IMPROVING D-XYLOSE AND D-GLUCOSE CO-CONSUMPTION FOR BIOETHANOL PRODUCTION IN Saccharomyces cerevisiae DS71054

• Main Author: DONNY RUDINATHA (NIM:20517017), YOSHUA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/31780
• Institution: Institut Teknologi Bandung

Bioethanol is an important renewable energy source that can be used to replace fossil fuels. <br /> <br /> Bioethanol has several advantages included reduced pollution, renewable energy, and easy <br /> <br /> starting material for production. First generation bioethanol production using agricultural stocks <br /> <br /> has drawn a negative connotation in “Food vs Fuel” issues. Second generation bioethanol <br /> <br /> production was used to avoid this controversy using lignocellulose raw materials. These are <br /> <br /> mainly composed of 40% hexose sugar (D-glucose) and 20% pentose sugar (D-xylose). <br /> <br /> The yeast Saccharomyces cerevisiae is used for bioethanol production but cannot naturally <br /> <br /> utilize D-xylose as carbon source. This problem was solved by introducing xylose isomerase <br /> <br /> from fungi and overexpression of genes involved in pentose phosphate pathway. This allowed <br /> <br /> for D-xylose consumption, but only when all the D-glucose has been consumed. To improve the <br /> <br /> simultaneous consumption of D-glucose and D-xylose, evolutionary engineering of S. cerevisiae <br /> <br /> was conducted and a new strain, Evo6, was generated. Evo6 shows improved D-xylose <br /> <br /> consumption compared to the parental strain, albeit with decreased D-glucose consumption and <br /> <br /> therefore reduced growth rate. The main purpose of this research is to increase D-glucose <br /> <br /> consumption in Evo6 for better bioethanol production. <br /> <br /> Evo6 is a flocculating strain caused by significant upregulation of Flo1, a gene involved in <br /> <br /> flocculation. Upon the deletion of FLO1 gene, the flocculation was abolished. Evo6&#916;Flo1, used <br /> <br /> in this research, shows improved sugar consumption rates compared to Evo6. Furthermore, <br /> <br /> RNAseq data indicates that in Evo6, Tsl1 and Tps3 are upregulated. These are genes involved in <br /> <br /> trehalose formation. One of the intermediates in this pathway is trehalose-6-phosphate which is <br /> <br /> an inhibitor of hexokinase that phosphorylates glucose into glucose-6-phosphate. Accumulation <br /> <br /> of trehalose-6-phosphate explains the lower D-glucose consumption by the Evo6&#916;Flo1 strain. <br /> <br /> Indeed, upon the deletion of TPS3 or TSL1, using CRISPR-Cas9 system, improved D-glucose <br /> <br /> consumption and ethanol production was observed. <br /> <br /> Furthermore in Evo6, Hxt1 is significantly down regulated compared to the parental strain. Hxt1 <br /> <br /> is a low affinity D-glucose transporter that is expressed at high concentrations of D-glucose. <br /> <br /> Overexpression of Hxt1 in Evo6 resulted in enhanced D-glucose metabolism but a lower Dxylose <br /> <br /> consumption. Hxt1 overexpression level was confirmed using qPCR compared to actin <br /> <br /> expression level. Overexpression of Hxt1 was done by changing the promoter using CRISPRCas9 <br /> <br /> system. To further elucidate the increased of D-xylose consumption in Evo6, the Hxt37 <br /> <br /> transporter carrying the N367I point mutation, as found in the Evo6 strain, was overexpressed in <br /> <br /> the parental strain. The mutation converts Hxt37 from a D-glucose into a specific D-xylose <br /> <br /> transporter. Overexpression of Hxt37 N367I resulted in a significant decreased in D-glucose <br /> <br /> metabolism however only a limited increased in D-xylose consumption was observed. <br /> <br /> Overexpression of Hxt37 was done using a plasmid based system. Overall, these data show that <br /> <br /> in strains that carry out D-glucose and D-xylose co-metabolism, D-xylose metabolism lead to <br /> <br /> increased trehalose-6-phosphate production that limits D-glucose consumption. <br />