Method for increasing streptomyces secondary metabolite yield

A technology for secondary metabolites and yield, applied in the fields of genetic engineering and fermentation engineering, can solve problems such as increased costs and difficulty in product purification, and achieve the effect of increased yield

Active Publication Date: 2018-03-16
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the industrial fermentation process, the use of inducers will increase the cost and the difficulty of product purification

Method used

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  • Method for increasing streptomyces secondary metabolite yield
  • Method for increasing streptomyces secondary metabolite yield
  • Method for increasing streptomyces secondary metabolite yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1 The construction of a kind of genetically engineered bacteria

[0016] Use primers P1-F / P1-R and plasmid pIJ8660::BsaI-sfgfp as a template to amplify the plasmid backbone; use the Streptomyces coelicolor M145 genome as a template and primers actII4-F / actII4-R to amplify actII-orf4 Gene, and the above-mentioned linear plasmid backbone using the Gibson assembly method to obtain the plasmid pActII-sfgfp. Using the Streptomyces coelicolor M145 genome as a template, the corresponding primers were used to amplify the physiological promoter. After digestion with BglII and EcoRV, it was digested with the corresponding enzymes and ligated with the plasmid pActII-sfgfp to obtain the plasmid pPn-actII-sfgfp in which the expression of the actII-orf4 gene is controlled by a physiological promoter. Using the plasmid pGCymRP21 as a template, use the primers cumate-F / cumate-R to amplify the cumate-inducible promoter, digest it with BglII and EcoRV, and then connect it int...

Embodiment 2

[0018] Embodiment 2 Construction of a kind of genetically engineered bacteria

[0019] Using the plasmid pIJ8660::BsaI-sfgfp as a template, the plasmid backbone was amplified using primers P1-F / P1-R. Using Streptomyces chamobilis genome as a template and primers otcR-F / otcR-R, the otcR gene was amplified, and combined with the above-mentioned linear plasmid backbone using the Gibson assembly method to obtain the plasmid pOtcR-sfgfp. Using Streptomyces coelicolor as a template, using the corresponding primers to amplify the appropriate physiological promoters, the plasmid pOtcR-sfgfp and these promoters were digested with BglII and EcoRV and ligated to obtain the plasmid pPn that uses the physiological promoter to control the otcR gene -otcR-sfgfp. Similarly, the digested kasO* and cumate promoter fragments were connected into the digested linear plasmid pOtcR-sfgfp to obtain plasmids pK-otcR-sfgfp and pCumate- otcR-sfgfp. Plasmids pPn-otcR-sfgfp and pCumate-otcR-sfgfp were ...

Embodiment 3

[0029] Example 3 Production of secondary metabolites actin and jodomycin B by fermentation and analysis of results

[0030] Utilize the genetically engineered bacteria M145-Pc prepared in Example 1, M145-OA, M145-Pn to ferment and produce the secondary metabolite actinhodine, the specific process is: Streptomyces coelicolor engineering strains are placed on MS solid plate (2% Mannitol, 2% soybean flour, 2% agar) were cultured at 28°C. After collecting the spores, press 4×10 8 The concentration of spores / 100ml was inoculated into 50mL SMM medium (81.9ml PEG6000 (6.1%, w / v), 2.5ml magnesium sulfate (24g / L), 10ml TES buffer (0.25M, pH7.2) , 2ml glucose (50%, w / v), 0.1ml trace elements (zinc sulfate, ferrous sulfate, manganese chloride, calcium chloride, sodium chloride each 0.1g / L), 1ml casein hydrolyzate (20% , w / v), 25ml glycine (20%, w / v)) was fermented in a 250mL shake flask at 28°C, 250rpm. Collect the bacteria, centrifuge at 10,000×g at 4°C for 3 minutes, collect the sup...

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Abstract

Belonging to the genetic engineering and fermentation engineering field, the invention provides a method for increasing streptomyces secondary metabolite yield. The method includes the steps of: utilizing an induction promoter to control the expression of a target secondary metabolite biosynthetic gene cluster, and determining the optimal induction condition by means of a response surface model; conducting transcriptome analysis to screen a physiological promoter with consistent control behavior to the induction promoter; and finally, preparing plasmid utilizing the physiological promoter to control the expression of the target secondary metabolite biosynthetic gene cluster, and transferring the plasmid into host bacteria for fermentation. The method provided by the invention can get rid of dependency on an inducer, and realizes self-regulation of target biosynthetic gene cluster expression and increase of the target product yield. The method provided by the invention regulates the expression of secondary metabolite biosynthetic gene cluster from the dimensions of time and intensity, makes the expression fit the physiological metabolic behaviors of the host, and is very important for increasing the yield of the target secondary metabolite in streptomyces.

Description

technical field [0001] The invention belongs to the field of genetic engineering and fermentation engineering, and in particular relates to a method for increasing the output of streptomyces metabolites. Background technique [0002] Streptomyces can produce a large number of secondary metabolites with important biological activities, many of which can be used as clinical, veterinary and agricultural drugs. The development of methods to increase the production of target secondary metabolites is an important research content of Streptomyces. During the fermentation process, Streptomyces undergoes a significant metabolic transformation process from rapid strain growth to massive accumulation of secondary metabolites, which is controlled by the complex and rigorous regulatory system of Streptomyces. However, the regulatory goal of this regulatory network is to optimize the survival of the strain, not the goal of maximizing the yield of metabolic engineering products. At prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/67C12N15/65C12P17/16C12P15/00C12P19/60C12R1/465C12R1/61
CPCC12N15/65C12N15/67C12P15/00C12P17/162C12P19/60
Inventor 李珊珊向文胜王为善王俊阳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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