Screening of the putative hexokinase genes from rhizopus oryzae

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2013
Alagöz, Eda
Rhizopus oryzae is a filamentous fungus which can ferment sugar to ethanol, and lactic acid. Increasing demand of the world for use of renewable carbon sources has put this and similar organisms into a position where the biotechnology industries are more and more interested with these organisms. R. oryzae can grow on some renewable carbon sources which makes it a good candidate for production of ethanol as well as lactic acid. The major use of lactic acid is in food industry and food- related application, which in the U.S., accounts for approximately 85% of demand. The rest (~15%) of the uses are for non-food industrial applications. Rhizopus oryzae produces only L-(+)-lactic acid form. Lactic acid is also produced by lactic acid bacteria which have at least 95 % yield on the basis of glucose converted into lactic acid (mainly lactobacillus species) and these bacteria produce both L –(+)-lactic acid and D-(-)-lactic acid form . L-(+)-lactic acid is preferred form of lactic acid since D-(-)-lactic acid can not been metabolized by human beings. Another advantage over lactic acid bacteria fermentations is that R. oryzae can grow in minimal medium which lowers the costs as well as it makes the downstream processing of lactic acid easier. However; the only disadvantage of R. oryzae for production of lactic acid is the low yield compared to the carbon consumed ~70 % (for Lactic acid bacteria this value is ~95 %). In order to increase the lactic acid yield of R. oryzae; understanding the mechanism of lactic acid production and other pathways e.g., glycolysis and ethanol production pathway and the regulation of the key enzymes of these pathways and increasing the flux through the lactic acid production branch is important. Glycolytic pathway lies at the centre of the energy metabolism and the intermediates of glycolytic pathway are channeled to different pathways like lactate production, ethanol production in different organisms which are industrially important. Glycolytic pathway is composed of 10 basic reactions that go from glucose to pyruvate. Hexokinase catalyse the first and irreversible step in glycolytic pathway and it is implicated as one of the major control point in this pathway. In this study; the putative hexokinase genes of R. oryzae were screened. The probable hexokinase genes were found from the genome database of R. oryzae by doing a blast search using the known protein sequences of closely related S. cerevisiae hexokinases. Seven of our ten probable hexokinase genes were obtained by using PCR. Sequencing results of three of them proved that these genes have no introns meaning they were expressed in R. oryzae. In the complementation study done in hexokinaless mutant of S. cerevisiae DFY632 for these genes, the growth of the transformant was observed on glucose or fructose containing media as a carbon source. The hexokinase activities of RoHXKs transformants determined and the inhibition effect of trehalose-6-phosphate on in RoHXKs transformants were investigated.

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Citation Formats
E. Alagöz, “Screening of the putative hexokinase genes from rhizopus oryzae,” Ph.D. - Doctoral Program, Middle East Technical University, 2013.