Improving ethanol production in a commercial yeast strain by evolutionary engineering
-
Fatemeh Sheikhi,1,* Khosrow Rostami,2 Mehrdad Azin,3 Mohammad Ali Asadollahi,4 Mansour Ebrahimi,5
1. Sugarcane Training and Research Institute, Khuzestan, Iran
2. Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
3. Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
4. Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan 81746-73441, Iran
5. Bioinformatics Research Group, Green Research Center, University of Qom, Iran
- Introduction: Saccharomyces cerevisiae is widely used for industrial production of ethanol. The tolerance to high ethanol concentrations, high-temperature is essential for process performance. To characterize ethanol-tolerant yeast strains, we performed adaptive laboratory evolution against higher concentrations of ethanol. Tolerance to ethanol is a key characteristic of the yeast Saccharomyces cerevisiae. Any increase in ethanol tolerance in the commercial strains led to faster and more complete fermentations, and may also allow the production of more alcohol. It appears that a large increase in ethanol tolerance requires complex changes in the yeast’s genome. Several approaches that rely on the effect of (random) variation generated by evolutionary engineering or mutagenesis have successfully yielded strains with increased ethanol tolerance.
- Methods: In the present study, to improve ethanol production the in the commercial “Raz170” strain, the parent strain was mutated physically and chemically. The mutants were screened using 1-butanol containing medium. The primary parent and the mutants were evolved within 144 days with evolutionary engineering strategy, while ethanol tolerance phenotype of selected strains was investigated.
- Results: According to the increase in the maximum growth rate, 8 strains were selected including parental strain and mutants, and the amounts of ethanol production of these strains were evaluated after evolutionary adaptation tests. Ethanol production of R111 and R113 which were mutated with EMS and UV before the adaptive evolution test and then evolved at 9 and 11% v/v ethanol, respectively, was improved from 93.25 ± 1.43 g/L to 102.25 ± 2.13 and 99.5 ± 0.93 g/L, respectively.
- Conclusion: Evolutionary engineering, is a narrow-experimental evolution that mimics this natural phenomenon in laboratory towards the desired phenotypes that has been used excessively since two decades ago, and it has an extensive capabilities in creating capable strains in order to increase ethanol tolerance in industrial strains. To increase the genetic diversity of the primary population, before starting the adaptive evolution experiments, mutation with ethyl methane sulfonate was used, which was more efficient than ultraviolet radiation in accelerating the evolution process to achieve the desired phenotype.
- Keywords: Evolutionary engineering, Ethanol production, Mutation, Saccharomyces cerevisiae
