1. Development of a Talaromyces emersonii ‘Molecular Toolkit’, enabling the efficient expression of designer enzymes/enzyme cocktails for industrial applications O’Donnell R. 1, Hernon A. 2, Williams. K. 3, Tuohy M. 4, Heneghan M. 1 1 School of life sciences, Department of science, Institute of technology Sligo, Ash Lane, Sligo, Ireland. ronan.odonnell@mail.itsligo.ie 2 NIBRT, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland. 3 N313, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1UG (UK) 4 Molecular Glycobiotechnology Group, Biochemistry, School of Natural Sciences, National University of Ireland, Galway, Ireland 3. Project Plan Task 1: Development of an efficient transformation method for T. emersonii. Phase 1: Identification of selectable markers for T. emersonii Phase 2: Development of plasmids for T. emersonii transformation. Phase 3: Establishment of an efficient transformation system for T. emersonii. Task 2: Construction of a T. emersonii ‘molecular toolkit’. Phase 1: Identification of T. emersonii regulatory elements. Phase 2: Construction of toolkit plasmids that will enable the transfer of genes between them Phase 3: Testing of the molecular toolkit 1. What is a ‘molecular toolkit’? A ‘Molecular toolkit’ is a collection of plasmids/vectors designed to facilitate the ease of gene expression in either homologous or heterologous hosts. This ‘molecular toolkit’ will be designed for fungal gene expression, specifically gene expression in Talaromyces emersonii. Vectors must contain regulatory sequences including promoters, terminators and multiple cloning sites. Each promoter, terminator and gene segment should be readily exchangeable via conserved restriction sites. A selectable marker for fungal expression must also be included within the plasmid. The toolkit plasmids used must also contain sequences for subcloning in E. coli, such as an origin of replication (ORI) and a bacterial selectable marker, under the control of regulatory sequences recognised by E. coli. Figure 1 illustrates the various different DNA sequences that can be incorporated into a fungal vector such as pMCSi004 (Figure 2), in order to facilitate efficient expression of genes and easy modification of the construct. Figure 1: A schematic of molecular toolkit components Figure 2: pMCSi004 1, a simple plasmid that will be used as the backbone in the construction of the T. emersonii molecular toolkit 2. Why use Talaromyces emersonii (T. emersonii)? T. emersonii is a thermophilic ascomycete, that has an optimal growth temperature of 45°C and “generally regarded as safe” (GRAS) status 2. It produces thermostable enzymes that are useful in industrial processing. The creation of a ‘molecular toolkit’ will allow for ease of strain development and future research. T. emersonii has been shown to express complete cellulase and hemicellulase enzyme systems; it has also been shown to display some pectinase activity 2-3. These thermostable enzymes are useful in the production of 2 nd generation bioethanol and can be extremely beneficial, where waste products are utilised to create a viable feedstock for fermentation by yeast. This process greatly increases the value of a crop such as maize or sugar beet, that is typically discarded after harvest. There are also secondary benefits of this, where the area of land required to produce the same amount of bioethanol is reduced, therefore resulting in a smaller carbon footprint, where there are less maintenance costs. Figure 3 depicts a sample of T. emersonii in solid culture. The centre turns green when spores are produced, while the actively growing hyphae remains white around the edges. Figure 3: T. emersonii grown at 45°C for 1 week on sabaraud’s dextrose agar 4. Funding This project is funded by the 2012 Presidents bursary award from IT Sligo. 6. References 1.Collins.C.M., Heneghan. M. N., Kilaru. S., Bailey. A.M., Foster. G. D., 2010. Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes. Journal of Microbiological Methods. 82:156–162 2.Tuohy. M. G., Puls. J., Claeyssens. M, Vrsanská. M., Coughlan. M. P., 1993. The xylan- degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans. Biochem J.; 290(Pt 2): 515–523 3.Fernandez. S.,. Murry. P. G, Tuohy. M. G., 2008.Enzyme systems from the thermophilic fungus Talaromyces emersonii for sugar beet bioconversion. BioResources. 3:3 4.http://www.toku-e.com/product/g418_disulfate_50mgml_in_water/ 5.http://www.toku-e.com/Assets/Protocols/Hygromycin%20Protocol.pdf 6.Fahey. G., Heneghan. M. N., may 2013. Development of a Molecular toolkit for Talaromyces emersonii. Unpublished 7.Iann Rancé, Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over-expression in plants. 2002. Plant Science. 162(5):833–842 5. Results Identification of selectable marker A growth curve was constructed to test T. emersonii’s sensitivity to G418 (figure 4). Concentrations were selected based on the manufacturers guidelines which state that G418 is effective against bacteria and algae at concentrations under 5µg/ml 4. Future work will focus on repeating the kill curve using higher concentrations of G418, such as those recommended for mammalian cell selection (200-1000µg/ml 4 ). A growth curve was also constructed to test T. emersonii’s sensitivity to Hygromycin B (figure 5). Fahey, (2013) tested concentrations ranging from 50–250 µg/mL and reported some inhibition of growth. This work was repeated but results showed only a minimal inhibition at a concentration of 250 µg/mL. Future work will focus on testing T. emersonii’s sensitivity at a higher concentration of Hygromycin B (100–1000 µg/mL). Development of plasmids Minipreps of plasmids pGFPi004 and pMCSi004 have been performed (Figure 6). Plasmid integrity was confirmed by a restriction digest with BamHI and HindIII (Figure 7). These plasmids will form the backbone of the proposed toolkit constructs. Both plasmids contain the Agaricus bisporus (A. bisporus) glyceraldehyde-3-phosphate dehydrogenase (gpdII) promoter, a 5’ intron and the Aspergillus nidulans tryptophan synthase (TrpC) terminator 1. pMCSi004 contains a multiple cloning site while pGFPi004 contains efficient green fluorescent protein (eGFP). Plasmid pGFPi004 will be used to identify if the A. bisporus gpdII promoter is recognised by T. emersonii through promoter- reporter gene fusion. Construction of ‘molecular toolkit’. Regulatory sequences that may be suitable to drive efficient gene expression in T. emersonii have been identified. Promoters such as A. bisporus gpdII 1 and the Cauliflower mozaic virus 35S (CaMV 35S) 7 and a T. marneffi terminator, along with endogenous T. emersonii regulatory sequences will be investigated. Design of primers to isolate these sequences is underway. These primers will be used on T. emersonii genomic DNA (gDNA) to PCR amplify regulatory sequences. Primer sequences have also been designed to amplify the Cellobiohydrolase II (CBHII) gene from gDNA extracts. CBHII and eGFP will be used as reference genes to establish the levels of gene expression using the toolkit plasmids. Figure 6: 1% agarose gel containing minipreps of cloned plasmids pMCSI004 (control Lane 6, Clone Lane 4) and pGFPi004 (control Lane 5, Clone Lane 3). 1Kb Molecular weight marker in lane 1 and 100bp marker in lane 2 Figure 7: BamHI and HindIII restriction digest products: pMCSI004 (control Lane 2, Clone Lane 4 and 6), pGFPi004 (control Lane 3, Clone Lane 5 and 7). 1Kb Molecular weight marker in lane 1 Figure 5: Hygromycin B growth curveFigure 4: G418 growth curve