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

A recombinant oxalate decarboxylase expressed in filamentous fungal host cells

A technology of oxalate decarboxylase and host cells, applied in the field of recombinant oxalate decarboxylase, can solve the problem that oxalate decarboxylase cannot be effectively recombinantly expressed

Active Publication Date: 2020-12-01
WUHAN KANGFUDE BIOTECH CO LTD
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the technical problem that the oxalate decarboxylase derived from fungi cannot be effectively recombinantly expressed in the prior art, the present invention has undergone long-term and large-scale experiments and efforts in the early stage, tried various expression systems, and used various biotechnological means , in order to achieve effective recombinant expression of fungal-derived oxalate decarboxylase

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 recombinant oxalate decarboxylase expressed in filamentous fungal host cells
  • A recombinant oxalate decarboxylase expressed in filamentous fungal host cells
  • A recombinant oxalate decarboxylase expressed in filamentous fungal host cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Codon optimization and artificial synthesis of oxalate decarboxylase (OXDC) gene

[0080] The present inventors have found through a large number of experimental studies that it can be used in the expression system of filamentous fungi to express oxalate decarboxylase derived from eukaryotes, preferably from tea tree mushroom, poplar mushroom, enoki mushroom, versicolor versicolor, brown rot fungus, aspergillus auricula, bisporus Oxalate decarboxylase from fungi such as mushrooms and jinfu mushrooms.

[0081] The gene encoding oxalate decarboxylase can be derived from Tea Tree Mushroom, the amino acid sequence of the oxalate decarboxylase is the amino acid sequence shown in SEQ ID NO.1, wherein the signal peptide sequence of the oxalate decarboxylase is 1- The 19th amino acid sequence, the mature peptide sequence is the 20-470th amino acid sequence shown in SEQ ID NO.1.

[0082] The OXDC gene derived from Tea tree mushroom was optimized according to the codo...

Embodiment 2

[0083]Embodiment 2: Trichoderma reesei Rut-C30 (pyr4 - ) construction of auxotrophic strain

[0084] The filamentous fungal host cells used to express the eukaryotic OXDC in the eukaryotic system are selected from Aspergillus, Coriolus, Mucor, White Rot, Acremonium, Cryptococcus, Fusarium, and Humicola Genus, Myceliophthora, Aureobasidium, Trametes, Pleurotus, Neurospora, Penicillium, Paecilomyces, Phaneroderma, Tobacillus, Cerexe, Thielavia Cells of the genus Coprinus, Chrysosporium, Schizophyllum, Coprinus, Magnaporthe oryzae, Neomystium, Curvularia, Talaromyces, Thermoascomyces, or Trichoderma, or their organisms Sexual or synonymous cells, but not limited thereto.

[0085] The Trichoderma host cell is Trichoderma harzianum, Trichoderma konningen, Trichoderma reesei, Trichoderma longistanum and Trichoderma viride, preferably Trichoderma reesei and Trichoderma viride. Take Trichoderma reesei as an example below to illustrate the present invention.

[0086] 1. Trichoderma...

Embodiment 3

[0109] Example 3: Construction of oxalate decarboxylase random integration recombinant expression vector

[0110] 1. Construction of random integration inducible expression vector pMGU-cbh1-TRA2

[0111] Construction of vector pMGU:

[0112]Using the plasmid vector pMDT05 prepared in Example 2 as a template, use the primers F1 and R1 in Table 3 as the upstream and downstream primers to PCR amplify a vector backbone fragment of about 6.6 kb, use the gel to recover and use the restriction endonuclease DpnI Digest for 3 hours, recover the target fragment, and set aside.

[0113] With reference to the method for Trichoderma reesei genome extraction in Example 2, extract Aspergillus niger CICC2439 genomic DNA, use this genomic DNA as a template, amplify about 2.9kb of Aspergillus niger pyrG with primers pyrG-F and pyrG-R in Table 3 Gene expression frame, gel recovery target fragment, spare. Using Trichoderma reesei genomic DNA as a template, the primers Pcbh-DR-F and Pcbh-DR-R i...

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

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a recombinant oxalate decarboxylase expressed by a filamentous fungal host cell, a construction method of the recombinant bacterial strain, a production method and application of the recombinant enzyme, and belongs to the technical field of genetic engineering. The recombinant filamentous fungal host cell comprises one or more copies of an oxalate decarboxylase expression cassette integrated in its genome, the oxalate decarboxylase expression cassette including a promoter, a signal peptide coding sequence, an oxalate decarboxylase coding gene and a terminator. The above-mentioned host cells can be constructed by random integration or site-specific integration. In addition, the present invention also optimizes the medium formulations for different recombinant filamentous fungal host cells. The production of oxalate decarboxylase, through the construction of expression cassettes, vector construction, host cell construction and the adjustment of the final culture positive formula, the final yield and enzyme activity are greatly improved, effectively solving the problem of oxalate decarboxylase in the prior art. Artificial production cannot be industrialized on a large scale.

Description

[0001] This application claims the priority of the Chinese patent application submitted to the China Patent Office on March 7, 2017, with the application number 201710130999.5, and the title of the invention is "Expression cassette, strain, method and application for expressing oxalate decarboxylase in eukaryotic microorganisms" , the entire contents of which are incorporated in this application by reference. technical field [0002] The invention relates to the technical field of genetic engineering, in particular to a recombinant filamentous fungal host cell capable of efficiently expressing oxalate decarboxylase, a recombinant oxalate decarboxylase, a production method and application of the recombinant oxalate decarboxylase. Background technique [0003] Oxalic acid, also known as oxalic acid, is a metabolite of organisms and widely exists in plants, animals and fungi in the form of oxalate. Many foods for humans and other mammals, such as spinach, strawberries, beets, c...

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): C12N9/88C12N1/15C12N15/80C12R1/645
CPCC12N9/88C12N15/80C12Y401/01002C12N1/14
Inventor 汪卫汪小锋刘艳红黄荷陈火晴陈先桥
Owner WUHAN KANGFUDE BIOTECH CO LTD
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