Novozymes, Inc.

Novozymes A/s

Novo Nordisk Biotech, Inc.

Novozymes Biotech, Inc

Howard Brody

Paul Vincent Harris

Suchindra Maiyuran

John C Royer

Gregory A Gambetta

Mariah Connelly

Michael Lamsa

Suzanne Otani

Ana Fidantsef

Wendy T Yoder

Lynne M Christianson

Kim Hansen

Deborah S Yaver

Hiroaki Udagawa

Donna L Moyer

Hiroshi Teramoto

Wendy Yoder

Lynne M Christanson

Suzanne M Otani

Amanda Fisher

Evee Ricacho

Donna Moyer

The present invention relates to methods for co-silencing expression of genes in a filamentous fungal strain by transitive RNA interference. The present invention also relates to methods for identifying a gene encoding a biological substance of interest.

Methods for co-silencing expression of genes in filamentous fungal strains and uses thereof

The present invention relates to methods of reducing or eliminating expression of a target gene in a filamentous fungal strain by transitive RNA interference.

Methods of eliminating or reducing expression of genes in filamentous fungal strains by transitive RNA interference

The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

Methods for increasing homologous recombination of a nucleic acid sequence

The present invention relates to methods for reducing or eliminating the expression of a target gene in a filamentous fungal strain, comprising: (a) inserting into the genome of the filamentous fungal strain a double-stranded transcribable nucleic acid construct comprising a first nucleotide sequence comprising a promoter operably linked to a homologous coding region of the target gene and a second nucleotide sequence comprising the homologous coding region, or a portion thereof, of the target gene, wherein the first and second nucleotide sequences are complementary to each other and the second nucleotide sequence is in reverse orientation relative to the first nucleotide sequence; and (b) inducing production of an interfering RNA encoded by the double-stranded transcribable nucleic acid construct by cultivating the filamentous fungal strain under conditions conducive for production of the interfering RNA; wherein the interfering RNA interacts with RNA transcripts of the target gene to reduce or eliminate expression of the target gene. The present invention also relates to the filamentous fungal strains and to methods of producing a biological substance of interest in such filamentous fungal strains.

Methods for eliminating or reducing the expression of a genes in a filamentous fungal strains

The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3' end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

Methods for producing secreted polypeptides

The present invention relates to methods of producing a heterologous biological substance, comprising: (a) cultivating a mutant of a parentstrain in a medium suitable for the production of the heterologous biological substance, wherein (i) the mutant strain comprises a first nucleotide sequence encoding the heterologous biological substance and one or more second nucleotide sequences comprising a modification of glaA and at least one of the genes selected from the group consisting of asa, amyA, amyB, prtT, and oah, and (ii) the mutant strain is deficient in the production of glucoamylase and at least one enzyme selected from the group consisting of acid stable alpha-amylase, neutral alpha-amylase A, and neutral alpha-amylase B, protease, and oxalic acid hydrolase compared to the parentstrain when cultivated under identical conditions; and (b) recovering the heterologous biological substance from the cultivation medium. The present invention also relates to enzyme-deficient mutants ofstrains and methods for producing such mutants.

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Methods for increasing homologous recombination of a nucleic acid sequences

The present invention relates to mutants cells comprising a marker-free modification of a gene, and methods for obtaining and using such mutant cells.

Methods for producing heterologous polypeptides in trichothecene-deficient filamentous fungal mutant cells

The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a mutant of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide, wherein (i) the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes involved in the production of a trichothecene and (ii) the mutant produces less trichothecene than the parent filamentous fungal cell when cultured under the same conditions; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells, isolated trichodiene synthases and nucleic acid sequences encoding the trichodiene synthases.

The present invention relates to methods for modifying the production of a polypeptide, comprising: (a) introducing a nucleic acid construct into a cell, wherein the cell comprises a DNA sequence encoding a polypeptide, under conditions in which the nucleic acid construct integrates into the genome of the cell at a locus not within the DNA sequence encoding the polypeptide to produce a mutant cell, wherein the integration of the nucleic acid construct modifies the production of the polypeptide by the mutant cell relative to the cell when the mutant cell and the cell are cultured under the same conditions; and (b) identifying the mutant cell with the modified production of the polypeptide.

Growing community of inventors

Davis, CA, United States of America

Howard Brody