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Recombinant oxalate decarboxylase obtained through mycelial fungus host cell expression

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: 2018-09-28
WUHAN KANGFUDE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

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  • Recombinant oxalate decarboxylase obtained through mycelial fungus host cell expression
  • Recombinant oxalate decarboxylase obtained through mycelial fungus host cell expression
  • Recombinant oxalate decarboxylase obtained through mycelial fungus host cell expression

Examples

Experimental program
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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 A. reesei was optimized according to the codon prefer...

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...

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Abstract

The invention discloses recombinant oxalate decarboxylase obtained through mycelial fungus host cell expression, construction method of recombinant bacterial strains, and a production method and applications of recombinant enzymes, and belongs to the technical field of gene engineering. The recombinant mycelial fungus host cell comprises one or a plurality of copies of oxalate decarboxylase expression cassette arranged in the genome through integration; the oxalate decarboxylase expression cassette comprises a promoter, a signal peptide coding sequence, an oxalate decarboxylase coding gene, and a terminator. The host cells can be constructed through two methods including random integration or site specific integration. The invention also discloses an optimized culture medium formula for different recombinant mycelial fungus host cells. The production of oxalate decarboxylase is realized through expression cassette construction, vector construction, host cell construction, and adjustingof a final culture active formula, so that the final yield and enzyme activity are increased greatly, and a difficult problem in the prior art that large-scale industrialized artificial production ofoxalate decarboxylase is impossible to realize is solved.

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

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Application Information

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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
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