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Gradient intensity promoter of gluconobacter oxydans

A technique for oxidizing glucose, a strong promoter, used in the field of genetic and metabolic engineering

Active Publication Date: 2014-08-06
JIANGNAN UNIV
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  • 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

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  • Gradient intensity promoter of gluconobacter oxydans
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  • Gradient intensity promoter of gluconobacter oxydans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Selection of promoters

[0023] G oxydans 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 (sorbitol dehydrogenase), membrane-bound aldehyde Promoters of dehydrogenase (aldehyde dehydrogenase), membrane-bound glucose dehydrogenase (membrane-bound glucose dehydrogenase) and the like. In addition, the P in the strongest Gluconobacter oxydans 621H reported in the literature was selected tuf-621 H served as a control. According to the G.oxydans WSH-003 genome sequence information (GenBank accession NO.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 ...

Embodiment 2

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

[0025] According to the genome sequence information of G.oxydans WSH-003 (GenBank accession NO.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 Terminator, the sequence is shown in SEQ ID NO.7), and then connected by fusion PCR to obtain promoter-egfp with restriction sites at both ends, perform T-A cloning and sequencing, and connect the positive clones with the same enzyme The broad-hosted vector pBBR1MCS2 with cutting sites 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. oxydans WSH-003 (preserved in the Industrial Microbiology Resource and Information Center of Chinese Universities, Jiangnan University, No. CICIM-CU B7004) by the method of three-parent hybridization. E.coli JM109 containing the recombinant expression vector was used as the donor bacterium, and E.coli HB101 containing the conjugative helper plasmid pRK2013 was used as the auxiliary bacterium. G.oxydans WSH-003 grows to OD 600 =0.9, E.coli grown to OD 600 = 0.8. Take 1ml of E.coli JM109 and E.coli HB101, mix them, centrifuge, mix and centrifuge with 2ml LB resuspended bacteria and 4ml G.oxydans WSH-003, and use 0.8ml Y-S medium (yeast extract 20g / L, Sorbet Alcohol 80g / L) to resuspend the bacteria, spread the bacteria solution on a Y-S solid medium plate containing filter paper but not c...

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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 (Gluconobacter oxydans) WSH-003 can use D-sorbitol (D-sorbitol) as a high-yielding strain of L-sorbose (L-sorbose) and is highly tolerant to D-sorbitol and L-sorbose , is widely used in the production of industrial vitamin C. With the successive publication of the genome sequencing of Ketogulonicigenium vulgare WSH-001 (a high-yield 2-KLG production strain using sorbose as a substrate) and G.oxydans WSH-003, the comprehensive application of genetic engineering and bioinformatics has made vitamin C one step ahead. The construction of bacteria is 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 improved...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/74C12N1/21C12R1/01
Inventor 陈坚周景文胡于东堵国成
Owner JIANGNAN UNIV
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