Concomitant knockout of target and transporter genes in filamentous
fungi by genome co-editing
Abstract
In most countries, genetically modified microorganisms are not approved
for use for fermentation in the food industry. Therefore, random
mutagenesis and subsequent screening are performed to improve the
productivities of valuable metabolites and enzymes as well as other
specific functions in an industrial microbial strain. In addition,
targeted gene knockout is performed by genetic recombination using its
enzyme genes as selectable markers to maintain self-cloning status.
However, random mutagenesis has a drawback as it does not guarantee
improvement of the targeted function. Conversely, self-cloning is rarely
used to breed an industrial microbial strain. This is probably because a
self-cloning strain is similar to a genetically modified strain, as both
undergo homologous recombination, although exogenous genes are not
introduced. In this article, I discuss the usefulness of genome editing
technology as a substitute for conventional techniques to breed
filamentous fungal strains. This article particularly focusses on
“genome co-editing,” a genome editing technology used for knocking out
two genes concomitantly, as reported in Magnaporthe grisea and
Aspergillus oryzae. Especially, when genome co-editing is applied to a
target gene and a membrane transporter gene that aid the entry of toxic
compounds into cells, the resulting clone can be categorized as an
autotrophic and non-genetically modified clone. Such a clone should
easily apply to industrial fermentation without being restricted by a
genetically modified status. Genome co-editing will also be used to
construct mutant strains with multiple target gene knockouts by
eliminating multiple membrane transporter genes. This could
substantially improve the productivities of valuable metabolites and
enzymes in a stepwise manner. Thus, genome co-editing is considered a
potentially powerful method to knock out single or multiple target genes
that can contribute to the breeding of filamentous fungal strains in the
food industry.