gene discovery, gene expression and product manufacturing using an

Gene discovery, gene expressionand product manufacturing usingan integrated fungal host systemyRichard P. Burlingame*Research & Development, Dyadic International, Inc., Jupiter, FL, USA

Proteins are an increasingly important source of newproducts for numerous industries, including human andanimal health, diagnostics, health and beauty aids,agricultural products, and chemicals. Because of thefunctional roles of proteins in living systems, proteinsrepresent a large fraction of all biologically active mol-ecules and the entire spectrum of biological activities. Toexploit these properties to bring new products to theseindustries, there is an increasing demand for biologicalsystems to discover, develop and manufacture proteins.Currently, there are a variety of systems available toperform discovery, development and manufacturing stepsindividually. However, some of these systems are limitedwith regards their abilities to discover and express genesfrom a large portion of the protein ‘universe’. Others areimpractical for commercial production of many proteinsbecause of restrictive conditions necessary for growth andcultivation of the biological systems or the inability ofthose systems to produce proteins economically at largecommercial scale.

Dyadic International is developing a system thatallows gene discovery, gene expression, productdevelopment and manufacturing in a single biologicalhost. This integrated system consists of gene discoveryand gene expression components, both of which utilizethe proprietary fungal host Chrysosporium lucknowense,or C1. C1 has differentiating advantages making ituniquely capable of functioning as an integrateddiscovery through manufacturing system. As a eukaryote,C1 is capable of faithfully expressing genes from virtuallyany source. Unlike bacteria, it is capable of splicingintrons, interrupting sequences present in manyeukaryotic genes. It is also capable of performingpost-translational modifications characteristic of manyeukaryotic proteins, for example glycosylation, with noevidence of the hyperglycosylation described for yeast.This allows access to portions of the biodiverse gene poolnot accessible to other screening systems. Since greaterthan 90% of the Earth’s species are eukaryotic by someestimates [1, 2], the C1 system will be able to discovergenes and gene products that bacterial and yeast systemswill not be able to.

C1 also has a culture morphology, unique among fungi,that confers the ability not only to grow in up to 384-well

microtitre dishes, but also for those microvolume culturesto be robotically transferred from well to well for culturereplication [3, 4]. Finally, C1 is capable of secretingand producing proteins under a variety of fermentationconditions at high yields, allowing cost-effective produc-tion of a large variety of proteins.

The gene-discovery engine utilizes robots for the prep-aration and assay of gene expression libraries in C1.The requisite genes can be from various sources—geno-mic or cDNA from individual organisms, DNA isolatedfrom environmental samples, individual gene variantsets generated in the laboratory. These genes are clonedinto expression vectors and introduced into C1. Theunique morphology of C1, described above, leads tothe formation of individual transferable elements, orpropagules. The formation of these propagules resultsin non-viscous growth in culture, allowing adequateaeration in microtiter wells. In addition, formationof propagules results in lack of pelleting, surface mattingand aerial sporulation. As shown in table 1, thiseliminates canula tip clogging in robotic liquid-handlingsystems. The lack of aerial sporulation, in addition,prevents well-to-well cross-contamination.

Using a Zymark (Hopkinton, MA, USA) Allegro system,the propagules are transferred to the wells of microtitredishes, each well containing on average one or asmall number of cloned genes. After cultivation in themicrotitre wells, the libraries of expressed genes arereplicated, again using the Allegro robotic system. It isthe lack of tip clogging and cross-contamination thatmakes the latter step possible.

The replicate, or daughter libraries, are then assayed forproteins or activities of interest. These can be enzymes,targets for fungicides, pharmaceutical targets or biother-apeutics. The assays are performed using a ZymarkStaccato system, allowing both high throughput andflexibility in the assay protocol.

Once activities are found, clones expressing the activitiesare ‘cherry-picked’ from a second set of daughter platesand re-assayed to verify the activity. The genes of interestare isolated from individual clones expressing the activ-ity. Since the method used for screening dictates that thegene is expressed at some level in C1, there is a highprobability of success that the gene can be overexpressedto commercially viable levels. Numerous tools to optimizeexpression, secretion and host strain optimization havebeen developed [5] and continue to be developed. Usingthese tools, a number of native and heterologous geneshave been overexpressed in C1 (table 2).

After a gene has been successfully overexpressed inthe laboratory setting, versatile culture conditions

Journal of Automated Methods & Management in ChemistryVol. 25, No. 3 (May–June 2003) pp. 67–68

Journal of Automated Methods & Management in Chemistry ISSN 1463–9246 print/ISSN 1464–5068 online # 2003 Taylor & Francis Ltdhttp://www.tandf.co.uk/journals

*e-mail: [email protected] paper was initially presented at the ISLAR 2002 Conference andis reproduced here by kind permission of Zymark Corporation.

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(table 3) allow the products of genes expressed in C1 tobe readily scaled up. The broad range of temperatureand pH, the low culture viscosity, and short cycletime offer numerous options to develop processes toproduce proteins economically that might otherwise beunstable or produced at lower yields in other productionsystems.

The C1 system is the first integrated system of itskind, allowing gene discovery, gene expression,process optimization and product manufacturing in asingle host organism. That these processes can all takeplace in a single organism increases the likelihood ofsuccess and concurrently reduces the cycle time betweenconception and commercialization. Reduced probabilityof failure and shorter development timeline will facilitate

development of numerous new industrial, pharmaceuticaland consumer products in a timely and cost-effectivemanner.

References

1. Bull, A. T., Goodfellow, M. and Slater, J. H., Annual Review ofMicrobiology, 46 (1992), 219.

2. Bank, D., Wall Street Journal, 22 ( January 2002).3. Emalfarb, M. A., Punt, P. J., van Zeijl, C. and van den Hondel,

C., PCT Application WO 01/79558, 2001.4. Emalfarb, M. A., PCT Application WO 01/25468, 2001.5. Emalfarb, M. A., Burlingame, R. P., Olson, P. T., Sinitsyn, A. P.,

Parriche, M., Bousson, J. C., Pynnonen, C. M. and Punt, P.J.,PCT Application WO 00/20555, 2000.

R. P. Burlingame Integrated fungal host system

Table 1. Suitability of fungi for robotic screening.

Strain Formation of propagules Phenotype

Chrysosporium lucknowense C1 þþþþþ no tip cloggingDyadic strain 2 þþþ no tip cloggingDyadic strain 3 þþ no tip cloggingOne fungal species þþ no tip clogging, but some large pelletsTen fungal species � to þþ tip clogging

Table 3. Fermentation parameters for C1 and other fungi.

Aspergillus Trichoderma C. lucknowense C1

pH 3.0–6.0 3.0–6.0 4.5–9.0Temperature (�C) 30–37 25–32 25–44Viscosity 1500–2000 200–1000 <10Fermentation (days) 8–9 6–7 5

Table 2. Examples of genes expressed in C1.

Gene product Gene source Level of expression

Endoglucanase 5 C1 15 g/lGlucoamylase Aspergillus 1 g/lInterleukin 6 human 10mg/lTNF receptor analogue human 50mg/l

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