Screening of the putative hexokinase genes from rhizopus oryzae

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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Alagoz, Eda; Acar, Seyda; Yucel, Meral; Hamamcı, Haluk (2012-09-23)
Rhizopus oryzae is a filamentous fungus which can grow on some renewable carbon sources and this makes it a good candidate for production of lactic acid and ethanol. However, in R. oryzae the production yields of these products are lower compared to alternative organisms. The utilization of the carbon source through glycolysis might end up in lactic acid or ethanol production. This makes the glycolytic pathway a focus of attention for our search for a way to increase the speed and efficiency of this pathway...
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Rhizopus oryzae is a filamentous fungus, which can produce high amounts of L(+)-lactic acid and produces ethanol as the main by-product. In an effort to understand the pyruvate branch point of this organism, fermentations under different inoculum and glucose concentrations were carried out. At low inoculum size (1x103 spores ml-1), high amount of lactate (78 g l-1) was produced, whereas high ethanol concentration (37 g l-1) was obtained at high inoculum sizes (1x106 spores ml-1). Decreasing working volume i...
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Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyr...
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Çekmecelioğlu, Deniz (2019-11-01)
Distillers dried grains with solubles (DDGS) are co-product of dry-grind ethanol plant, which can be used as a feedstock for fermentation, e.g. for biofuels or other value-added products. However, DDGS loading is a critical factor in the pretreatment, hydrolysis, and fermentation, where the low DDGS loading lowers sugar concentration in the hydrolysate and yields low product concentrations, which can result in high energy cost at the recovery step, while too high loading may have inhibitory effects on the m...
Citation Formats
E. Alagöz, “Screening of the putative hexokinase genes from rhizopus oryzae,” Ph.D. - Doctoral Program, Middle East Technical University, 2013.