoxypropylene glycerin and polyoxyethylene oxypropylene glycerin.
本发明另一方面提供一种生产红霉素或硫氰酸红霉素的方法，包括将保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02进行发酵，得到红霉素发酵液。Another aspect of the present invention provides a method for producing erythromycin or erythromycin thiocyanate comprising a strain KLCNDX-01 having a deposit number of CCTCC NO: M 2017647 or a strain KLCNDX having a deposit number of CCTCC NO: M 2017648- 02 Fermentation was carried out to obtain an erythromycin fermentation broth.
在某些实施方式中，本发明红霉素的生产方法包括：1)培养本发明的微生物以获得细胞培养物；和2)从细胞培养物或微生物收集红霉素发酵液，通过对红霉素发酵液进行处理获得红霉素或硫氰酸红霉素成品。In certain embodiments, the method of producing erythromycin of the present invention comprises: 1) cultivating the microorganism of the present invention to obtain a cell culture; and 2) collecting erythromycin fermentation broth from a cell culture or microorganism, by passing on a red mold The fermentation broth is processed to obtain a finished erythromycin or erythromycin thiocyanate.
在某些实施方式中，本发明红霉素的生产方法包括：将红霉素生产菌株发酵液通过陶瓷膜过滤，菌渣经环保处理，滤液经进一步纳滤、结晶、干燥，获得红霉素产品。In some embodiments, the method for producing erythromycin of the present invention comprises: filtering the fermentation broth of the erythromycin producing strain through a ceramic membrane, the slag is subjected to environmental protection, and the filtrate is further subjected to nanofiltration, crystallization, and drying to obtain erythromycin. product.
在某些实施方式中，本发明硫氰酸红霉素的生产方法包括：将红霉素发酵液通过陶瓷膜过滤，菌渣经环保处理，滤液经过纳滤膜进行浓缩得到红霉素浓缩液，向浓缩液中加入适量硫氰酸钠溶液，结晶得到硫氰酸红霉素粗品，通过二次结晶、干燥获得硫氰酸 红霉素成品。In some embodiments, the method for producing erythromycin thiocyanate according to the present invention comprises: filtering the erythromycin fermentation broth through a ceramic membrane, the slag is subjected to environmental protection, and the filtrate is concentrated through a nanofiltration membrane to obtain an erythromycin concentrate. Adding an appropriate amount of sodium thiocyanate solution to the concentrated solution to obtain a crude erythromycin thiocyanate, and obtaining a finished erythromycin thiocyanate by secondary crystallization and drying.
在某些实施方式中，本发明硫氰酸红霉素的生产方法包括如下步骤：In certain embodiments, the method of producing erythromycin thiocyanate of the present invention comprises the steps of:
(1)发酵液浓缩：将红霉素发酵液调pH为7.5-9.0，采用50nm-100nm孔径的陶瓷膜过滤，过滤液用200分子量的纳滤膜进行浓缩,得到红霉素浓缩液；(1) Concentration of fermentation broth: The erythromycin fermentation liquid is adjusted to a pH of 7.5-9.0, filtered through a ceramic membrane having a pore size of 50 nm to 100 nm, and the filtrate is concentrated with a 200 molecular weight nanofiltration membrane to obtain an erythromycin concentrate;
(2)结晶：将红霉素浓缩液置于结晶罐,加入适量5～20％硫氰酸钠溶液，调pH为5.5～7.0,结晶析出硫氰酸红霉素，固液混合物经离心分离得硫氰酸红霉素粗品；在硫氰酸红霉素粗品中加入适量丙酮，调pH为9.5～10.5，将硫氰酸红霉素转化成红霉素碱，静置；取丙酮溶液，加入适量硫氰酸钠溶液，调pH至5.5～7.5，按溶解液体积的1～3倍加入45～60℃纯化水，控制结晶温度20-35℃，搅拌10分钟，静置降温0.5～2小时，离心获得硫氰酸红霉素湿品,进一步通过丙酮、45～60℃纯化水淋洗,离心分离直到母液排出,干燥,得硫氰酸红霉素成品。(2) Crystallization: The erythromycin concentrate is placed in a crystallization tank, and an appropriate amount of 5-20% sodium thiocyanate solution is added to adjust the pH to 5.5-7.0 to crystallize erythromycin thiocyanate, and the solid-liquid mixture is centrifuged. Obtaining crude erythromycin thiocyanate; adding appropriate amount of acetone to the crude erythromycin thiocyanate, adjusting the pH to 9.5~10.5, converting erythromycin thiocyanate into erythromycin base, and standing; taking acetone solution, Add appropriate amount of sodium thiocyanate solution, adjust the pH to 5.5 ~ 7.5, add 45 ~ 60 ° C purified water 1 to 3 times the volume of the solution, control the crystallization temperature 20-35 ° C, stir for 10 minutes, let stand to cool down 0.5 ~ 2 After hours, the erythromycin thiocyanate wet product was obtained by centrifugation, further rinsed with acetone, 45-60 ° C purified water, centrifuged until the mother liquid was discharged, and dried to obtain a erythromycin thiocyanate product.
本发明另一方面提供保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02在生产红霉素中的应用。Another aspect of the present invention provides the use of the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 for the production of erythromycin.
本发明另一方面提供一种降低红霉素发酵生产中恶臭气味的方法，所述方法包括以保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M2017648的菌株KLCNDX-02进行发酵生产红霉素。Another aspect of the present invention provides a method for reducing malodor odor in erythromycin fermentation production, the method comprising the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX with the accession number CCTCC NO: M2017648 02 Fermentation to produce erythromycin.
在某些优选的实施方式中，本发明降低红霉素发酵生产中恶臭气味的方法还包括对发酵产生的尾气、废水和/或废渣进行处理，降低其中的恶臭气味。In certain preferred embodiments, the method of the present invention for reducing malodors in erythromycin fermentation production further comprises treating exhaust gas, wastewater, and/or waste generated by fermentation to reduce malodors therein.
在某些优选的实施方式中，本发明对发酵产生的尾气、废水和/或废渣进行处理的方法包括采用物理和/或化学的方法处理尾气、废水和/或废渣，降低其中的恶臭气味。In certain preferred embodiments, the method of the present invention for treating tail gas, wastewater, and/or waste generated by fermentation includes treating the tail gas, waste water, and/or waste residue by physical and/or chemical means to reduce the malodor odor therein.
在某些优选的实施方式中，所述物理的方法包括：吸附法(如：采用疏水性活性炭、硅胶、活性氧化铝、沸石分子筛等吸附)、等离子体法等。In certain preferred embodiments, the physical methods include adsorption methods (eg, adsorption using hydrophobic activated carbon, silica gel, activated alumina, zeolite molecular sieves, etc.), plasma methods, and the like.
在某些优选的实施方式中，所述化学的方法包括：吸收法(如：碱水吸收)、燃烧法(如：直接燃烧法、催化燃烧法、热力燃烧法)、光催化氧化等。In certain preferred embodiments, the chemical method includes an absorption method (e.g., alkali water absorption), a combustion method (e.g., direct combustion method, catalytic combustion method, thermal combustion method), photocatalytic oxidation, and the like.
红色糖多饱菌在发酵过程中会形成特征性尾气，但由于发酵气体多为多种异味气体的混合物，不容易识别，因此需要一种红色糖多饱菌的标准发酵尾气作为参照，来识别发酵气体中是否存在红色糖多饱菌发酵产生的特征性尾气。The red sugar multi-saturated bacteria will form a characteristic tail gas during the fermentation process, but since the fermentation gas is mostly a mixture of various odor gases, it is not easy to identify, so a standard fermentation tail gas of red sugar multi-saturated bacteria is needed as a reference to identify Whether there is a characteristic tail gas produced by fermentation of red sugar polysaturated bacteria in the fermentation gas.
本发明另一方面提供一种应用，将保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02的发酵尾气用作标准发酵尾气，应用于红色糖多孢菌发酵特征性尾气的识别。Another aspect of the present invention provides an application of the fermentation tail gas of the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 as standard fermentation tail gas, applied to red Identification of characteristic tail gas from Saccharopolysaccharide fermentation.
本发明另一方面提供保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02在红色糖多孢菌发酵特征性尾气识别中的应用。In another aspect, the invention provides the use of the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 in the characteristic tail gas recognition of S. erythraea fermentation.
本发明另一方面提供一种红色糖多孢菌发酵特征性尾气的识别方法，以保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02的发酵尾气作为标准发酵尾气，识别红色糖多孢菌发酵特征性尾气。Another aspect of the present invention provides a method for identifying characteristic tail gas of Saccharopolyspora erythraea, the strain KLCNDX-01 with the accession number CCTCC NO:M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO:M 2017648 The fermentation tail gas is used as a standard fermentation tail gas to identify the characteristic tail gas of the fermentation of S. cerevisiae.
本发明另一方面提供一种红色糖多孢菌标准发酵尾气，该尾气由保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02发酵收集获得。Another aspect of the present invention provides a standard fermentation tail gas of Saccharopolyspora erythraea which is obtained by fermentation collection of strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or strain KLCNDX-02 with the accession number CCTCC NO: M 2017648. .
在某些实施方式中，红色糖多饱菌发酵产生尾气的识别可参照GB/T14675-93《空气质量-恶臭的测定-三点式比较式臭袋法》方法进行。本发明的红色糖多孢菌标准发酵尾气可用于嗅辨员的技术培训或嗅辨员的嗅辨识别，使嗅辨员了解红色糖多孢菌发酵特征性尾气的气味特性，提高对相应尾气的嗅辨能力。本发明的红色糖多孢菌标准发酵尾气和特征性尾气的识别方法，也可用于非专业嗅辨人员识别红色糖多孢菌发酵特征性尾气或辨别红色糖多孢菌发酵尾气。In some embodiments, the identification of tail gas by red sugar multi-fermentation fermentation can be carried out according to GB/T 14675-93 "Air quality - malodor measurement - three-point comparative odor bag method" method. The standard fermentation tail gas of the genus Saccharomyces cerevisiae of the invention can be used for the technical training of the olfactory discriminator or the olfactory identification of the olfactory discriminator, so that the scent finder can understand the odor characteristic of the characteristic tail gas of the fermentation of S. cerevisiae and improve the corresponding exhaust gas. The ability to sniff. The method for identifying the standard fermentation tail gas and characteristic tail gas of the Saccharopolyspora erythraea of the present invention can also be used for identifying non-professional olfactory personnel to identify the characteristic tail gas of S. erythraea fermentation or to distinguish the fermentation tail gas of S. cerevisiae.
本发明的红色糖多孢菌KLCNDX-01和KLCNDX-02由红霉素工业化生产中使用的红色糖多孢菌经基因改造和驯化育种获得，所述菌株的菌落呈菊花型，菌苔色素呈鼠灰色，基内菌丝色素呈红褐色。所述菌株相对于改造和驯化前的原始菌株有效降低了红霉 素发酵生产中恶臭气味的产生。同时，改造后的驯化育种进一步提高了红霉素的生产能力，KLCNDX-01为基因改造后菌株经两代驯化获得，KLCNDX-02为进一步传代驯化到***获得。其中KLCNDX-01比驯化前菌株摇瓶效价提高了64％，KLCNDX-02比驯化前菌株摇瓶效价提高了67.8％；两菌株相对于基因改造前的生产菌株红霉素生产能力提高了14-16.7％。The S. erythraea KLCNDX-01 and KLCNDX-02 of the present invention are obtained by genetic modification and domestication breeding of Saccharopolyspora erythraea used in the industrial production of erythromycin, and the colonies of the strain are chrysanthemum-type, and the bacteriophre pigment is The mouse is gray, and the mycelium pigment is reddish brown. The strain effectively reduced the generation of malodorous odors in the fermentation production of the red mold relative to the original strain before transformation and acclimation. At the same time, the domesticated breeding after transformation further improved the production capacity of erythromycin. KLCNDX-01 was obtained after genetic modification of the strain after two generations of domestication, and KLCNDX-02 was obtained for further passage and domestication to the fourth generation. Among them, KLCNDX-01 increased the titer of shake flask by 64% compared with the strain before acclimation, and the potency of KLCNDX-02 was 67.8% higher than that of the strain before acclimation. The production of erythromycin by the two strains was higher than that of the strain before the genetic modification. 14-16.7%.
本发明的红色糖多孢菌KLCNDX-01和KLCNDX-02在发酵过程中，种子罐和发酵罐的尾气排放能够达到直排标准，减少了发酵尾气的处理成本。同时，由于尾气直排，从而可以减轻发酵尾气管道***压力，减少染菌风险。In the fermentation process of the present S. erythraea KLCNDX-01 and KLCNDX-02, the tail gas discharge of the seed tank and the fermenter can reach the straight discharge standard, and the treatment cost of the fermentation tail gas is reduced. At the same time, due to the straight exhaust gas, the pressure of the fermentation tail gas pipeline system can be reduced and the risk of contamination can be reduced.
综上，本发明菌株即解决了发酵生产中的恶臭气味问题又提高了红霉素的生产能力，具有非常重大的工业生产价值和环保价值。In summary, the strain of the present invention solves the problem of malodorous odor in fermentation production and improves the production capacity of erythromycin, and has very important industrial production value and environmental protection value.
生物材料保藏Biomaterial preservation
本发明的红色糖多孢菌(Saccharopolyspora erythraea)KLCNDX-01已于2017年11月1日保藏于中国典型培养物保藏中心(CCTCC，地址：中国，武汉，武汉大学)，保藏编号为CCTCC NO:M 2017647。The Saccharopolyspora erythraea KLCNDX-01 of the present invention has been deposited with the China Center for Type Culture Collection (CCTCC, China, Wuhan, Wuhan University, China) on November 1, 2017, under the accession number CCTCC NO: M 2017647.
本发明的红色糖多孢菌(Saccharopolyspora erythraea)KLCNDX-02已于2017年11月1日保藏于中国典型培养物保藏中心(CCTCC，地址：中国，武汉，武汉大学)，保藏编号为C CCTCC NO:M 2017648。The Saccharopolyspora erythraea KLCNDX-02 of the present invention has been deposited with the China Center for Type Culture Collection (CCTCC, China, Wuhan, Wuhan University, China) on November 1, 2017, under the accession number C CCTCC NO :M 2017648.
具体实施方式Detailed ways
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. For a better understanding of the invention, definitions and explanations of related terms are provided below.
本发明中，术语“培养”指允许微生物在人工控制的条件下生长。多种本领域熟知的方法可用于培养本发明的菌株，可以用分批方法或连续方法如补料分批方法或重复补料分批方法培养，但本发明不限于此。In the present invention, the term "cultivating" means allowing the microorganism to grow under artificially controlled conditions. A variety of methods well known in the art can be used to culture the strain of the present invention, and can be cultured by a batch method or a continuous method such as a fed-batch method or a repeated fed-batch method, but the present invention is not limited thereto.
用于培养的培养基必须满足所使用菌种的要求。本领域熟知适用于培养放线菌的培 养基如高氏一号培养基和面粉琼脂培养基。培养基的碳源可以是：可溶性淀粉、糊精、液化糖、葡萄糖等。用于培养本发明菌株的氮源可以是：黄豆饼粉、蛋白胨、玉米浆、硫酸铵、硝酸钾等。培养基的其他成分可包括氯化钠、硫酸镁、磷酸氢二钾等。上述这些材料可以单独或组合使用。The medium used for the culture must meet the requirements of the species used. Culture media suitable for the cultivation of actinomycetes such as Gaussian No. 1 medium and flour agar medium are well known in the art. The carbon source of the medium may be: soluble starch, dextrin, liquefied sugar, glucose, and the like. The nitrogen source for cultivating the strain of the present invention may be: soybean cake powder, peptone, corn syrup, ammonium sulfate, potassium nitrate or the like. Other components of the medium may include sodium chloride, magnesium sulfate, dipotassium hydrogen phosphate, and the like. These materials may be used singly or in combination.
本发明中，“具有[规定菌株的]鉴定特征的菌株”，或“具有[规定菌株的]鉴定特征的培养物”，包括规定菌株的同源物或突变体，与规定菌株紧密相关(即与其享有共同祖先)或来源于规定菌株，但通常与规定菌株在一种或多种基因型或表型特征上有差异。通常通过遗传差异的评估，可鉴定突变体。具有鉴定特征的菌株，包括具有规定菌株的所有鉴定特征(例如，对应于规定菌株的DNA指纹的基于DNA分析的DNA指纹)的同源菌株或突变菌株。In the present invention, "a strain having an identification characteristic of [prescribed strain]", or a "culture having an identification characteristic of [specified strain]", including a homologue or a mutant of a prescribed strain, is closely related to a prescribed strain (ie, It has a common ancestor or is derived from a prescribed strain, but usually differs from the prescribed strain in one or more genotypes or phenotypic characteristics. Mutants can usually be identified by assessment of genetic differences. A strain having an identifying characteristic includes a homologous strain or a mutant strain having all the identifying characteristics of a prescribed strain (for example, a DNA analysis-based DNA fingerprint corresponding to a DNA fingerprint of a prescribed strain).
本发明中，术语“培养物”指通过将微生物进行生长和/或扩增而获得的产物，其可以是微生物的生物学纯培养物，也可以含有一定量的培养基、代谢物或培养过程中产生的其他成分，只要这些成分不会实质上影响该培养物生产产物的活性。术语“培养物”还包括通过将微生物传代而获得的传代培养物，其可以是某一代的培养物，也可以是若干代的混合物。所述(传代)培养物产生红霉素的能力与本发明的KLCNDX-01或KLCNDX-02基本上相同。In the present invention, the term "culture" refers to a product obtained by growing and/or amplifying a microorganism, which may be a biologically pure culture of microorganisms, or may contain a certain amount of medium, metabolite or culture process. Other ingredients produced as long as they do not substantially affect the activity of the product produced by the culture. The term "culture" also includes subcultures obtained by passage of microorganisms, which may be a generation of cultures, or a mixture of several generations. The ability of the (passage) culture to produce erythromycin is substantially the same as KLCNDX-01 or KLCNDX-02 of the present invention.
本发明中，术语“生物学纯的培养物”指基本没有生物污染的并具有遗传一致性从而使得从中取得的不同传代培养物将呈现基本相同的基因型和表型的培养物(例如，培养物具有至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、最高100％纯的纯度)。In the present invention, the term "biologically pure culture" refers to a culture (eg, cultured) that is substantially free of biological contamination and has genetic consistency such that different subcultures derived therefrom will exhibit substantially the same genotype and phenotype (eg, culture) At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99%, up to 100% pure purity).
本发明中，通过“衍生”获得的菌株指通过任何方式的转化，例如通过一次或更多次的杂交和/或通过变异和/或通过转基因而衍生出的菌株。In the present invention, a strain obtained by "derived" refers to a strain which is transformed by any means, for example, by one or more crosses and/or by mutation and/or by transgene.
通过杂交衍生出的菌株可通过将根据本发明的菌株与相同菌株或另一种根据本发明的菌株，或者任何其他的菌株进行杂交来获得。A strain derived by hybridization can be obtained by hybridizing a strain according to the present invention with the same strain or another strain according to the present invention, or any other strain.
通过变异衍生出的菌株可以为经过至少一次其基因组中的自发变异或经过通过例如致突变而诱发的至少一次变异的菌株。所述衍生菌株的变异是或不是无声的。用表述“致突变”表示通过辐射(例如紫外线)或致突变化学品得到的标准的致突变，和通过转座或外源DNA片段的整合实现的***诱变。通过射线致突变包括使用紫外线、X射线或伽马射线。致突变化学物质例如EMS(乙基-甲基磺酸酯)、EES(乙基-乙基磺酸酯)、亚硝基胍、亚硝酸、黄曲霉素B1，羟胺、5-溴-尿嘧啶、2-氨基-嘌呤、前黄素、吖啶橙。The strain derived by the variation may be a strain that undergoes at least one spontaneous mutation in its genome or undergoes at least one variation induced by, for example, mutagenesis. The variation of the derived strain is or is not silent. The expression "mutagenic" means a standard mutagen obtained by irradiation (for example, ultraviolet light) or a mutagenic chemical, and insertional mutagenesis by integration of a transposition or a foreign DNA fragment. Mutagenicity by radiation involves the use of ultraviolet light, X-rays or gamma rays. Mutagenic chemicals such as EMS (ethyl-methylsulfonate), EES (ethyl-ethylsulfonate), nitrosoguanidine, nitrous acid, aflatoxin B1, hydroxylamine, 5-bromo-urine Pyrimidine, 2-amino-indole, pre-flavin, acridine orange.
通过转基因衍生出的菌株为引入外源DNA的菌株。所述外源DNA优选通过质粒引入或直接整合到基因组中。The strain derived by the transgene is a strain into which foreign DNA is introduced. The foreign DNA is preferably introduced by a plasmid or directly integrated into the genome.
本发明中，术语“同源性”指多核苷酸或多肽与另一序列有一定百分数的“序列同一性”(sequence identity)。在比较两个序列时，当联配时，这些百分数的碱基或氨基酸是相同的。可以用本领域中已知的软件程序来确定这种联配和同源性百分数或序列同一性，例如描述于CURRENT PROTOCOLS INMOLECULAR BIOLOGY(F.M.Ausubel等人编辑，1987)增刊30，7.7.18部分中的那些程序。优选的联配程序是ALIGN Plus(Scientific andEducational Software，Pennsylvania)，优选地应用缺省参数，如下：错配＝2；开放空位(open gap)＝0；延伸空位(extend gap)＝2。可以应用的另一序列软件程序是TFastA Data Searching Program，可以从SequenceAnalysis Software Package版本6.0(Genetic Computer Group，Universityof Wisconsin，Madison，WI)得到。In the present invention, the term "homology" refers to a certain percentage of "sequence identity" of a polynucleotide or polypeptide to another sequence. When comparing two sequences, these percentage bases or amino acids are identical when conjugated. Such mating and homology percentages or sequence identity can be determined using software programs known in the art, as described, for example, in CURRENT PROTOCOLS INMOLECULAR BIOLOGY (FMAusubel et al., eds., 1987) Supplement 30, Section 7.7.18. Those programs. A preferred mating procedure is ALIGN Plus (Scientific and Educational Software, Pennsylvania), preferably using default parameters as follows: mismatch = 2; open gap = 0; extend gap = 2. Another sequence of software programs that can be applied is the TFastA Data Searching Program, available from the Sequence Analysis Software Package version 6.0 (Genetic Computer Group, University of Wisconsin, Madison, WI).
本发明中，术语“同等或更高的红霉素生产能力”中同等的红霉素生产能力指在相同的条件下培养时，与衍生前的放线菌宿主细胞相比，改变的放线菌细胞中的红霉素的产生量或摇瓶效价与改变前相同，或变化量不大于±5％、±4％、±3％、±2％、±1％或更小；更高的红霉素生产能力指上述变化为增加至少6％、10％、15％、20％、30％、40％、50％、60％或更多。In the present invention, the equivalent erythromycin production capacity in the term "equivalent or higher erythromycin production capacity" means that when cultured under the same conditions, the changed payout is compared with the actinomycete host cell before derivatization. The amount of erythromycin produced in the bacterial cells or the shake flask titer is the same as before the change, or the amount of change is not more than ± 5%, ± 4%, ± 3%, ± 2%, ± 1% or less; The erythromycin production capacity means that the above change is an increase of at least 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60% or more.
本发明中，术语“在培养或发酵中不产生或产生更少的恶臭气味”中不产生恶臭气味指在培养或发酵中不产生恶臭气体，或者恶臭气体的产生量低于嗅觉阈值。所述嗅觉阈值包括可以嗅觉气味存在的感觉阈值或能够定出气味特性的识别阈值(见国家标准 GB/T14675-93)。所述产生更少的恶臭气味是指培养或发酵中产生的恶臭气体浓度更低。所述恶臭气体浓度指单位体积内恶臭气体的质量数。In the present invention, the term "no odor is produced or produced in a culture or fermentation" does not produce malodorous odors, meaning that no malodorous gas is generated in culture or fermentation, or the amount of malodorous gas generated is lower than the olfactory threshold. The olfactory threshold includes a sensory threshold at which an olfactory odor can exist or an identification threshold capable of determining odor characteristics (see National Standard GB/T 14675-93). The production of less malodorous odor means that the concentration of malodorous gas produced in the culture or fermentation is lower. The malodorous gas concentration refers to the mass of the malodorous gas per unit volume.
在本发明中，恶臭气体浓度也可以参照国家标准GB/T14675-93所述《空气质量-恶臭的测定-三点比较式臭袋法》中的方法测定。其中恶臭气体浓度指根据嗅觉器官试验法对臭气气味的大小予以数量化表示的指标，用无臭的清洁空气对臭气样品连续稀释至嗅辨员阈值时的稀释倍数。In the present invention, the malodorous gas concentration can also be measured by the method in "Air Quality - Measurement of malodor - three-point comparative odor bag method" described in the national standard GB/T 14675-93. The malodorous gas concentration refers to an index indicating the magnitude of the odor odor according to the olfactory organ test method, and the dilution factor when the odorous sample is continuously diluted to the sniffer threshold by the odorless clean air.
在本发明中，红霉素生产能力及恶臭气味产生水平或能力的比较，可选择在相同条件下培养待比较的两种或多种微生物宿主细胞，并且使用相同条件、优选使用相同测定方法分别测定红霉素的产生量或恶臭气味的大小。In the present invention, the comparison of the erythromycin production capacity and the level or ability of malodorous odor generation may be carried out by culturing two or more microbial host cells to be compared under the same conditions, and using the same conditions, preferably using the same measurement method. The amount of erythromycin produced or the magnitude of the malodor odor was measured.
本发明中，术语“恶臭气味”指红霉素发酵生产过程中产生的刺激嗅觉器官引起人们不愉快的气体气味，可以是多种气体异味的集合，或单种气体的气味。所述气味可以通过人们的感知思维进行分析判断，也可以根据红色糖多孢菌标准发酵尾气进行对比识别，或者由专业的嗅辨员根据国家标准GB/T14675-93所规定的三点比较式臭袋法进行识别。In the present invention, the term "malodor" means that the stimulating olfactory organ produced during the erythromycin fermentation production process causes an unpleasant gas odor, and may be a collection of various gas odors or a single gas odor. The odor can be analyzed and judged by people's perceptual thinking, or can be compared and identified according to the standard fermentation tail gas of Saccharopolyspora erythraea, or a three-point comparison formula prescribed by the professional olfactory discriminator according to the national standard GB/T14675-93. The odor bag method is used for identification.
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制。本领域技术人员根据本发明的教导，可以做出各种修改或改进，而不脱离本发明的基本思想和范围。The invention is further illustrated by the following description of specific embodiments, but this is not to be construed as limiting. A person skilled in the art can make various modifications or improvements in accordance with the teachings of the present invention without departing from the basic spirit and scope of the invention.
实施例1菌株的发酵液的发酵方法Fermentation method of fermentation broth of Example 1 strain
本发明菌株KLCNDX-01或KLCNDX-02发酵液的发酵方法可参考本公司公开的发明专利CN102409071A进行，该专利公开内容作为本发明的一部分引入本发明。The fermentation method of the fermentation strain KLCNDX-01 or KLCNDX-02 of the present invention can be carried out by referring to the invention patent CN102409071A disclosed in the company, which is incorporated herein by reference.
作为其中一个实施例，该培养和发酵方法可包括如下步骤：As one of the embodiments, the culture and fermentation method may include the following steps:
(1)红霉素种子液培养：①母斜面孢子制备：将冷冻保存在砂土管中的红霉素链霉菌孢子，接种于经灭菌的斜面培养基上,在温度34℃条件下,培养9天；②子斜面孢子制备：将母斜面孢子,接种于经灭菌的斜面培养基上,在温度34℃条件下,培养9天；③种子培养： 将子斜面孢子,接种于经灭菌的种子培养基,经过三级种子扩大培养,得到红霉素种子液；(2)发酵：将红霉素种子液按10％体积比接种于经灭菌的培养基中,通入无菌空气并搅拌20小时,添加营养物质,经过8天培养,得红霉素发酵液。(1) Erythromycin seed culture: 1 preparation of mother slant spores: Spores of Streptomyces erythromycin stored in a sand tube, inoculated on sterilized slant medium, cultured at a temperature of 34 ° C 9 days; preparation of 2 slanted spores: inoculation of mother slant spores on sterilized slant medium, cultured for 9 days at 34 °C; 3 seed culture: inoculation of slanted spores, sterilized Seed culture medium, after three-stage seed expansion culture, to obtain erythromycin seed liquid; (2) Fermentation: inoculate erythromycin seed liquid in a sterilized medium at a ratio of 10% by volume, and pass through sterile air. The mixture was stirred for 20 hours, nutrients were added, and after 8 days of culture, an erythromycin fermentation broth was obtained.
其中，母斜面培养基组成为：淀粉0.5g，玉米浆0.1g，琼脂0.5g，葡萄糖0.5g，氯化钠1g，氯化钙0.5g，用自来水定容到100ml；子斜面培养基组成为：淀粉0.4g，玉米浆0.2g，琼脂0.4g，葡萄糖0.4g，氯化钠1.5g，氯化钙1.0g，用自来水定容到100ml；发酵培养基组成为：黄豆粉4.5g，淀粉3.0g，硫酸铵0.12g，CaCO 30.8g，大豆油0.7g，聚氧丙烯甘油0.01g，用自来水定容到100ml；营养物质为40％葡萄糖溶液20g，大豆油4g，60％正丙醇溶液2g；一级种子培养基为：黄豆粉3g，淀粉1.5g，CaCO30.7g，糊精1.5g，硫酸铵0.3g，玉米浆2g，大豆油0.7g，用自来水定容到100ml；二、三级种子培养基为：黄豆粉3.5g，淀粉3.2g，CaCO30.6g，氯化钠0.5g，糊精3.2g，硫酸铵0.2g，玉米浆1.5g，大豆油1.1g，聚氧丙烯甘油0.005g，用自来水定容到100ml。 Among them, the composition of the mother slant medium is: starch 0.5g, corn syrup 0.1g, agar 0.5g, glucose 0.5g, sodium chloride 1g, calcium chloride 0.5g, toluene to 100ml with tap water; : starch 0.4g, corn syrup 0.2g, agar 0.4g, glucose 0.4g, sodium chloride 1.5g, calcium chloride 1.0g, with tap water to 100ml; fermentation medium composition: soybean powder 4.5g, starch 3.0 g, ammonium sulfate 0.12g, CaCO 3 0.8g, soybean oil 0.7g, polyoxypropylene glycerol 0.01g, dilute to 100ml with tap water; nutrient is 40% glucose solution 20g, soybean oil 4g, 60% n-propanol solution 2g; the first seed medium is: soybean powder 3g, starch 1.5g, CaCO30.7g, dextrin 1.5g, ammonium sulfate 0.3g, corn syrup 2g, soybean oil 0.7g, with tap water to 100ml; two, three The seed culture medium is: soybean powder 3.5g, starch 3.2g, CaCO30.6g, sodium chloride 0.5g, dextrin 3.2g, ammonium sulfate 0.2g, corn syrup 1.5g, soybean oil 1.1g, polyoxypropylene glycerin 0.005 g, make up to 100ml with tap water.
实施例2硫氰酸红霉素的提取方法Example 2 Extraction method of erythromycin thiocyanate
本发明从菌株KLCNDX-01或KLCNDX-02发酵液中制备硫氰酸红霉素的方法，可参考本公司公开的发明专利CN102408462A进行，该专利公开内容作为本发明的一部分引入本发明。The method for producing erythromycin thiocyanate from the fermentation broth of the strain KLCNDX-01 or KLCNDX-02 of the present invention can be carried out by referring to the invention patent CN102408462A disclosed in the company, the disclosure of which is incorporated herein by reference.
作为其中一个实施例，该制备方法可包括如下步骤：As one of the embodiments, the preparation method may include the following steps:
(1)发酵液浓缩：红霉素发酵液除渣后，用浓度为15％的氢氧化钠溶液调pH为7.8-8.2，采用50nm-100nm孔径的陶瓷膜过滤，过滤液用200分子量的纳滤膜进行浓缩，得到红霉素浓缩液。(1) Concentration of fermentation broth: After removing slag from erythromycin fermentation broth, adjust the pH to 7.8-8.2 with a sodium hydroxide solution with a concentration of 15%, filter with a ceramic membrane with a pore size of 50 nm-100 nm, and filter the molecular weight with 200 molecular weight. The filter was concentrated to give an erythromycin concentrate.
(2)重结晶：将红霉素浓缩液置于结晶罐，加入15％的硫氰酸钠溶液(每十亿单位红霉素加入0.15kg的15％硫氰酸钠溶液)，用40％的冰醋酸溶液调节pH为6.0-6.5，结晶析出硫氰酸红霉素，固液混合物经离心分离得硫氰酸红霉素粗品，硫氰酸红霉素粗品按照1：3比例加入丙酮，用浓度为15％的氢氧化钠溶液调节pH为9.6-9.8，将硫氰酸 红霉素转化成红霉素碱，静置，取丙酮溶液，加入15％的硫氰酸钠溶液(每十亿单位红霉素加入0.15kg的15％硫氰酸钠溶液)，用40％冰醋酸溶液调pH至6.5，按溶解液体积的1.0倍加入50-55℃纯化水，控制结晶温度25-30℃，停搅拌，静置2小时进入离心机分离得硫氰酸红霉素湿品，硫氰酸红霉素湿品分别用丙酮、50-55℃纯化水淋洗，干燥，即得硫氰酸红霉素成品。(2) Recrystallization: Place the erythromycin concentrate in the crystallization tank, add 15% sodium thiocyanate solution (0.15kg of 15% sodium thiocyanate solution per billion units of erythromycin), use 40% The glacial acetic acid solution adjusts the pH to 6.0-6.5, crystallizes erythromycin thiocyanate, and the solid-liquid mixture is centrifuged to obtain crude erythromycin thiocyanate. The crude erythromycin thiocyanate is added in acetone at a ratio of 1:3. Adjust the pH to 9.6-9.8 with a 15% sodium hydroxide solution, convert erythromycin thiocyanate into erythromycin base, let stand, take acetone solution, add 15% sodium thiocyanate solution (per ten 100 million units of erythromycin added 0.15kg of 15% sodium thiocyanate solution), adjusted to pH 6.5 with 40% glacial acetic acid solution, added 50-55 ° C purified water 1.0 times the volume of the solution, control crystallization temperature 25-30 °C, stop stirring, let stand for 2 hours into the centrifuge to obtain erythromycin thiocyanate wet product, erythromycin thiocyanate wet product with acetone, 50-55 ° C purified water rinse, dry, that is, thiocyanate Finished acid erythromycin.
实施例3 50L发酵恶臭气味测定Example 3 Determination of malodor odor in 50L fermentation
恶臭气味的测定方法采用的是“三点比较式臭袋法”，根据中华人民共和国国家标准GB/T14675-93《空气质量-恶臭的测定-三点比较式臭袋法》的方法进行测定，该标准全部内容作为本发明的一部分引入本发明。The method for measuring the foul odor is to use the "three-point comparative odor bag method", which is determined according to the method of the People's Republic of China national standard GB/T14675-93 "Air quality - measurement of malodor - three-point comparative odor bag method". The entire content of this standard is incorporated into the present invention as part of the present invention.
三点比较式臭袋法测定恶臭气体浓度的方法是：先将三只无臭袋中的二只充入无臭空气、另一只则按一定稀释比例充入无臭空气和被测恶臭气体样品供嗅辨员嗅辨，当嗅辨员正确识别有臭气袋后，再逐级进行稀释、嗅辨，直至稀释样品的臭气浓度低于嗅辨员的嗅觉阈值时停止实验。每个样品由若干名嗅辨员(通常不少于6名)同时测定，最后根据嗅辨员的个人阈值和嗅辨小组成员的平均阈值，求得臭气浓度。The three-point comparative odor bag method for measuring the concentration of malodorous gas is: firstly, two of the three odorless bags are filled with odorless air, and the other is charged with odorless air and the measured malodorous gas according to a certain dilution ratio. The sample is sniffed by the sniffing discriminator. When the sniffing staff correctly identifies the odorous bag, it is diluted and smeared step by step until the odor level of the diluted sample is lower than the olfactory threshold of the sniffer. Each sample is simultaneously determined by a number of sniffers (usually no less than 6), and finally the odor concentration is determined based on the individual threshold of the sniffer and the average threshold of the members of the sniffing panel.
针对本发明红霉素生产菌株KLCNDX-01和KLCNDX-02不同发酵时间的尾气进行采集，采样方法参照《恶臭培训教材》(由国家环境保护恶臭污染控制重点实验室编制)第二章恶臭样品的采集，通过“三点比较式臭袋法”进行检测。The tail gas of different fermentation time of the erythromycin producing strains KLCNDX-01 and KLCNDX-02 of the present invention is collected, and the sampling method is referred to the "bad smell training material" (prepared by the national key laboratory for environmental odor pollution control). Chapter 2 malodorous samples The collection was carried out by the "three-point comparison odor bag method".
以本发明菌株KLCNDX-01为例，当其发酵136小时时平均臭气浓度为220，基因工程改造前原生产菌种发酵136小时臭气浓度为549，经测定KLCNDX-01菌株同期发酵恶臭气味与基因工程改造前的原生产菌株相比，臭气浓度降低了60％。Taking the strain KLCNDX-01 of the present invention as an example, when the fermentation time is 136 hours, the average odor concentration is 220, and before the genetic engineering transformation, the original production strain is fermented for 136 hours, and the odor concentration is 549, and the odor of the KLCNDX-01 strain is measured at the same time. The odor concentration was reduced by 60% compared to the original production strain before genetic engineering.
实施例4红霉素的生产能力测定Example 4 Determination of the production capacity of erythromycin
通过化学显色法、高效液相色谱法对菌株的生产能力进行测定，方法如下：The productivity of the strain was determined by chemical colorimetry and high performance liquid chromatography as follows:
化学显色法：Chemical color development method:
(1)原理：红霉素与磷酸应生成黄色物质，其最大吸收波长为483nm。用分光光度 计进行比色测定，能够定量分析。(1) Principle: Erythromycin and phosphoric acid should form a yellow substance with a maximum absorption wavelength of 483 nm. Colorimetric measurement using a spectrophotometer enables quantitative analysis.
(2)试剂，混合显色液：将磷酸与纯化水按7:3的配比进行配制，混合摇匀后待用。(2) Reagent, mixed coloring solution: Phosphoric acid and purified water are prepared in a ratio of 7:3, mixed and shaken, and used.
(3)标准曲线的绘制，分别量取红霉素标准溶液1.0ml、1.5ml、2.0ml、2.5ml、3.0ml、3.5ml、4.0ml于7个25ml容量瓶中(约64U/ml-256U/ml),使用煮沸后冷却的纯化水定容，分别制得红霉素工作标准溶液。置2-8℃冰箱中冷藏。分别精密量取各个红霉素工作标准溶液2.0ml于7支(30×200mm)试管中，精密加入混合显色溶液8ml,摇匀，放置80℃水浴中加热6min，取出后，立即用水冷至室温，使用分光光度计以纯化水为空白，在483nm波长处测定吸光度。以加入红霉素效价含量(U)对吸光度值绘制标准曲线，求得回归线方程。(3) Drawing of the standard curve, respectively, taking erythromycin standard solution 1.0ml, 1.5ml, 2.0ml, 2.5ml, 3.0ml, 3.5ml, 4.0ml in 7 25ml volumetric flasks (about 64U/ml-256U) /ml), using a purified water cooled by boiling, to make a standard solution of erythromycin. Store in a refrigerator at 2-8 °C. Separately measure 2.0ml of each erythromycin working standard solution into 7 (30×200mm) tubes, accurately add 8ml of mixed coloring solution, shake well, place in a water bath at 80°C for 6min, and immediately remove it with water until it is cooled. At room temperature, the absorbance was measured at a wavelength of 483 nm using a spectrophotometer with purified water as a blank. A standard curve was prepared by adding the erythromycin titer content (U) to the absorbance value, and the regression line equation was obtained.
(4)化学效价的测定：取经滤纸过滤或离心分离后的各工序提炼液上清液适量，用稀释剂稀释使成约100-200U/ml红霉素的溶液，作为供试品溶液。精密量取供试液2.0ml，置30×200mm试管中，再精密加入混合显色溶液8ml，摇匀，放入80℃水浴中加热6min，取出后，立即用水冷至室温，使用分光光度计以纯化水为空白，在483nm波长处测定吸光度。同时利用标准曲线项下火的的回归线方程。(4) Determination of chemical potency: The supernatant of the extract liquid obtained by filtration or centrifugation of the filter paper is diluted with a diluent to form a solution of about 100-200 U/ml erythromycin as a test solution. Precisely measure the test solution 2.0ml, place it in a 30×200mm test tube, then accurately add 8ml of the mixed color development solution, shake it, put it in a water bath of 80°C for 6min, remove it, and immediately cool it to room temperature with water, using a spectrophotometer. The absorbance was measured at a wavelength of 483 nm with purified water as a blank. At the same time, the regression equation of the fire under the standard curve term is used.
计算公式：发酵液效价(U/ml)＝回归线方程效价计算值×稀释倍数Calculation formula: Fermentation solution titer (U/ml) = regression line equation titer calculation value × dilution factor
高效液相色谱法：High performance liquid chromatography:
(1)色谱条件，色谱柱：C18柱(4.6*250㎜,5μm)，柱温：35℃，检测波长：215nm，流动相：0.025mol/l磷酸氢二钾：乙腈＝59:41，流速：1.0ml/min。(1) Chromatographic conditions, column: C18 column (4.6*250mm, 5μm), column temperature: 35°C, detection wavelength: 215nm, mobile phase: 0.025mol/l dipotassium hydrogen phosphate: acetonitrile=59:41, flow rate : 1.0 ml/min.
(2)样品制备，①标准品溶液制备：取红霉素标准品0.04克，精密称定，置10ml容量瓶中，用流动相溶解并稀释至刻度。②供试品溶液制备：取5克红霉素发酵液滤液至25ml容量瓶中，加甲醇适量，超声溶解，静置，用甲醇定容。取该溶液用离心机(3500r/min,20min)离心后取上清液，过0.22um的有机系滤膜，待用。(3)测定取10ul标准品溶液和供试品溶液，分别注入液相色谱仪。按外标法，以A组分标准品峰面积计算供试品红霉素A组分、B组分、C组分和E组分。(2) Sample preparation, 1 standard solution preparation: Take erythromycin standard 0.04 g, accurately weighed, placed in a 10 ml volumetric flask, dissolved with mobile phase and diluted to the mark. 2 Preparation of test solution: Take 5 grams of erythromycin fermentation broth filtrate into a 25ml volumetric flask, add appropriate amount of methanol, dissolve in ultrasound, let stand, and dilute with methanol. The solution was centrifuged (3500 r/min, 20 min), and the supernatant was taken. The organic membrane was passed through 0.22 um and used. (3) Measure 10 ul of the standard solution and the test solution, and inject them into the liquid chromatograph. According to the external standard method, the erythromycin A component, the B component, the C component and the E component of the test sample were calculated from the peak area of the A component standard.
红霉素A含量＝红霉素A峰面积*标准品A组分浓度*稀释倍数/红霉素标准品A组分峰面积Erythromycin A content = erythromycin A peak area * standard product A component concentration * dilution factor / erythromycin standard product A component peak area
红霉素B含量＝红霉素B峰面积*标准品A组分浓度*稀释倍数/红霉素标准品B组分峰面积Erythromycin B content = erythromycin B peak area * standard product A component concentration * dilution factor / erythromycin standard product B component peak area
红霉素C含量＝红霉素C峰面积*标准品A组分浓度*稀释倍数/红霉素标准品C组分峰面积Erythromycin C content = erythromycin C peak area * standard product A component concentration * dilution factor / erythromycin standard product C component peak area
红霉素E含量＝红霉素E峰面积*标准品A组分浓度*稀释倍数/红霉素标准品E组分峰面积Erythromycin E content = erythromycin E peak area * standard product A component concentration * dilution factor / erythromycin standard product E component peak area
其结果如表1所示：The results are shown in Table 1:
表1红霉素生产菌株摇瓶效价及各组分含量Table 1 erythromycin production strain shake flask titer and the content of each component
Figure PCTCN2019072500-appb-000001
Figure PCTCN2019072500-appb-000001
(注：效价为化学显色发测定结果。总组分、红霉素A、红霉素B、红霉素C、红霉素E为高效液相色谱法测定结果)(Note: The titer is the result of chemical coloration. The total components, erythromycin A, erythromycin B, erythromycin C, and erythromycin E are the results of high performance liquid chromatography)
由表1可知，KLCNDX-01发酵生产红霉素总组分效价较基因工程改造前生产菌株提高了14％，其中主要活性成分红霉素A组分提高9.68％，且红霉素B组分降低了36％；与经过基因工程改造但未经驯化筛选的菌株相比，KLCNDX-01发酵生产红霉素总组分效价提高了64％，主要活性成分红霉素A组分提高了70.4％，且红霉素B组分降低了28％。It can be seen from Table 1 that the total component titer of erythromycin produced by KLCNDX-01 is 14% higher than that of the strain before genetic engineering modification, and the main active ingredient erythromycin A component is increased by 9.68%, and erythromycin B group The fraction was reduced by 36%; compared with the strains that were genetically engineered but not domestically screened, the total component titer of erythromycin produced by KLCNDX-01 was increased by 64%, and the main active ingredient erythromycin A was improved. 70.4%, and the erythromycin B component was reduced by 28%.
KLCNDX-02发酵生产红霉素总组分效价较基因工程改造前生产菌株提高了16.7％，其中主要活性成分红霉素A组分提高9.66％，且红霉素B组分降低了44％；与经过基因工程改造但未经驯化筛选的菌株相比，KLCNDX-02发酵生产红霉素总组分效价提高了 67.8％，主要活性成分红霉素A组分提高了70.34％，且红霉素B组分降低了36.8％。The total component titer of erythromycin produced by KLCNDX-02 was increased by 16.7% compared with the strain before genetic engineering. The main active ingredient erythromycin A was increased by 9.66%, and the erythromycin B component was reduced by 44%. Compared with the strains that were genetically engineered but not domesticated, the total component titer of KLCNDX-02 fermented to produce erythromycin increased by 67.8%, and the main active ingredient erythromycin A increased by 70.34%. The mycin B component was reduced by 36.8%.
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (25)

  1. 一种红色糖多孢菌(Saccharopolyspora erythraea)，其名称为KLCNDX-01，保藏编号为CCTCC NO:M 2017647；或名称为KLCNDX-02，保藏编号为CCTCC NO:M 2017648。A species of Saccharopolyspora erythraea, named KLCNDX-01, with the accession number CCTCC NO: M 2017647; or the name KLCNDX-02, with the accession number CCTCC NO: M 2017648.
  2. 一种由保藏编号为CCTCC NO:M 2017647的菌株KLCNDX-01，或保藏编号为CCTCC NO:M 2017648的菌株KLCNDX-02培养得到的红色糖多孢菌。A strain of Saccharopolyspora erythraea cultured from strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  3. 一种红色糖多孢菌，其具有保藏编号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏编号为CCTCC NO:M 2017648的菌株KLCNDX-02的鉴定特征。A S. erythraea having the identification characteristics of strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  4. 一种放线菌培养物，其含有权利要求1、2或3所述的红色糖多孢菌。An actinomycete culture comprising the Saccharopolyspora erythraea of claim 1, 2 or 3.
  5. 根据权利要求4的放线菌培养物，其特征在于，所述培养物为生物学纯的培养物。The actinomycete culture according to claim 4, wherein the culture is a biologically pure culture.
  6. 根据权利要求4或5的放线菌培养物，其特征在于，所述培养物由菌株KLCNDX-01或KLCNDX-02培养获得；或所述培养物具有KLCNDX-01或KLCNDX-02的鉴定特征。The actinomycete culture according to claim 4 or 5, wherein the culture is obtained by culturing the strain KLCNDX-01 or KLCNDX-02; or the culture has the identifying characteristic of KLCNDX-01 or KLCNDX-02.
  7. 一种红色糖多孢菌或其培养物，其由保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02衍生获得。A P. erythropolis or a culture thereof, which is obtained by the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  8. 根据权利要求7所述的红色糖多孢菌或其培养物，所述红色糖多孢菌相对于衍生前具有同等或更高的红霉素生产能力，和/或在培养或发酵中不产生或产生更少的恶臭气味。The P. erythropolis or the culture thereof according to claim 7, wherein the S. erythraea has an equal or higher erythromycin production capacity before derivatization, and/or does not produce in culture or fermentation. Or produce less stench.
  9. 一种红色糖多孢菌，其与CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02具有相同的或基本相同的基因组序列，所述基本相同的基因组序列指菌株基因组内仅存在自然条件下增值传代中发生的自发突变。A Saccharopolyspora erythraea having the same or substantially the same genomic sequence as the strain KLCNDX-01 of CCTCC NO: M 2017647 or the strain KLCNDX-02 deposited under the accession number CCTCC NO: M 2017648, said substantially identical genome Sequence refers to the spontaneous mutations that occur in the value-added passages under natural conditions in the genome of the strain.
  10. 根据权利要求9的红色糖多孢菌，其特征在于，所述红色糖多孢菌与KLCNDX-01或KLCNDX-02具有基本相同的红霉素生产能力，和/或在培养或发酵中不产生或少产生恶臭性气味能力。The Saccharopolyspora erythraea according to claim 9, wherein the Saccharopolyspora erythraea has substantially the same erythromycin production capacity as KLCNDX-01 or KLCNDX-02, and/or does not produce in culture or fermentation. Or less odorous odor.
  11. 根据权利要求9或10的红色糖多孢菌，其特征在于，所述红色糖多孢菌基因 组序列与KLCNDX-01或KLCNDX-02具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。The Saccharopolyspora erythraea according to claim 9 or 10, wherein the S. erythraea genomic sequence has at least 90%, 91%, 92%, 93%, 94% with KLCNDX-01 or KLCNDX-02 , 95%, 96%, 97%, 98%, 99% or higher homology.
  12. 一种发酵方法，其以保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02进行发酵。A fermentation method which is carried out by the strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  13. 根据权利要求12所述的发酵方法，其特征在于，所述发酵包括：将低温保存的KLCNDX-01或KLCNDX-02菌株孢子进行斜面培养，斜面培养获得的孢子悬浮液经多级种子罐培养，最终接入发酵罐中发酵。The fermentation method according to claim 12, wherein the fermentation comprises: incubating the cryopreserved KLCNDX-01 or KLCNDX-02 strain spores, and the spore suspension obtained by the slant culture is cultured in a multi-stage seed tank. Finally, the fermentation is carried out in the fermenter.
  14. 根据权利要求13所述的发酵方法，其中所述多级种子罐培养为一级种子罐培养，或两级、三级或更多级种子罐连续培养；优选的，所述种子罐培养为三级培养。The fermentation method according to claim 13, wherein the multi-stage seed tank culture is a first-stage seed tank culture, or a two-stage, three-stage or more seed tank continuous culture; preferably, the seed tank culture is three Grade culture.
  15. 根据权利要求12-14任一所述的发酵方法，其中菌株的培养和发酵温度为28-37℃，优选30-35℃。The fermentation method according to any one of claims 12 to 14, wherein the culture and fermentation temperature of the strain is 28 to 37 ° C, preferably 30 to 35 ° C.
  16. 根据权利要求12-15任一所述的发酵方法，其发酵步骤包括：(1)红霉素种子液培养：①母斜面孢子制备：将冷冻保存在砂土管中的红霉素链霉菌孢子，接种于灭菌的斜面培养基上，在温度32-34℃条件下，培养7-9天；②子斜面孢子制备：将母斜面孢子，接种于灭菌的斜面培养基上，在温度32-34℃条件下，培养7-9天；③种子培养：将子斜面孢子，接种于灭菌的液体种子培养基中，经过三级种子扩大培养，得到红霉素种子液；(2)红霉素发酵：将红霉素种子液按10-20％体积比接种于灭菌的培养基中，通入无菌空气并开启搅拌，添加营养物质，在温度32-34℃条件下，培养7-10天，得红霉素发酵液。The fermentation method according to any one of claims 12-15, wherein the fermentation step comprises: (1) erythromycin seed liquid culture: 1 mother slant spore preparation: erythromycin streptavidin spores frozen in a sand tube, Inoculated on sterilized slant medium, cultured at a temperature of 32-34 ° C for 7-9 days; 2 slant spore preparation: mother slant spores, inoculated on sterilized slant medium at temperature 32- Incubation at 34 ° C for 7-9 days; 3 seed culture: seed slant spores, inoculated in sterilized liquid seed culture medium, and expanded by three-stage seed to obtain erythromycin seed liquid; (2) red mold Fermentation: The erythromycin seed solution is inoculated into the sterilized medium at a volume ratio of 10-20%, and the sterile air is introduced and stirred, the nutrient is added, and the culture is carried out at a temperature of 32-34 ° C. 10 days, get erythromycin fermentation broth.
  17. 一种生产红霉素或硫氰酸红霉素的方法，包括将保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02进行发酵，得到红霉素发酵液。A method for producing erythromycin or erythromycin thiocyanate, comprising fermenting a strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or a strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 to obtain red Fermentation broth.
  18. 根据权利要求17的方法，其特征在于，所述方法包括：1)培养所述微生物以获得细胞培养物；和2)从细胞培养物或微生物收集红霉素发酵液，通过对红霉素发酵液进行处理获得红霉素或硫氰酸红霉素成品。The method according to claim 17, wherein said method comprises: 1) cultivating said microorganism to obtain a cell culture; and 2) collecting erythromycin fermentation broth from a cell culture or microorganism, by fermenting erythromycin The liquid is treated to obtain a finished erythromycin or erythromycin thiocyanate.
  19. 根据权利要求17或18的方法，其特征在于，所述获得硫氰酸红霉素成品方法具体包括：将红霉素发酵液通过陶瓷膜过滤，菌渣经环保处理，滤液经过 纳滤膜进行浓缩得到红霉素浓缩液，向浓缩液中加入适量硫氰酸钠溶液，结晶得到硫氰酸红霉素粗品，通过二次结晶、干燥获得硫氰酸红霉素成品。The method according to claim 17 or 18, wherein the method for obtaining the finished erythromycin thiocyanate comprises: filtering the erythromycin fermentation broth through a ceramic membrane, the slag is subjected to environmental protection, and the filtrate is subjected to a nanofiltration membrane. Concentration to obtain erythromycin concentrate, adding appropriate amount of sodium thiocyanate solution to the concentrate, crystallization to obtain crude erythromycin thiocyanate, and obtaining erythromycin thiocyanate by secondary crystallization and drying.
  20. 一种降低红霉素发酵生产中恶臭气味的方法，包括以保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02进行发酵。A method for reducing malodor odor in erythromycin fermentation production, comprising carrying out fermentation with strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  21. 根据权利要求20的方法，所述方法还包括对发酵产生的尾气、废水和/或废渣进行除味处理的方法，以降低其中的恶臭气味；优选的，所述处理的步骤包括采用物理和/或化学的方法处理；更优选的，所述物理方法包括：吸附法(如：疏水性活性炭、硅胶、活性氧化铝、沸石分子筛)、等离子体法；所述化学的方法包括：吸收法(如：碱水吸收)、燃烧法(如：直接燃烧法、催化燃烧法、热力燃烧法)、光催化氧化。The method according to claim 20, further comprising a method of deodorizing the tail gas, waste water and/or waste generated by the fermentation to reduce the malodor odor therein; preferably, the step of treating comprises using physical and/or Or chemically treated; more preferably, the physical method comprises: adsorption method (such as: hydrophobic activated carbon, silica gel, activated alumina, zeolite molecular sieve), plasma method; the chemical method includes: absorption method (such as : Alkali water absorption), combustion method (such as: direct combustion method, catalytic combustion method, thermal combustion method), photocatalytic oxidation.
  22. 一种红色糖多孢菌发酵特征性尾气的识别方法，以保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02的发酵尾气作为标准发酵尾气，识别红色糖多孢菌发酵特征性尾气。A method for identifying characteristic tail gas of S. erythraea fermentation, using the fermentation tail gas of the strain KLCNDX-01 with the preservation number CCTCC NO: M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO: M 2017648 as the standard fermentation tail gas Identify the characteristic tail gas of the fermentation of S. cerevisiae.
  23. 保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02在红色糖多孢菌发酵特征性尾气识别中的应用。The application of the strain KLCNDX-01 with the accession number CCTCC NO:M 2017647 or the strain KLCNDX-02 with the accession number CCTCC NO:M 2017648 in the characteristic tail gas recognition of S. cerevisiae fermentation.
  24. 一种红色糖多孢菌标准发酵尾气，该尾气由保藏号为CCTCC NO:M 2017647的菌株KLCNDX-01或保藏号为CCTCC NO:M 2017648的菌株KLCNDX-02发酵收集获得。A standard fermentation tail gas of S. erythraea obtained by fermentation collection of strain KLCNDX-01 with the accession number CCTCC NO: M 2017647 or strain KLCNDX-02 with the accession number CCTCC NO: M 2017648.
  25. 根据权利要求24所述的标准发酵尾气在嗅辨识别中的应用。The use of a standard fermentation tail gas according to claim 24 for olfactory identification.
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