Abstract
The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.
Highlights
The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol
We report a detailed genomic and physiological characterization of the best-performing evolved strains, and demonstrate that the improved consumption of xylose is associated with a high copy-number of xylose isomerase (xylA) and inactivation of the ISU1 and SSK2 genes
In order to build a robust xylose-fermenting S. cerevisiae strain, we dissected a tetrad from the industrial diploid strain PE-2 and chose a MATα haploid spore (LVYA1) for further genetic manipulations
Summary
The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In Brazil, first-generation ethanol is produced from the fermentation of sucrose-rich sugarcane extract, using a process in which Saccharomyces cerevisiae yeast cells are continuously recycled through successive batches, in some cases for up to 250 days in a row[6] This prolonged cell recycling regimen combined with the harsh environment found in the bioethanol distilleries require strains that display traits such as high tolerance to abiotic stress and robust growth to outcompete microbial contaminants. It combines high fermentative capacity and environmental stress tolerance, with the ability to prevent other yeast invaders from getting established in the fermentation tanks, providing stability to the distilleries’ industrial operations These desirable traits make PE-2 an ideal biological platform for the delivery of new technologies essential for second-generation (2 G) bioethanol production[8,9]. With a better comprehension of C5-metabolism, we point out iron as a new additive that can induce improvement on performance of xylose fermenting strains, suggesting new directions to increase the economic viability of second-generation technologies
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