Fungal Cell Factories - Food and Industrial Bioproducts and Bioprocessing - page 209

Molecular, genetic and cell biology analysis will aid in illuminating the functions and
features of these annotated gene products in enzyme or acid synthesis. Gene targeting
methods, including replacing the coding sequence with selectable markers or fusing it to
fluorescent labels, can be used to evaluate the protein's cellular role. Fast and efficient
high throughput gene deletions are facilitated by the recent development of the A. niger
kusA ( ku 70) non-homologous end joining-deficient mutant that suppresses double
stranded break repair (Meyer et al ., 2007 ; Carvalho et al ., 2010 ). Polarized growth is
thought to be an important feature of fermentative output, and imaging analysis has
demonstrated that productive citric acid overflow is dependent on cell morphology (Cox
and Thomas, 1992 ; Papagianni, 2004 ). Microscopy imaging of A. niger during
fermentative growth will allow investigation of recognized key polarization components,
including the Spitzenkörper, F-actin, GTPases and other signaling molecules for
metabolite production and secretion (Harris et al ., 2005 ). A detailed understanding of
individual genes combined with high throughput characterization of the metabolome,
proteome and transcriptome will improve targeted strain development of this industrial
workhorse.
Systems biology approaches to understanding metabolism and accelerating strain
improvement are gaining momentum across the genus Aspergillus (Andersen and Nielsen,
2009). In 2008, a comprehensive metabolic reconstruction for Aspergillus niger based on
published literature (aka the “bibliome”), genome sequence, metabolomic and transcriptomic
data was published; it lays a solid foundation for a systems biology approach to strain
improvement (Andersen et al ., 2008). This model has been used to predict essential genes
(Thykaer et al ., 2009) as well as generate a deeper understanding of the biochemical
pathways that feed production of various metabolites (Andersen et al ., 2009 ; Panagiotou
et al ., 2008 ; Sorensen et al ., 2009 ).
8.4.2 Cellulase production
Enzyme production costs present a considerable barrier to economic lignocellulose fuel
ethanol. A clearer understanding of the molecular mechanisms of cellulase synthesis will
accelerate industrial strain development for the generation of more economical enzymes.
Le Crom and colleagues performed massively parallel sequencing to identify mutations in
the genomes of two cellulase hyper-producing mutants NG14 and RUT C30 (Le Crom
et al ., 2009). They detected a surprisingly high number of mutagenic events: 223 single
nucleotide variants, 15 small deletions or insertions, and 18 larger deletions, leading to the
loss of more than 100 kb of genomic DNA. The authors confirmed previously reported
mutations and uncovered novel mutations in 43 genes that are involved in nuclear transport,
mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This hetero-
geneity of functional categories suggests that multiple changes may be necessary to
improve cellulase production (Le Crom et al ., 2009). The results of Martinez and colleagues
and Le Crom and colleagues build a genomic foundation to test a large number of
hypotheses about the mechanisms underlying T. reesei protein secretion, carbon catabolite
repression and cellulose induction of cellulase (Le Crom et al ., 2009 ; Martinez et al .,
2008). Their findings also highlighted previously unexplored targets for directed strain
improvement, including nucleocytoplasmic transport, vacuolar protein trafficking and
mRNA turnover.