Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A Gradient Strength Promoter of Gluconobacter oxidans

A Glucose Oxidation, Gradient Intensity Technique for Genetic and Metabolic Engineering

Active Publication Date: 2016-04-13
JIANGNAN UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few domestic studies on the transcriptional regulation level of the promoter of Gluconobacter oxydans, but most of them focus on the improvement and mutagenesis of the bacteria, and the research on the properties of important dehydrogenases

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A Gradient Strength Promoter of Gluconobacter oxidans
  • A Gradient Strength Promoter of Gluconobacter oxidans
  • A Gradient Strength Promoter of Gluconobacter oxidans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The selection of embodiment 1 promoter

[0023] Goxydans contain a variety of important dehydrogenases related to the metabolism of glucose, glycerol, sorbitol, etc. These enzymes may have potential strong constitutive expression promoters, such as sorbitol dehydrogenase (sorbitoldehydrogenase), membrane-bound aldehyde dehydrogenase Promoters of enzymes (aldehydedehydrogenase), membrane-bound glucose dehydrogenase (membrane-boundglucosedehydrogenase), etc. In addition, the P in the strongest Gluconobacteroxydans621H reported in the literature was selected tuf-621 H served as a control. According to the G.oxydansWSH-003 genome sequence information (GenBankaccessionNO.AHKI00000000), select all nucleotide fragments between the target gene and the ORF (open reading frame) of the previous gene, and then according to the promoter software ( http: / / www.softberry.com ) to evaluate the score of the promoter region, select a promoter with a higher score, and use EGFP as a repo...

Embodiment 2

[0024] Example 2 Construction of expression vector pBBR1MCS2-promter-egfp

[0025] According to the genome sequence information of G.oxydansWSH-003 (GenBankaccessionNO.AHKI00000000), fusion PCR primers were designed to amplify the highly scored promoter nucleotide sequence and the gene sequence of enhanced green fluorescent protein egfp (egfp gene sequence includes a terminator, The sequence is shown in SEQIDNO.7), and then connected by fusion PCR to obtain the promoter-egfp with restriction sites at both ends, perform T-A cloning and sequencing, and connect the positive clones with the same restriction sites to the wide The host type vector pBBR1MCS2 was constructed into the expression vector pBBR1MCS2-promter-egfp ( figure 1 ).

Embodiment 3

[0026] Example 3 Construction of Fluorescent Protein Recombination Detection Strain

[0027] The cloned expression vector pBBR1MCS2-promoter-egfp verified by sequencing was introduced into G.oxydansWSH-003 (preserved in the Industrial Microbiology Resource and Information Center of Chinese Universities, Jiangnan University, No. CICIM-CUB7004) by three-parent hybridization. E.coliJM109 containing the recombinant expression vector was used as the donor bacterium, and E.coliHB101 containing the conjugation helper plasmid pRK2013 was used as the auxiliary bacterium. G.oxydansWSH-003 grows to OD 600 =0.9, E.coli grown to OD 600 = 0.8. Take 1ml each of E.coliJM109 and E.coliHB101, mix them, centrifuge, mix and centrifuge with 2ml LB resuspended bacteria and 4ml G.oxydansWSH-003, and use 0.8ml Y-S medium (yeast extract 20g / L, sorbitol 80g / L ) to resuspend the bacteria, spread the bacteria solution on a Y-S solid medium plate containing filter paper but not containing antibiotics, ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a gradient intensity promoter of gluconobacter oxydans and belongs to the field of genetic and metabolic engineering. Sequences between constitutively expressed genes and previous genes are selected from the gluconobacter oxydans and preliminarily screened by promoter prediction software, an obtained high-score potential strong promoter is subjected to qualitative and quantitative detection through a fluorescent microscope and a fluorescence microplate reader respectively by using enhanced green fluorescence protein (EGFP) as a reporter gene, and finally promoters with an intensity sequence of P[tuf-WSH003]>P[tuf-621H]>P[sldh]-P[psldh]-P[sod]>P[aldh] are selected from G.oxydans WSH-003. A series of gradient promoters and a novel strong promoter are screened out, and great help is provided for genetic and metabolic engineering reform of the industrial production strain G.oxydans WSH-003, especially adjustment of gene transcription level.

Description

technical field [0001] The invention relates to a gradient-strength promoter of Gluconobacter oxydans, belonging to the field of gene and metabolic engineering. Background technique [0002] Gluconobacter oxydans (Gluconobacteroxydans) WSH-003 can use D-sorbitol (D-sorbitol) as a high-yielding strain of L-sorbose (L-sorbose) as a substrate, and is highly tolerant to D-sorbitol and L-sorbose, It is widely used in the production of industrial vitamin C. With the successive publication of the genome sequencing of Ketogulonicigenium vulgareWSH-001 (a high-yield 2-KLG production strain with sorbose as substrate) and G.oxydansWSH-003, the comprehensive application of genetic engineering and bioinformatics has enabled the construction of vitamin C one-step bacteria. be greatly promoted. Among them, through the construction of one-step genetic engineering bacteria, the direct production from D-sorbitol to 2-KLG can be realized in shake flask fermentation, but the yield needs to be...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/74C12N1/21C12R1/01
Inventor 周景文陈坚胡于东堵国成
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com