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

A kind of Aureobasidium pullulans polyketide synthase gene and its application

A technology of polyketide synthase and Aureobasidium pullulans, which is applied in the field of genetic engineering, can solve the problems of limited sources and few polyketide synthases, and achieve the effects of increasing production, wide application range, and optimizing reaction conditions

Active Publication Date: 2017-09-29
SHANDONG UNIV +1
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the reports on polyketide synthase gene and DHN melanin synthesis are mostly concentrated in bacteria, and there are few reports on polyketide synthase gene and DHN melanin synthesis in fungi. Aureobasidium pullulans belongs to mold in biological taxonomy, and is a fungal A type of polyketide synthase related to DHN melanin synthesis in Aureobasidium pullulans
Moreover, there are few polyketide synthases for melanin production research in the world, and the sources are limited. Therefore, it is necessary to develop new enzyme sources for efficiently synthesizing melanin.

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 kind of Aureobasidium pullulans polyketide synthase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: PCR amplification of the Aureobasidium pullulans polyketide synthase gene

[0036] Use 5 mL of Aureobasidium pullulans culture solution with the strain number As3.3984 from the General Microbiology Center (CMGCC) of China Committee for Culture Collection of Microorganisms, and use the RNA extraction kit to extract total RNA. The extraction steps refer to the RNA extraction reagent of Takara Company box instructions. The extracted total RNA was used as a template to carry out reverse transcription with a reverse transcription kit to obtain cDNA. The reverse transcription steps refer to the instructions of the reverse transcription kit from Takara Company.

[0037] The nucleotide sequences of polyketide synthase genes of different strains of the same genus were retrieved from GenBank, and a pair of primers F-I and R-I were designed. The primer sequences are as follows:

[0038] F-I: 5'-ATGTCTAACGTTCTTCTTTT-3' SEQ ID NO.3;

[0039] R-I: 5'-GATCTGAAGACCTTCTTGG...

Embodiment 2

[0042] Example 2: Expression of the Aureobasidium pullulans polyketide synthase gene in Pichia pastoris SMD1168H

[0043] Primers F-I-A and R-I-A were designed according to the sequence shown in SEQ ID No.1, respectively introduced into the SacII and XbaI restriction sites (shown underlined) that can be inserted into the pGAPZα-A plasmid (purchased from Invitrogen, USA), and the sequences are as follows

[0044] F-I-A: 5'-GCG CCGCGG CATGTCTAACGTTCTTCTTTT-3' SEQ ID NO. 5;

[0045] R-I-A: 5'-GCG TCTAGA ATGATCTGAAGACCTTCTTGGA-3' SEQ ID NO. 6;

[0046] Using the reverse-transcribed Aureobasidium pullulans cDNA as a template, the above primers were used for PCR amplification. PCR amplification conditions and PCR product recovery method were the same as in Example 1. After the PCR product was recovered, NdeI and SalI (NEB, USA) were added to digest at 37°C for 1 hour to purify the DNA, and the pGAPZα-A plasmid vector was treated with the same digestion method. The prepared en...

Embodiment 3

[0050] Example 3: Synthesis of melanin catalyzed by Aureobasidium pullulans polyketide synthase in vitro

[0051] The recombinant Pichia pastoris SMD1168H-alb obtained in Example 2 was inoculated into YPD liquid medium, cultured on a shaker at 30° C. for 3 days, and the rotation speed was 200 rpm. The cultured fermentation broth was centrifuged at 8000rpm for 5 minutes to collect the supernatant, ammonium sulfate was slowly added to the supernatant to a concentration of 80%, and the protein precipitate was collected by centrifugation at 8000rpm for 5 minutes. The protein precipitate was redissolved with 50 mM sodium phosphate buffer (pH 8.0) and then desalted and concentrated with a 100KD ultrafiltration tube. Finally, the protein solution was concentrated 1000 times in volume compared with the above fermentation supernatant. The protein solution obtained through desalination and concentration is the recombinant polyketide synthase crude enzyme solution.

[0052] Aureobasidiu...

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 relates to a polyketide synthase gene, in particular relates to a polyketide synthase gene related to melanin synthesis of fungi and an application thereof, and belongs to the technical field of gene engineering. The nucleotide sequence of the polyketide synthase gene is as shown in SEQ ID No.1. The amino acid sequence of the polyketide synthase coded by the gene is as shown in SEQ ID NO.2. The polyketide synthase gene can be used for synthesizing melanin in vitro. Through detection, the content of melanin in in-vitro enzyme reaction liquid is 35.6g / L.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a polyketide synthase gene of Aureobacidium pullulans and an application thereof. Background technique [0002] Melanin is a protective pigment that exists widely in organisms. It has functions such as biological semiconductors, scavenging free radicals, preventing ultraviolet radiation, and chelating heavy metal ions. It plays an important role in many fields such as daily cosmetics industry, food industry, pharmaceutical industry, and agriculture. use. According to the source, melanin can be divided into animal melanin, plant melanin and microbial melanin; according to its chemical composition, it can be divided into eumelanin, phaeomelanin and allomelanin. Eumelanin contains carbon, hydrogen, oxygen, nitrogen, does not contain sulfur atoms, and is dark brown or black in color. It mainly exists in animals, poultry black or blue feathers, eyes, skin and related ti...

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
IPC IPC(8): C12N15/53C12N9/04C12N15/81C12N1/19C12P17/10C12R1/84
CPCC12N9/0006C12P17/10C12Y101/01252
Inventor 刘飞张金华王凤山朱希强于林艳张林军袁超张祥奎侯重文凌沛学
Owner SHANDONG 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