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Actinoplanes spp. genetic manipulation system

A kind of acarbose, genetic manipulation technology, applied in the field of genetic manipulation system, can solve the problems of research limitation, lack of genetic manipulation system, etc.

Active Publication Date: 2017-04-19
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of an effective genetic manipulation system, the research on acarbose biosynthesis and its regulatory mechanism, rational design and transformation of high-yielding strains is limited

Method used

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  • Actinoplanes spp. genetic manipulation system
  • Actinoplanes spp. genetic manipulation system
  • Actinoplanes spp. genetic manipulation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The establishment and optimization of embodiment 1 mycelia conjugation transfer system

[0047] A. Antibiotic susceptibility analysis

[0048] After activating the three strains of acarbose-producing bacteria Actinoplanes sp.SE50, SE50 / 110 and HDC preserved in the laboratory, take fresh mycelia and streak them on a plate containing different antibiotics, culture them at 30°C for 3-5 days, and observe the bacteria of growth. The results are shown in Table 1. Actinoplanes spp. is insensitive to thiostrepton (Thio), spectinomycin (Spec) and trimethoprim (TMP), but to apramycin (Apr), kanamycin (Kan), chloramphenicol Sensitivity to hormone (Cml), streptomycin (Str) and nabumexate.

[0049] Table 1 Sensitivity analysis of Actinoplanes sp. to commonly used antibiotics

[0050]

[0051] Note: "+" means sensitive; "-" means insensitive;

[0052] B. Culture of mycelium

[0053] Streak the hyphae preserved in glycerol into the activation medium, after culturing for 48 ho...

Embodiment 2

[0066] Example 2. Enhanced expression of acarbose biosynthesis gene cluster

[0067]A. Construction and screening of a fosmid plasmid containing a complete acb cluster: extract the genomic gDNA of Actinoplanes sp., blow it with a yellow pipette tip, randomly interrupt the gDNA, fill in the end, and use low melting point agar after 5' phosphorylation Sugar recovery fragments with a length of 40kb were ligated and embedded with Copycontrol pCC1FOS vector, and transfected into EPI300-TI R Plating cells, using the genes (acbM, acbZ, acbD) located in the middle and both ends of the acb cluster as selection markers, were screened to obtain the Fosmid plasmid 31F6 containing the complete acarbose biosynthesis gene cluster. The attP-int-oriT-aac(3) IV element from pSET152 was integrated into fosmid plasmid 31F6 by PCR targeting to obtain pLQ666, which was transformed into E.coli ET12567 (pUZ8002).

[0068] Conjugative transfer of B.E.coli ET12567::pLQ666 to Actinoplane spp.

[0069]...

Embodiment 3

[0094] Example 3. Construction of replicating plasmid pLQ-752

[0095] A. Replacement of thiostrepton resistance selection marker

[0096] Using apr-1278-F / R as a primer and pSET152 as a template, the apramycin resistance gene aac(3)IV and its promoter fragment were obtained by PCR amplification, with AflII and NheI restriction enzymes at both ends At the same time, using OriT-F / R as primers and pJTU1278 as a template, amplify a 986bp fragment including the oriT element, two segments containing NheI and AflII restriction sites, and insert them with NdeI after digestion and pJTU1278 digested with NheI to obtain recombinant plasmid pLQ750.

[0097] B. Insert reverse screening marker CodA(sm)

[0098] The codA(sm) gene was amplified from pWHU2573 with codA-F / R primers, both ends contained AflII restriction sites, and inserted into pLQ750 after AflII restriction to obtain recombinant plasmid pLQ752. The primer sequences are shown in Table 6, and the construction process is as f...

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Abstract

An Actinoplanes spp. genetic manipulation system introduces a phi derivative plasmid and a pIJ101 derived replicating plasmid into Actinoplanes spp. In a mycelium conjugal transfer mode in order to realize gene expression and knockout. The Actinoplanes spp. strain genetic manipulation system is established and systemically optimized, and a genetic manipulation tool suitable for like strains is developed and is successfully applied to accurate genome editing to improve the acarbose output and lays a foundation for realization of rational design and reconstruction of the Actinoplanes spp. strain. The system is adopted to reinforce expression of acarbose biosynthesis gene cluster (acb cluster) in order to increase of the final fermentation output of acarbose by 35%.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to a genetic operation system of the acarbose-producing bacterium Actinoplanes spp. Background technique [0002] Acarbose is a pseudotetrasaccharide of C7 cyclic polyols, which can compete with α-glucosidase and reduce the effect of α-glucosidase on disaccharides and oligosaccharides in small intestinal wall cells. And the hydrolysis of polysaccharides to reduce the production, absorption and utilization of glucose and fructose, so as to achieve the purpose of reducing postprandial blood sugar in patients with type 2 diabetes. Because of its good pharmacokinetic properties and low toxicity, it has become an ideal drug for the treatment of type Ⅱ diabetes, and its sales share has surpassed traditional hypoglycemic drugs such as sulfonylureas and biguanides. [0003] At present, the industry mainly uses Actinoplanes spp. to ferment and produce acarbose. The gene cluster responsible for i...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/03C12P19/26C12R1/045
CPCC07K14/365C12N9/00C12N15/03C12N15/74C12N2800/60C12P19/26
Inventor 白林泉赵芹芹
Owner SHANGHAI JIAO TONG UNIV
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