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Methylophilus pyrroloquinoline quinine synthesis-related gene cluster and encoding protein system thereof

A technology of pyrroloquinoline quinone and gene clusters of methyl-eating bacteria, which is applied in the field of new synthetic gene clusters of methyl-eating bacteria pyrroloquinoline quinone and its coding protein system, can solve the problems that other gene functions are not very clear, and achieve improved yield effect

Inactive Publication Date: 2011-10-19
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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  • Methylophilus pyrroloquinoline quinine synthesis-related gene cluster and encoding protein system thereof
  • Methylophilus pyrroloquinoline quinine synthesis-related gene cluster and encoding protein system thereof
  • Methylophilus pyrroloquinoline quinine synthesis-related gene cluster and encoding protein system thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Pick a single colony of Methylobacterium MP688 and inoculate it into MPQ liquid medium (0.2g MgSO in 1L MPQ medium) 4 ·7H 2 O, 3g(NH 4 ) 2 SO 4 , 1.4g KH 2 PO 4 , 3gNa 2 HPO 4 , Sterilize at 121℃, 20min. Then add 1 mL of vitamin solution, 1 mL of metal ion solution, and 9.5 mL of methanol. Metal ion solution: 30g iron citrate, 30g calcium chloride, 5g manganese chloride, 5g zinc sulfate, 0.5g copper sulfate, use H 2 Make the volume of O to 1L, and filter and sterilize after the solution is completely dissolved. Vitamin solution: 2mg biotin, 400mg calcium pantothenate, 400mgVB 1 , 400mg VB 6 , 200mg p-aminobenzoic acid, 2mg folic acid, 2g inositol, 400mg niacin, 200mg riboflavin, use H 2 Make the volume of O to 1L, filter and sterilize) in a test tube, culture and activate at 200r / min at 30°C for 48h. The volume ratio of 1% was inoculated into 500mL MPQ medium, 200r / min, 30°C for 48h. Extract microbial genome according to the literature ("Compiled Molecular Biology Exper...

Embodiment 2

[0032] Example 2 Sequencing of the genome of MP688 strain

[0033] MP688 was sequenced by SOLEX, with a total read length of 369Mbp and a genome coverage of 129 times. The genomic DNA of MP688 consists of a circular chromosome with a total length of 2,862,391bp and a G+C content of 55.44%. Using Glimmer 3.0 for gene prediction of bacterial genome, it is predicted that there are 2730 coding frames and the percentage of coding region is 90.41%. 46 tRNAs and 2 rRNA operons were found in the genome ( figure 2 ).

Embodiment 3

[0034] Example 3 PQQ synthetic gene in genome annotation of MP688 strain

[0035] According to the MP688 whole-genome determination sequence, Glimmer 3.0 software gene prediction, comparison with COG, KEGG, SWISSPROT, TrEMBL database, gene annotation, and manual correction. It was found that the gene sequence related to PQQ synthesis in the MP688 genome includes 1 pqqABCDE and 1 pqqFG gene cluster ( image 3 ).

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Abstract

The invention discloses methylophilus pyrroloquinoline quinine synthesis gene clusters and encoding protein systems thereof, wherein the gene clusters are pqqABCDE and pqqFG, respectively. The invention also discloses encoded pyrroloquinoline quinine synthesis gene clusters, and the nucleotide sequences are shown in SEQ ID No.1 and SEQ ID No.12. The gene clusters provided by the invention can promote the synthetic quantity of methylophilus pyrroloquinoline quinine, and the introduction of both the two gene clusters has synergistic interaction for the promotion of the synthesis of pyrroloquinoline quinine.

Description

Technical field [0001] The invention belongs to the field of microbial biotechnology, and specifically relates to a novel methylotrophic pyrroloquinoline quinone synthetic gene cluster and its coding protein system. Background technique [0002] Pyrroloquinoline quinone (PQQ) is the third oxidoreductase coenzyme discovered in the 1970s after pyridine nucleotides and riboflavin. It is considered a new B vitamin. So far, all organisms known to be able to synthesize PQQ are Gram-negative bacteria, of which methylotrophic bacteria have the highest yield. Studies have shown that PQQ has a variety of physiological functions such as stimulating the growth of certain microorganisms, animals and plants, preventing liver damage, promoting the production of nerve growth factors, regulating the level of free radicals in the body, and regulating immunity. It may be used for Alzheimer’s disease and cardiovascular disease. Prevention and treatment of diseases and diabetes. [0003] Known organi...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P17/18
Inventor 熊向华张惟材汪建华杨璐李淼鑫智静娟杨延新韦娜安佳佳
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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