Trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof

A host cell, Trichoderma reesei technology, applied in the direction of biochemical equipment and methods, glycosylase, the use of vectors to introduce foreign genetic material, etc.

Pending Publication Date: 2015-08-26
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Aspergillus fumigatus is highly allergic and pathogenic to humans and plants

Method used

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  • Trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof
  • Trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof
  • Trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof

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Experimental program
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preparation example Construction

[0220] 3. Preparation of AfGA and its variants

[0221] AfGA or a variant thereof can be isolated from the host cell, eg, by secretion of the AfGA or variant from the host cell. Cultured cell material comprising AfGA or a variant thereof may be obtained following secretion of AfGA or a variant thereof from a host cell. AfGA or variants thereof are optionally purified prior to use. The AfGA gene can be cloned and expressed according to methods well known in the art. Suitable host cells include bacterial, plant or yeast cells, such as filamentous fungal cells. Particularly useful host cells include Trichoderma reesei. T. reesei host cells express AfGATR at higher or at least comparable levels compared to naturally expressed AfGA A. fumigatus.

[0222] In some embodiments, AfGA is heterologously expressed in the host at at least 10 g / L. In some embodiments, AfGA is heterologously expressed at at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 ...

example 1

[0432] Example 1: Cloning of AfGA1 .

[0433] Genomic DNA of A. fumigatus Af293 was purchased from the Fungal Genetics Stock Center, Kansas City, MO (FGSC A1100). The genome of Aspergillus fumigatus was sequenced. The nucleotide sequence of the AfGA1 gene (within the disclosed genome in NCBI reference sequence NC_007195), and the predicted glucan 1,4-alpha-glucosidase (NCBI accession number XP_749206) encoded by the AfGA1 gene were obtained in the NCBI database ) amino acid sequence. AfGA1 is homologous to other fungal glucoamylases as determined from BLAST searches. See Figure 1. The nucleotide sequence of the AfGA1 gene contains three introns, and the nucleotide sequence is shown in SEQ ID NO:8.

[0434] The AfGA1 gene was amplified from genomic DNA of Aspergillus fumigatus using the following primers: Primer 1: AfGA1-Fw 5'-GCG GCGGCCGC ACC atgcctcgcctttcctacgc-3' (SEQ ID NO: 9), and Primer 2: AfGA1-Rv 5'-cc ggcgcgccc TTA tcactgccaagtatcattctcg-3' (SEQ ID NO: 10). Th...

example 2

[0435] Example 2: Expression and purification of AfGA1TR .

[0436] The plasmid pJG222(Trex3gM-AfGA1) was transformed into a quadruple deletion Trichoderma reesei strain (described in WO 05 / 001036) using the gene gun method (Te'o et al., J. Microbiol. Methods ("Journal of Microbiology Methods") "), 51:393-99, 2002). Transformed colonies (about 50) appeared within about 1 week. After 5 days of growth on acetamide plates, colonies were inoculated in 250 ml shake flasks containing 30 ml glucose / agarose defined medium for protein expression. The protein AfGA1TR was secreted into the extracellular medium, and the filtered medium was used for SDS-PAGE and glucoamylase activity assay on DP7 to confirm enzyme expression.

[0437] The stable strains were then grown in defined medium in 7L fermentors. The fermentation broth was collected by centrifugation. After centrifugation, filtration and concentration, a 450 ml concentrated sample was obtained. By using the BCA method (Prote...

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Abstract

Fungal glucoamylases from Aspergillus fumigatus - expressed in Trichoderma reesei host cells (AfGATR) are provided. Trichoderma reesei host cells express AfGATRs at higher, or at least comparable, levels to natively expressed AfGA Aspergillus fumigatus. AfGATRs, including AfGA1TR and AfGA2TR, exhibit high activity at elevated temperatures and at low pH, so AfGATRs can be used efficiently in a process of saccharification in the presence of alpha-amylase, such as Aspergillus kawachii alpha-amylase (AkAA). AfGATRs advantageously catalyze starch saccharification to an oligosaccharide composition significantly enriched in DP1 (i.e., glucose) compared to the products of saccharification catalyzed by Aspergillus niger glucoamylase (AnGA) or native AfGA expressed in Aspergillus fumigatus. AfGATRs such as AfGA1TR, AfGA2TR or a variant thereof can be used at a lower dosage than AnGA and natively expressed AfGAs to produce comparable levels of glucose.

Description

[0001] Cross references to related patent applications [0002] This patent application claims priority from International Patent Application no. PCT / CN2012 / 086349 filed on December 11, 2012, which is hereby incorporated by reference in its entirety. [0003] Attached is a sequence listing comprising SEQ ID NOs: 1-14, which is hereby incorporated by reference in its entirety. technical field [0004] The present invention provides Trichoderma reesei host cells expressing glucoamylase (AfGATR) from Aspergillus fumigatus (AfGATR) or variants thereof, and methods of use thereof. Background technique [0005] The starch consists of a mixture of amylose (15-30% w / w) and amylopectin (70-85% w / w). Amylose consists of linear chains of alpha-1,4-linked glucose units with a molecular weight (MW) of about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units; its MW may be as high as 100,000,000. [0006] Sugars derived f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/34C12N15/80C12P19/14
CPCC12N9/2428C12C7/00C12P19/14C12Y302/01003
Inventor 葛晶L·华S·H·李J·李J·K·舍蒂Z·唐B·张K·钟
Owner DANISCO US INC
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