1. Field of the Invention
The present invention relates to eliminating or reducing expression of a gene in a filamentous fungal strain.
2. Description of the Related Art
Filamentous fungal strains are widely used for the production of biological substances of commercial value. However, filamentous fungal strains with the desirable traits of increased expression and secretion of a biological substance may not necessarily have the most desirable characteristics for successful fermentation. The production of the biological substance may be accompanied by the production of other substances, e.g., enzymes, that degrade the biological substance or co-purify with the biological substance, which can complicate recovery and purification of the biological substance.
One solution to these problems is to inactivate the gene(s) involved in the production of the undesirable substance. Inactivation can be accomplished by deleting or disrupting the gene(s) using methods well known in the art. However, in some cases, inactivation of the gene may be difficult because of poor targeting to homologous regions of the gene. Inactivation can also be accomplished by random mutagenesis, but random mutagenesis is not always specific for the intended target gene and other mutations are often introduced into the host organism. In other situations, the gene and its product may be required for survival of the filamentous fungal strain. Where multiple genes are to be inactivated by deletion or disruption, the task can be very cumbersome and time-consuming. When highly homologous members of gene families exist, deletion or disruption of all members can be extremely tedious and difficult.
In recent years various forms of epigenetic gene regulation have been described (Selker, 1997, Trends Genet. 13: 296-301; Matzke and Matzke, 1998, Cell. Mol. Life. Sci. 54: 94-103). These processes influence gene expression by modulating the levels of messenger RNA (Hammond and Baulcombe, 1996, Plant Mol. Biol. 32: 79-88; Xi-song Ke et al., 2003, Current Opinion in Chemical Biology 7: 516-523) via micro RNAs (Morel et al., 2000, Curr. Biol. 10: 1591-4; Bailis and Forsburg, 2002, Genome Biol. 3, Reviews 1035; Grewal and Moazed, 2003, Science 301: 798-802).
Based on genetic studies of Drosophila and Caenorhabditis elegans, RNA interference (RNAi), also known as post-transcriptional gene silencing (in plants), is understood to involve silencing the expression of a gene by assembly of a protein-RNA effector nuclease complex that targets homologous RNAs for degradation (Hannon, 2002, Nature 418: 244-251). The processing of double-stranded RNA (dsRNA) into small interfering RNAs is accomplished by a family of enzymes known as Dicer (Bernstein et al., 2001, Nature 409: 363). Dicer, a member of the RNase III family of endonucleases that specifically cleaves dsRNA, is responsible for digestion of dsRNA into siRNAs ranging from 20-25 nucleotides (Elbashir et al., 2001, Nature 411: 494). These siRNAs denature with the anti-sense strand and then associate with the RNA Induced Silencing Complex (RISC) (Elbashir et al., 2001, Genes and Dev. 15: 188; NyKanen et al., 2001, Cell 197: 300; Hammond et al., 2001, Science 293: 1146.). Although not well understood, RISC targets the mRNA from which the anti-sense fragment was derived followed by endo and exonuclease digestion of the mRNA effectively silencing expression of that gene. RNAi has been demonstrated in plants, nematodes, insects, and mammals (Matzke and Matzke, 1998, supra; Kennerdell et al., 2000, Nat. Biotechnol. 18: 896-8; Bosher et al., 1999, Genetics 153: 1245-56; Voorhoeve and Agami, 2003, Trends Biotechnol. 21: 2-4; and McCaffrey et al., 2003, Nat Biotechnol. 21: 639-44).
WO 98/53083 discloses constructs and methods for enhancing the inhibition of a target gene within a plant by inserting a silencing vector comprising an inverted repeat sequence of all or part of the target gene into the genome of a plant.
WO 01/49844 describes an inverted repeat gene construct encoding an inverted repeat gene, comprising a promoter element operably linked in a 5′ to 3′ direction to a first coding sequence and a second sequence in an antisense orientation for disrupting gene expression in targeted organisms, such as Caenorhabditis elegans, yeast, Dictostelium, Drosophila, mice, plants, insects, human cells, and nematodes.
WO 03/050288 discloses methods of silencing a target gene in a plant by providing a recombinant DNA construct comprising a promoter operably linked to a chimeric nucleotide sequence encoding all or part of the target gene and a transgene, transforming the plant with the DNA construct such that the expression cassette is inserted into the genome, and initiating post-transcriptional gene silencing of the transgene in the plant, whereby initiation of post-transcriptional gene silencing of the transgene causes silencing of the target gene.
It would be an advantage in the art to have alternative methods for eliminating or reducing the expression of one or more genes for strain development and improvement, functional genomics, and pathway engineering of filamentous fungal strains.
It is an object of the present invention to provide alternative methods for eliminating or reducing the expression of one or more genes in a filamentous fungal strain.