Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSFriday, March 15 3:00 PM–6:00 PMNautilusSynthetic BiologyCo-chairs: Nancy Keller and Peter PuntEngineering Aspergillus oryzae for high level production of L-malic acid. Debbie S Yaver 1 , S. Brown 2 , A. Berry 2 . 1) Expression Technology, Novozymes, Inc.,Davis, CA; 2) Microbial Physiology, Novozymes, Inc., Davis, CA.In the last decade, there has been widespread interest and investment in developing processes for the production of bulk and specialty chemicals fromrenewable feedstocks by fermentation. During this period, Novozymes has successfully developed technology for production of a specialty molecule(hyaluronic acid) by Bacillus fermentation and has been very active in developing technologies for the production of bulk chemicals by metabolicengineering and fermentation using several different microorganisms. An example of the latter is L-malic acid. In the literature it is reported that somewild-type Aspergillus strains produce high levels of malic acid under specific cultivation conditions. Concentrations up to 113 g/L malate (94% w/w fromglucose) reported for A. flavus in fed-batch fermentations (Battat, et. al., 1991. Biotechnol. Bioeng. 37:1108-1116). The goal of our work was to improvemalic acid production in the natural malic acid producing filamentous fungus Aspergillus oryzae NRRL 3488 by overexpression of cloned genes and classicalmutagenesis. More than 75 different recombinant strains were tested containing combinations of overexpression of genes as well as deletions. A highthrough put screen was developed and used to screen mutagenized strains. Combined genetic engineering and mutagenesis/HTS was used to increase themalic acid production rate of A. oryzae NRRL3488 by 4-fold with final C4 acid totals of 340 g/l at 8 days in lab scale fermentations.When synthetic biology meets metabolic engineering: in vivo pathway assembly in Saccharomyces cerevisiae. Niels Kuijpers 1,2 , Daniel Solis Escalante 1,2 ,Jack T. Pronk 1,2,3 , Jean-Marc Daran 1,2,3 , Pascale Daran-Lapujade 1,2 . 1) Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BCDelft, The Netherlands; 2) Kluyver Centre for Genomics of Industrial Fermentation, PO Box 5057, 2600 GA Delft, The Netherlands; 3) Platform GreenSynthetic Biology, Julianalaan 67, 2628 BC Delft, The Netherlands.The yeast Saccharomyces cerevisiae is a powerful and versatile workhorse intensively exploited for a wide range of biotechnological applications. Besidesthe large scale production of endogenous products, such as the biofuel ethanol, S. cerevisiae has been genetically engineered to produce manyheterologous compounds, including half of the worldwide insulin market. The past decade has been marked by the conversion of S. cerevisiae into acomplex cell factory with remarkable new capabilities such as the production of the anti-malaria drug precursor artemisinic acid. The ever-increasingdemand for cheap and sustainable production of complex molecules combined with its attractiveness as a host for pathway engineering will inevitablyintensify the exploitation of S. cerevisiae as cell factory in the future. Even in the genetically accessible bakers yeast, expression of dozen of genes is stilllargely based on laborious classical techniques involving successive restrictions and ligations, complemented with the creative application of PCR.However, the increasing size and complexity of today’s constructs in metabolic engineering has made design and construction of plasmids by theseclassical techniques increasingly complicated and time consuming. Although uncovered nearly three decades ago, the high efficiency of S. cerevisiaehomologous recombination is only beginning to reveal its full potential for the assembly of large DNA constructs (Gibson et al., 2008). In vivo assembly inyeast is predicted to have a large impact on laboratory practices, ranging from simple plasmid construction to engineering of complex pathways viaautomated high-throughput strain construction. Despite those promising prospects, in vivo assembly has not yet become a standard technique in mostacademic laboratories. This offers unique possibilities for standardization and, simultaneously, for further optimization. In the present work we describe anapproach designed to improve the efficiency of in vivo assembly and to make a robust, versatile in vivo assembly strategy for multi-component plasmids.As a proof of principle, the method was used to assemble a 21 kb plasmid from 9 overlapping fragments, using only PCR and yeast transformation. GibsonD.G. et al. (2008), Science, 319, 1215-1220.Analysis of the intracellular galactoglycom of Trichoderma reesei grown on lactose. Levente Karaffa 1 , Leon Coulier 2 , Erzsébet Fekete 1 , Karin M.Overkamp 2 , Irina S. Druzhinina 3 , Marianna Mikus 3 , Bernhard Seiboth 3 , Levente Novák 4 , Peter J. Punt 2 , Christian P. Kubicek 3 . 1) Department of BiochemicalEngineering, University of Debrecen, H-4032, Debrecen, Hungary; 2) TNO, P.O. Box 360, 3700 AJ Zeist, The Netherlands; 3) Research Area Biotechnologyand Microbiology, Institute of Chemical Engineering, TU Wien, Gumpendorferstrasse 1a, A-1060 Wien, Austria; 4) Department of Colloid andEnvironmental Chemistry, Faculty of Science and Technology, University of Debrecen, H-4032, Debrecen, Hungary.Lactose (1,4-0-b-D-galactopyranosyl-D-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for - among otherpurposes - in the biofuel and biorefinery industries. However, the mechanism how lactose induces cellulase formation in T. reesei is still enigmatic.Previous results raised the hypothesis that intermediates from the two D-galactose catabolic pathway may give rise to the accumulation of intracellularoligogalactosides that could act as inducer. We have therefore used HPAEC-MS to study the intracellular galactoglycome of T. reesei during growth onlactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivity. Lactose, allo-lactose and lactulose weredetected in the highest amounts in all strains, and two trisaccharides (Gal-b-1,6-Gal-b-1,4-Glc/Fru; and Gal-b-1,4-Gal-b-1,4-Glc/Fru) also accumulated tosignificant levels. D-Glucose and D-galactose, as well as two further oligosaccharides (Gal-b-1,3/1,4-Gal; Gal-b-1,2/1,3-Glc) were only detected in minoramounts, In addition, one unknown disaccharide and four trisaccharides were also detected. The unknown hexose disaccharide to correlate with cellulaseformation in the improved mutant strains as well as the galactose pathway mutants, and Gal-b-1,4-Gal-b-1,4-Glc and two other unknown hexosetrisaccharides to correlate with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulaseinduction by lactose.78