VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

Results: 4 of 9 parent strains were not able to form ascospores in theexperiment conditions. 198 spores were isolated after asci dissection. Sporesurvival ranged between 10 and 85% depending on the strain. After multistepselection, 16 haploid strains were assessed during fermentation of 1L(25°Blg) medium prepared from concentrated sugar beet juice and theresults were compared with those obtained for initial diploid strains. Ethanolyield ranged between 60-77% - for haploids and 72-90% for parent strains.Conclusions: Selected haploids will be used during obtaining yeast hybridsdesigned for dynamic and efficient fermentation of sugar beet juices forethanol.This work was financed from funds for Polish Science in 2009-2010 yearsas a part of projects: R&D N R12 0062 06 and R&D N312 301037.GWP040Chitin deacetylases - towards patterns of acetylationS. Kolkenbrock*, F. Bernard, B. MoerschbacherInstitute of Plant Biology and Biotechnology (IBBP), AK Moerschbacher,Münster, GermanyChitosan, the linear heteropolysaccharide of glucosamine and N-acetylglucosamine residues, is the only naturally occurring polycationicpolymer. As such, it has a number of highly attractive physico-chemicalproperties and, through interaction with the mostly anionic components ofliving cells, interesting biological activities.Today’s commercially available chitosans are produced chemically fromchitin isolated from shrimp shell wastes. They can be well definedconcerning their degree of polymerization and degree of acetylation, butthey are invariably characterized by a random pattern of acetylation (PA).The biological activities of chitosan, such as antimicrobial, plantstrengthening or immuno-stimulatory activities, may be greatly influencedby their PA. However, no methods are available today for the production ofchitosans with defined non-random PA.A chitin deacetylase from the phytopathogen fungus Puccinia graminis f. sp.triticum was heterologously synthesized in the fission yeastSchizosaccheromyces pombe. The hexahistidin tagged protein was purifiedto electrophoretic purity and used for kinetics with chitin trimers, tetramers,pentamers and hexamers. The resulting products were analyzed by massspectrometry and high performance thin layer chromatography (HP-TLC).After hydrolysis the resulting products of the substrates ranging fromtetramer to hexamer retained two acetylated units (A) at the non-reducingends, whereas the following units were completely deacetylated (D). Theseproducts (AADD, AADDD, AADDDD) were identified by electrosprayionization mass spectrometry, whereas the PA of the hydrolysis product ofthe trimer was ambiguous. Polymeric substrates treated with this chitindeacetylase may also contain a non-random blockwise PA as NMR datasuggests.A putative peptidoglycan deacetylase from Bacillus licheniformis DSM13was heterologously synthesized as a strepII-tagged protein in Escherichiacoli Rosetta 2 (DE3) [pLysSRARE2] with a pET-22b(+) vector constructand enriched to electrophoretic purity. This new enzyme shows strongdeacetylating activity towards chitin and may also generate a PA. In ananalogous way to the P. graminis enzyme this enzyme should also beanalyzed towards the potentially generated PA.GWP041Systems metabolic engineering of biotechnologicaldiaminopentane production by Corynebacteriumglutamicum for bio-based polyamidesS. Kind* 1 , W.K. Jeong 2 , H. Schröder 2 , C. Wittmann 11 Institute of Biochemical Engineering, University of Technology,Braunschweig, Germany2 Research Fine Chemicals & Biotechnology, BASF SE, Ludwigshafen,GermanyPolyamides, nowadays produced mainly from petrochemically derivedmonomers, display an important fraction in the field of polymers. Due to thevery durable material properties they are used e. g. in the automotiveindustry or in high value consumer products. The growing shortage of fossilresources, connected to rising oil price, escalating CO 2 production andglobal warming, has led to a search for suitable monomers derived fromrenewable resources. A very promising candidate in this context is thebiotechnologically produced C5 diamine diaminopentane (DAP), a keybuilding block for novel bio-based polyamides like PA 5.4 or PA 5.10.Here, we describe the biotechnological production of diaminopentanethrough systems-wide metabolic engineering of Corynebacteriumglutamicum. Lysine-overproducing strains of C. glutamicum were used aspromising starting point, as diaminopentane is directly available from lysineby decarboxylation. This was realized by expression of lysine decarboxylase(ldcC) from E. coli. Subsequent studies revealed this reaction as key targetfor efficient production, since in vitro activity and in vivo flux towardsdiaminopentane closely correlated in mutants expressing different variantsof E. coli ldcC. High-level expression from a single genomic copy wasachieved by combining the use of the strong constitutive promoter EFTUand a codon usage optimized for the translation system of C. glutamicum.The corresponding strain, C. glutamicum P eftuldcC opt , produceddiaminopentane on glucose with a yield of 0.2 mol mol -1 (1). Using theniterative rounds of systems-wide pathway analysis and engineeringsupporting pathways were successfully engineered. Metabolome analysis,however, revealed substantial formation of a so far unknown by-product,which was identified as an acetylated variant, N-acetyl-diaminopentane. Theundesired by-product reached levels of more than 20% of that ofdiaminopentane. By identification and elimination of the competing pathwaytowards N-acetyl-diaminopentane in P eftuldcC opt , the yield for DAP wasfurther increased (2).[1] Kind, S. et al (2010): Metab Eng 12(4), 341-51.[2] Kind, S. et al (2010): Appl Environ Microbiol 76(15), 5175-80.GWP042Effect of yeast strains and nitrogen content on efficiencyof alcoholic fermentation of raw juice based wortsK. Pielech-Przybylska* 1 , M. Balcerek* 1 , P. Patelski 1 , P. Dziugan 21 Institute of Fermentation Technology and Microbiology, Department ofSpirit and Yeast Technology, Technical University of Lodz, Lodz, Poland2 Institute of Fermentation Technology and Microbiology, Department ofFermentation Technology, Technical University of Lodz, Lodz, PolandIntroduction: Production of bioethanol from by-products of sucrose factoriessuch as raw and thick juices or molasses is a promising alternative for Polishsugar industry. In distilleries these raw materials can be directly fermentedafter pH adjustment and inoculation with yeast (raw and thin juices) oreasily converted to fermentation worts (thick juice, molasses) only throughdilution, supplementation with some nutrients and inoculation with yeastcells.Aim: The aim of this study was to evaluate the effect of various strains ofyeast S. cerevisiae on dynamics and efficiency of alcoholic fermentation ofraw sugar beet juice based worts.Materials and methods: Four yeast strains were used: M1, M2, M3 (derivedfrom Pure Culture Collection of the Institute of Fermentation Technologyand Microbiology of the Technical University of Lodz) and As-4(commercial yeast preparation). As the raw material, the raw sugar beetjuice was used. Raw juice based worts were prepare with and withoutaddition mineral nitrogen source (NH 4) 2HPO 4.Results: It was found that all used yeast strains fermented raw juice basedworts dynamically and efficiently. Total time of fermentations not exceeded48 hours. Addition of mineral nitrogen nutrient has not affected onfermentations capacity and their dynamic.Analysis of the chemical composition of distillates obtained, showedcorrelation between wort supplementation with mineral nutrient andconcentration of fermentation by-products, especially higher alcohols.Supplementation of raw juice based wort with mineral nitrogen contributedto a significant decrease of over 30% content of 2-methyl-1-butanol, 3-methyl-1-butanol and 2-methyl-1-propanol, and more than 13% of n-propanol and n-butanol in distillates.Application of molasses yeast strains (M1, M2, M3) for raw juice wortsfermentation had significant impact on by-products concentration indistillates. The content of higher alcohols (in particular isoamyl ones) in themajority of distillates from the raw juice (worts with mineral nitrogen)obtained by using yeast strains: M1, M2 and M3 were much lower than indistillates from the worts fermented with yeast As-4. Contents of 2-methyl-1-butanol and 3-methyl-1-butanol in these distillates ranged between91.3±6.5 - 348.4±23.6 mg/l spirit 100% v/v and 692.5±37.5 - 1191.9±83.7mg/l spirit 100% v/v respectively. In the relation to trials withoutsupplementation, contents of higher alcohols obtained from worts withadditional mineral nitrogen were reduced in distillates in ranges from 7%(M2) to 55% (M3).The study was financed by the Polish Ministry of Science and HigherEducation under R&D Grant No N R12 0062 06.spektrum | Tagungsband 2011