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University of Mitrovica “Isa Boletini” Complex media and genetic manipulation for improvement and production of new product by Kluyveromyces marxianus Bahtir Hyseni^~ , Rifat Morina^, Alush Musaj^ ^Univerity of Mitrovica, Food Technology Department, Faculty of Food Engineering ~Yeditepe University, Graduate School of Natural And Applied Sciences GREDIT2016 Skopje 01.04.2016 • Outlines • Kluyveromyces marxianus • Range of metabolites production by K.marxianus in different variety of medium • Ethanol production • Other products of K. marxianus • Genetic analysis and manipulation of K.marxianus and their expression • Conclusion Role of Kluyveromyces marxianus in Biotechnology • Kluyveromyces marxianus is well known yeast, and it has many known qualities which make it a desirable tool in biotechnology • Capabilities, which makes it a desirable organism for industry like high temperature fermentation and different kind of medium assimilation. • The wide range of the medium assimilation is a suitable feature for usage a complex media which have cheaper source. The main characteristics of K.marxianus There are many products that are analyzed to be produced by K.marxianus among them are: – Ethanol, β-galactosidase , Inulinase Other characteristics of K. marxianus are; – high capacity of converting substrate to biomass, growth in high temperature, – high production of enzymes,aroma compound production – good expression of heterologous protein production – have been labeled as fastest growing eukaryote on the planet Ethanol production from K.marxianus The ethanol could be produced from Opuntia ficus-indica and Jerusalem artichoke juice. Ethanol production also was found to be successful by using the juice from Henequen (agave fourcrodyes lem) leaf which are used as a source of natural fibers. Mixture of beet molasses and cheese whey was used as substrate for cultivation of strains of K.marxianus and S.cerevisiae, the substrate was converted efficiently to ethanol The results shows a low biomass yield, but the protein content of the medium was improved. Different complex medium used for growth of K.marxianus Reference Type of medium Maximum production of Ethanol g/l [24] [20] K.marxianus 5.6 initial 102 S.cerevisiae / / [21] D-xylose Jerusalem artichoke extract with 250 g/l sugar addition Jerusalem Artichoke irrigated with 25% sea water 57.5 / [25] Mixture of beet molasses and cheese whey 72.6 70.8 [22] Jerusalem artichoke at High gravity condition with addition of pectinase and K2HPO4 Opuntia ficus-indica YDP medium Henequen juice acid hydrolyzed Henequen juice enzyme hydrolyzed juice 6.5g/l Apple chokeberry pomace, free cells, without hydrolysis 6.0g/l Apple cranberry pomace, free cells, without hydrolysis 6.5g/l Apple chokeberry pomace, immobilized cells, without hydrolysis 6.0g/l Apple cranberry pomace, immobilized cells, 94.2 / 19.5 69 13.74 16.5 1.37 20.6 / No growth No growth / 1.65 / 1.08 / 0.14 / [17] [26] [27] [27] [28] [28] [28] [28] Different other products of K. marxianus • β-galactosidase which is produced by K.marxianus MTCC1388 by using whey. • It is a great opportunity to use this strain in βgalactosidase production form whey, when is know the big concern of whey in environment. • From their results was concluded, that this process can be applied in industrial scale. Different other products of K. marxianus • Inulinase is another product produced by K.marxianus by using Dahlia tuber extract, which was fortified with yeast extract as carbon and nitrogen source. The combined medium shows potential for inulinase production. • Strains of K.marxianus were successfully used to ferment Theobroma cacao seeds. • Another positive effect was seen in the color of seeds. Genetic analysis and manipulation of K.marxianus and their expression • Although with great ability for many metabolite production K.marxianus was also subject to many gene transformation for better expression, adding foreign gene features for production of new products. • A good example is development of gene integration method by non homologous end joining pathway, by using PCRamplified DNA molecules. • For testing its heterologous expression ability of K.marxianus, authors Siekstele et. al., analyzed the expression of the EPG gene (endo-poly-galacturonase) in the yeast by cloning. Genetic analysis and manipulation of K.marxianus and their expression • K.marxianus genes were found to be useful for transformation of the other species, GAP1 gene which is responsible for production of the p37 protein, mostly found in the cell of K.marxianus was found to be doubtful for the flocculation of the cells. • Feature of GAP1 gene was proved when non-flocculation cells of Sccharomyces transformed by GAP1 gene. cerevisiae were genetically Genetic analysis and manipulation of K.marxianus and their expression • Another gene of the K.marxianus which was used for cloning in Sccharomyces cerevisiae was PHO610, which encodes an acid phosphatase, which is a novel cell-wall protein having an enzyme activity. • The high evaluation of the yeast K.marxianus for the ethanol production was the reason that Nonklang et. al, construct a flocculation type of the yeast by introducing the gene of S.cerevisiae FLO1, FLO5, FLO9 and FLO10 by using TDH3 promoter. Conclusion • K.marxianus is known yeast recently for its potential to replace some known “horse work microorganism”. • High potential for using lignocellulosic and food waste materials by K.marxianus, makes it known for biotechnology industry. • The catabolic ability to break down the unused components from producing process and to turn them in valuable pharmaceutical or food components is a great strength of these yeasts. • With improvements in the biotechnology research techniques, many features of K.marxianus were highlights, with a hope that with a continuous work in the future this microorganism will be an important tool for bio-products.