New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)

ACIB-5

Esterases from Clostridium are involved in anaerobicdegradation of synthetic polyester

Veronika Perz1,∗ , Veronika Perz2, Armin Baumschlager2, KlausBleymaier2, Andrzej Łyskowski2, Altijana Hromic2, Karl Gruber3,Carsten Sinkel4, Ulf Küper4, Melanie Bonnekessel 4, Doris Ribitsch2,Georg Guebitz5

1 ACIB GmbH, Austria2 ACIB GmbH, Petersgasse 14, 8010 Graz, Austria3 Institute of Molecular Biosciences, University of Graz, Austria4 BASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, Germany5 Institute of Environmental Biotechnology, University of Natural Resources andLife Sciences, Austria

The anaerobic degradation of synthetic aliphatic–aromaticpolyesters used in food packaging is of great importance duringanaerobic digestion. Several studies have proven the biodegrad-ability of PBAT (poly(butylene adipate-co-butylene terephthalate))under aerobic conditions while there is only little information onanaerobic degradation.

In this study, imaging analysis (CLSM, SEM) and quantifica-tion of degradation products indicated anaerobic hydrolysis ofPBAT in biogas sludge. However, the detected hydrolysis ratesare still too low for efficient PBAT degradation in industrial bio-gas plants. Consequently, hydrolysis of PBAT by enzymes fromdifferent anaerobic organisms (Clostridium species) was investi-gated. Therefore, various hydrolases from these organisms weresuccessfully heterologously expressed in E. coli BL21-Gold(DE3).The kinetic parameters on the standard substrate p-nitrophenylacetate were determined and revealed high activity of up to 700U/mg (vmax). Analysis of the crystal structure of one esterase fromC. botulinum disclosed the presence of a Zn2+ metal ion that liesdeep beneath the protein surface.

The degradation of synthesized oligomeric and polymericmodel substrates was studied in order to get a deeper insight intothe reaction mechanisms of these new hydrolases.

Esterases from C. hathewayi and C. botulinum were indeedable to hydrolyze aliphatic-aromatic polyesters like PBATand different model substrates as indicated by HPLC/MSquantification of the hydrolysis products terephthalic acid,adipic acid, mono(4-hydroyxbutyl)terephthalate and bis(4-hydroxybutyl)terephthalate. We could demonstrate that theesterases show activity under mild conditions as well as in biogassludge.

http://dx.doi.org/10.1016/j.nbt.2014.05.1619

ACIB-6

Designing robust Saccharomyces cerevisiae strainsagainst stresses encountered during bioethanol fermen-tations from lignocellulosic biomass

Vinod Kumar1,∗ , Darren Greetham1, Tithira Wimalasena2

1 The University of Nottingham, United Kingdom2 Kingston Research Limited (BP and DuPont JV), United Kingdom

During the pre-treatment of lignocellulosic biomass inhibitorycompounds are released which can exert adverse effects on cellulargrowth, metabolism and ethanol production. In addition, osmoticstress caused by the high concentrations of available sugars andend-stage ethanol toxicity reduce overall rates of bioethanol fer-mentation. In previous studies, F1 hybrid segregants derived fromclean lineage Saccharomyces cerevisiae strains were assessed fortolerance to a range of stresses encountered during industrialbioethanol fermentations. Using a systems biology approach (QTL-Quantitative Trait Locus) chromosomal loci conferring resistanceagainst weak acid and osmotic stress were identified, and withinthose loci genes COX20 (acetic acid) and RCK2 (osmotic) weredetermined as important for stress response.

In the present study, strains in which COX20 or RCK2 haveeither been deleted or inserted into on tetracycline induced vec-tors. Phenotypic microarray assessment (Biolog, US) of thesestrains under acetic acid, formic acid, furfural, HMF, vanillin andsorbitol stress have been determined and compared against appro-priate controls. Results have highlighted that presence of COX20improves tolerance to weak acids while RCK2 gene conferred resis-tance to osmotic stress. The presence of either gene also enhancedthe tolerance against furanic compounds such as furfural and HMF.

http://dx.doi.org/10.1016/j.nbt.2014.05.1620

ACIB-7

Systems biology of Pichia pastoris

Brigitte Gasser

Dep. of Biotechnology, BOKU University of Natural Resources and Life SciencesVienna, Austria

Pichia pastoris is the most frequently used yeast system forheterologous protein production today, however, the toolbox ofavailable genetic elements is rather limited. To enable more robustand cost-effective production processes for biopharmaceutical pro-teins and for industrial enzymes, it is crucial to understand themolecular physiology of the host, and the specific limitations thatthe product may exert on expression.

Instead of classical genetic approaches, we applied systems biol-ogy tools to improve several aspects of the P. pastoris productionplatform. Combined transcriptomics, proteomics, metabolomicsand flux data were used to investigate the interplay between pro-tein production and cell metabolism. Thereby we gained insightin key regulatory effects exerted during heterologous protein pro-duction processes. These genome scale data were then furtherexploited for the identification of novel regulatory elements and

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