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Mutant cells for protein secretion and lignocellulose degradation

a technology of lignocellulose and protein, applied in the field of protein secretion and lignocellulose degradation, can solve the problems of significant technical challenge, major bottleneck in the biofuel production process, and less effective soluble inducers, and achieve the effect of reducing expression, reducing expression, and increasing the expression of cre-1 genes

Inactive Publication Date: 2014-02-13
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes mutant cells that increase the secretion of proteins and degrate lignocellulosic biomass. These mutant cells contain inactivating mutations in β-glucosidase genes and the catabolite repressor gene, cre-1. When contacted with cellulosic biomass, the mutant cells secrete more proteins. The patent also provides methods for increasing secretion of proteins and degrading lignocellulosic biomass using these mutant cells. The technical effects of the patent include increasing the efficiency of protein production and degrading lignocellulosic biomass.

Problems solved by technology

However, the initial conversion of insoluble lignocellulosic biomass into cell-permeable and readily fermentable sugars presents a significant technical challenge and major bottleneck in the biofuel production process.
However, whereas cellulase secretion in filamentous fungi is effectively induced by insoluble plant cell wall components, such as cellulose, hemicellulose, or xylan, soluble inducers are much less effective.
However, one problem with insoluble inducers is that cellulase can adhere to insoluble inducers, resulting in reduced yields of secreted enzyme activity.
The processing of insoluble biomass matter is a heterogeneous process and access to biomass surfaces is limiting for fungal cells.

Method used

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  • Mutant cells for protein secretion and lignocellulose degradation
  • Mutant cells for protein secretion and lignocellulose degradation
  • Mutant cells for protein secretion and lignocellulose degradation

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0145]The following example relates to the characterization of the cellulase transcription and cellulolytic enzyme production induced in the N. crassa triple β-glucosidase gene deletion strain and the triple β-glucosidase and cre-1 gene deletion strain.

[0146]Materials and Methods

[0147]Strains

[0148]Strains were obtained from the Fungal Genetics Stock Center (FGSC) including the Neurospora crassa wild-type (WT) (FGSC 2489), the cre-1 gene deletion (Δcre-1) (FGSC 10372), and deletion strains for the intracellular β-glucosidase NCU00130 (FGSC 11822 and FGSC 11823), and extracellular β-glucosidases: NCU08755 (FGSC 18387 and FGSC 18388) and NCU04952 (FGSC 13731 and FGSC 13732).

[0149]The quadruple deletion produced by performing sequential crosses of the single deletions using the method described by the FGSC (http: / / www.fgsc.net / neurosporaprotocols / How%20to%20make%20a%20cross-2.pdf). The genotype of all deletion strains was confirmed by using a gene-specific primer and a common primer for...

example 2

[0178]The following example relates to the identification of orthologues of the N. crassa β-glucosidase genes NCU00130, NCU04952, and NCU08755.

Materials and Methods

[0179]BLASTp searches were conducted using the National Center for Biotechnology Information (NCBI) non-redundant amino acid database using the NCU00130, NCU04952, and NCU08755 amino acid sequences as queries. Sequence hits from the BLASTp searches were aligned in MEGA5 using ClustalW2.

[0180]Phylogenetic trees were generated using the Neighbor-Joining method (Saitou N. and Nei M., 1987). The evolutionary distances were computed using the Poisson correction method (Zuckerkandl E. and Pauling L., 1965) and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA5 (Tamura K., Dudley J., Nei M., and Kumar S., 2007).

Results

[0181]The results of the ClustalW amino acid sequence alignments for NCU00130, NCU04952, and NCU08755 orthologues in closely related fungi are shown i...

example 3

[0185]The following example relates to the identification and characterization of the proteins secreted at higher levels from the triple β-glucosidase gene deletion N. crassa strain, and the triple β-glucosidase and cre-1 N. crassa deletion strain.

Materials and Methods

[0186]Strains

[0187]Strains obtained from the Fungal Genetics Stock Center (FGSC) include the wild type (FGSC 2489), and deletion strains for the intracellular β-glucosidase NCU00130 (FGSC 11822 and FGSC 11823), and extracellular β-glucosidases: NCU08755 (FGSC 18387 and FGSC 18388) and NCU04952 (FGSC 13731 and FGSC 13732). The homokaryon cre-1 deletion strain (NCU08807) is described in (44). Multiple deletion strains were made by performing sequential crosses. The genotype of each multiple deletion strain was confirmed using a gene-specific primer and a common primer for the hygromycin (hph) cassette. The hph forward primer used was SEQ ID NO: 4 from Example 1. The reverser primers used for NCU00130, NCU004953, NCU08755...

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Abstract

The present disclosure provides mutant cells for the secretion of proteins and for the degradation of lignocellulosic biomass. Methods for the use of these cells are also provided. Specifically, the utility of combined genetic deletions of β-glucosidases and the catabolite repressor gene creA / cre-1 for protein secretion in fungal and yeast cells is disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 453,086, filed Mar. 15, 2011, which is hereby incorporated by reference, in its entirety.SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE[0002]The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 677792001640SEQLIST.txt, date recorded: Mar. 14, 2012, size: 22 KB).FIELD[0003]The present disclosure relates to mutant cells for the production of proteins, such as cellulases, and for the degradation of lignocellulosic biomass. In particular, mutant cells and methods for the production of proteins, such as cellulases, are provided.BACKGROUND[0004]Lignocellulosic biomass is an abundant and renewable raw material for biofuel production. However, the initial conversion of insoluble lignocellulosic biomass into cell-permeable and readily fermentable ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/24
CPCC12N9/2402C12N9/2437C12N9/2445C12P19/14C12Y302/01021C12P19/02C12P21/00C12N9/0006C12N9/18C12N9/24C12Y101/99018C12Y302/01004C12N15/80
Inventor ZNAMEROSKI, ELIZABETH A.DOUDNA CATE, JAMES H.GLASS, N. LOUISE
Owner RGT UNIV OF CALIFORNIA
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