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Gluconobacter oxydans-pyrroloquiniline quinone (PQQ) synthetic protein system and gene cluster for coding same

A technology of pyrroloquinoline quinone and gene clusters, applied in genetic engineering, plant gene improvement, bacterial peptides, etc., can solve problems such as unclear functions of other genes, and achieve the effect of improving transformation efficiency

Inactive Publication Date: 2011-08-10
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to confirming that the polypeptide encoded by pqqA is the precursor of PQQ synthesis and pqqC is the enzyme that catalyzes the last step in the synthesis reaction of PQQ, the functions of other genes are still not very clear.

Method used

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  • Gluconobacter oxydans-pyrroloquiniline quinone (PQQ) synthetic protein system and gene cluster for coding same
  • Gluconobacter oxydans-pyrroloquiniline quinone (PQQ) synthetic protein system and gene cluster for coding same
  • Gluconobacter oxydans-pyrroloquiniline quinone (PQQ) synthetic protein system and gene cluster for coding same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Extraction of Gluconobacter oxydans 1.110 Genomic DNA

[0023] Pick a single colony of Gluconobacter oxidans 1.110 and inoculate it into 5 mL of HJY liquid medium (1L medium contains 15g sorbose, 3g yeast extract, 3g corn steep liquor, 15g peptone, 4g urea, 2g glucose, 0.2g MgSO 4 12H 2 O, 4g CaCO 3 , 5gKH 2 PO 4 , pH 6.8) in test tubes, cultured and activated at 200r / min, 30°C for 48h. Inoculate into 500mL HJY medium at a volume ratio of 1%, culture at 30°C for 48h at 200r / min. The microbial genome was extracted according to the literature ("Guidelines for Molecular Biology Experiments" by F. Osper, R. Brent, R.E. Kingston, translated by Yan Ziying and Wang Hailin. 2.4 (39~40)). Genomic DNA requires an OD value between 1.8 and 2.0; sample concentration > 100ng / μL ( figure 1 ).

Embodiment 2

[0024] Example 2 Genome sequencing of Gluconobacter oxydans 1.110

[0025] Gluconobacter oxydans 1.110 was sequenced by SOLEXA, with a total read length of 221Mbp and a genome coverage rate of 67.2 times. The total genome size of Gluconobacter oxydans 1.110 is 3,288,404bp, and the G+C content is 61.76%. Using Glimmer 3.0 for gene prediction of the bacterial genome, there are 3359 coding frames predicted, and the percentage of coding regions is 91.12%. There are 59 tRNA and 5 rRNA operons found in the genome.

Embodiment 3

[0026] Example 3 The PQQ synthetic gene in the genome annotation of Gluconobacter oxydans 1.110

[0027] According to the whole genome sequence of Gluconobacter oxydans 1.110, the gene was predicted by Glimmer 3.0 software, compared with COG, KEGG, SWISSPROT, TrEMBL database, gene annotation was carried out, and manual correction was carried out. It was found that the PQQ synthesis gene pqqABCDE in the genome of Gluconobacter oxydans 1.110 was arranged in a gene cluster ( figure 2 ).

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Abstract

The invention discloses a pyrroloquiniline quinone (PQQ) synthetic gene cluster and a coded protein system thereof. The protein system consists of the following proteins: 1) proteins shown in SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 and SEQ ID No.11; or 2) proteins which are derived from the proteins 1) with the amino acid sequences shown in SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 and SEQ ID No.11 through replacing, deleting or adding one or more of amino acid residues, and have the same activities. The invention also provides the gene cluster for coding the protein system, wherein the sequence of the gene cluster is shown in SEQ ID No.1. The PQQ synthetic gene cluster and coded protein system provided by the invention play important roles in the PQQ biosynthesis, the improvement and construction of PQQ producing strains, the increase of small bacterium saccharic acid conversion efficiency and the construction of alcohol saccharic acid one-step conversion engineering bacteria.

Description

technical field [0001] The invention relates to a novel pyrroloquinoline quinone synthetic gene cluster and its coding protein system. Background technique [0002] Vitamin C (vatamin C, Vc) is a water-soluble vitamin, also known as L-ascorbic acid (L-ascorbic acid), which can participate in various hydroxylation reactions and redox reactions in the body, and is an important redox compound in cell metabolism , plays an essential physiological role in the human body, and is widely used in many fields such as medicine, cosmetics, food and feed. With the improvement of people's living standards and the continuous development of the use of vitamin C, the market demand for vitamin C is increasing day by day. [0003] The production of vitamin C in the early days mainly used the chemical synthesis of "Leys method" (Helvetica chimica Acta, volume 17, page 311, 1934). This method has good product quality and high conversion efficiency, but the process is complicated and the enviro...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N5/10C12N15/63C12P17/18
Inventor 熊向华张惟材汪建华杨延新智静娟杨璐李淼鑫韦娜安佳佳
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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