Genomics and proteomics of fungi.
Researchers increasingly are using high-throughput methods for genomic sequencing and proteomic analysis to investigate the
fundamental biology of fungi. To date, about 80 fungal strains (including both yeasts and filamentous fungi) have been or
are being sequenced, including fungi of medical, agricultural, and industrial importance.10
The sequencing and annotation of fungi facilitates, for example, identification of genes and proteins involved in protein
synthesis, protein secretion, protein degradation, morphology control, and post-translational modifications. Furthermore,
whole genome transcriptional analysis using microarrays will be possible when specific genomes are sequenced and annotated.
Armed with this knowledge, investigators will be able to rapidly discover, characterize, and modify genes involved with the
basic cellular processes of protein production. Differential analysis of gene transcription and protein production patterns
under various conditions will enable researchers to identify the factors controlling these basic cellular processes. As more
genomic and proteomic information becomes available, this knowledge-based improvement of fungal host strains and fermentation
processes for the commercial production of proteins and metabolites is becoming more and more common.
INTEGRATING SCREENING, PRODUCTION, AND MANUFACTURING SYSTEMS
To satisfy the protein expression needs of the various stages of drug discovery and development, an ideal fungal expression
system should have the ability to (i) express a wide variety of eukaryotic proteins; (ii) express proteins in a biologically
active form; (iii) produce proteins in a rapid and inexpensive fashion; (iv) seamlessly interface with laboratory automation;
(v) produce sufficient quantities of protein to support the confirmation of hits and the many other activities involved in
transforming a hit into a drug; and (vi) scale up to commercial drug production. With these needs in mind, an integrated
technology platform is currently under development using Chrysosporium lucknowense gene expression technology coupled with high-throughput screening.11 The morphology of this fungal strain enables this technology platform. Its low viscosity in culture and formation of propagules
allows the growth of this fungus in microtiter cultures and the manipulation of cultures in liquid handling systems utilizing
robotics. These characteristics enable high-throughput screening of gene expression libraries, either for gene discovery or
for screening variants created using molecular evolution.12 The ability to grow in microtiter plates can also facilitate gene expression and strain development processes. This miniaturization
allows large numbers of transformants to be screened for the presence of genes and gene products, using high-throughput hybridization
and blotting methods. Finally, as described earlier, the low-viscosity phenotype allows more versatile and robust culture
conditions to optimize protein manufacturing by fermentation.
Using a single organism for gene discovery, gene evolution, protein engineering, gene expression, and product manufacturing
offers significant advantages in the product development area by increasing the probability of success and decreasing development
time. Such an integrated system will be anticipated to eliminate bottlenecks in the drug development process that are inherent
when different organisms are used for gene discovery, protein evolution, early expression for preclinical testing, and product
manufacturing. Screening by expression will likely ensure that improved variants will also be well expressed, and successful
expression at the laboratory scale will more than likely lead to successful expression in the final manufacturing process.
The downside of integrating the technologies is that creating the platform requires the development and seamless integration
of several complex technologies. Filamen-tous fungi are genetically and physiologically less tractable than bacteria or yeast.
However, the physiological advantages of filamentous fungi—high level protein production and secretion, post-translational
modification, and proper folding—suggest that once the technical obstacles are overcome, the technology platform will be especially
valuable as the number of biopharmaceutical candidates continues to grow.
Richard P.Burlingame,PhD, is the director of R&D for Dyadic International, 140 Intracoastal Pointe Dr., Jupiter, FL 33477; tel 561.743.8333; firstname.lastname@example.org
Jan C.Verdoes,PhD, Dyadic Nederland, BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; tel. +31.30.6944015, email@example.com