Abstract
SUMMARYBioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication.
Highlights
IntroductionThe requirement for the fuel is constantly increasing and the production of biofuels (bioethanol, biodiesel and biobutanol) from renewable sources has become more frequent in the last several decades
The requirement for the fuel is constantly increasing and the production of biofuels from renewable sources has become more frequent in the last several decades
We constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively
Summary
The requirement for the fuel is constantly increasing and the production of biofuels (bioethanol, biodiesel and biobutanol) from renewable sources has become more frequent in the last several decades. Biofuels are usually classified depending on the source of material used for production [1]. Biofuels of the first generation are made by fermentation of raw starch-containing material that can be used for human consumption, resulting in both expensive and unethical production. Lignocellulosic waste consists of cellulose (35-50 %), hemicellulose (20-35 %) and lignin (5-30 %), its exact composition depends on the origin of the material [2]. Since microorganisms are not able to ferment raw lignocellulosic material directly, it is first pretreated to release compounds that can be further hydrolysed and used by yeasts for fermentation. There are different methods of pretreatment (chemical, physical, physicochemical and enzymatic), the choice of method is still one of the main challenges resulting in hydrolysates whose chemical composition depends both on the type of starting material and the type of pretreatment. The pretreatment does not result in a complete degradation of a starting material releasing
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