ENGINEERING YEAST FOR IMPROVED ETHANOL PRODUCTION
Posted on Wednesday, April 9th, 2025
Saccharomyces cerevisiae is essential to the industrial production of bioethanol, a sustainable substitute for fossil fuels. However, metabolic pathways like the GPD2 gene, which regulates the synthesis of glycerol as a competitive pathway for carbon flow, and the ADH2 gene, which promotes ethanol oxidation in aerobic conditions, limit the amount of ethanol that may be produced. Improving the efficiency of ethanol production requires addressing these metabolic drawbacks. Even while genetic alterations such as ADH2 knockouts have shown favorable results, simultaneous GPD2 knockouts have still not been extensively researched for maximizing the production of ethanol from economical feedstocks like maize starch.
The goal of this study is to enhance the production of bioethanol by using homologous recombination to knock out the ADH2 and GPD2 genes in S. cerevisiae. Nourseothricin (NAT) will be used as a selection marker for the ADH2 knockout, and hygromycin will be used for the GPD2 knockout. To evaluate the effects of both genetic deletions on ethanol yield, the study compares the ethanol yields of wild-type and mutant yeast strains grown on glucose produced from maize.
PCR was used to create templates for knocking out the ADH2 and GPD2 genes. Transformation methods based on lithium acetate will be used to convert these templates into S. cerevisiae. Using colony PCR, nourseothricin (NAT) and hygromycin resistance will verify the successful knockouts of ADH2 and GPD2, respectively. As a substrate, glucose produced from maize will be used to ferment both mutant and wild-type strains. Using a spectrophotometer-based NADH-linked assay, which tracks the conversion of NAD+ to NADH during ethanol oxidation by alcohol dehydrogenase (ADH), the amount of ethanol produced will be measured. To verify the samples’ ethanol concentrations, standard curves will be generated.
It is anticipated that the dual knockout approach will significantly raise ethanol yield by removing ethanol oxidation and decreasing carbon flux towards undesirable pathways like glycerol synthesis. Results agree with earlier research that demonstrated ADH2 knockouts could increase yield by up to 74.7%, more gains are anticipated when GPD2 deletion is included. This research is intended to promote the use of renewable feedstocks, like corn starch, in industrial biofuel applications and provide important insights for optimising yeast strains for efficient bioethanol production.