Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

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.

Mushroom cultivation in the circular economy

“…Chemical and enzymatic treatment resulted in about 40% SMS residue and 25% total reducing sugars, most of which being glucose and xylose. One gram of these sugars can be converted to about 0.5 g ethanol (Vieira dos Santos et al 2016 ; Gutiérrez-Rivera et al 2012 ). Thus, 1 ton of SMS in these examples would yield up to 150 kg of ethanol.…”

The yeasts of the genus Spathaspora: potential candidates for second‐generation biofuel production

“…In contrast, modified S. cerevisiae strains expressing XYL1 genes from Sc. stipitis , the most widely used eukaryotic donor of xylose pathway‐encoding genes used to engineer S. cerevisiae (Moysés et al ., ; Kwak and Jin, ), or xylose isomerase (XI)‐encoding genes ( xylA ), responsible for converting xylose to xylulose directly, have shown ethanol yields up to 0.39 and 0.46 g g −1 , respectively (Li et al ., ; dos Santos et al ., ; Kwak and Jin, ). These works obtained the transformants through more elaborate techniques, such as codon‐optimization, mutagenesis and adaptative evolution, even using, in some cases, robust industrial S. cerevisiae strains as host systems.…”

“…However, despite extensive knowledge of the regulatory networks controlling glucose metabolism in yeast, less is known about the regulation of xylose metabolism and how to reprogramme S. cerevisiae to ferment this pentose at rates comparable with those of glucose (Sato et al ., ). Engineered and evolved S. cerevisiae strains depend upon epistatic interactions among specific genes to rapidly metabolize xylose aerobically or anaerobically, and genetic mutations in these genes are responsible for synergistically altering metabolic pathways to improve the rate of xylose conversion (dos Santos et al ., ; Sato et al ., ). Nonetheless, these mutations cause loss of function in important genes, such as HOG1 and IRA2 , both of which are known to result in reduced stress tolerance (Sato et al ., ).…”

Genomic and phenotypic characterization of a refactored xylose-utilizing Saccharomyces cerevisiae strain for lignocellulosic biofuel production

“…Therefore, manganese ions accumulated in the cells via this transporter could be more easily incorporated into manganese-requiring enzymes such as xylose isomerase [ 25 ]. In addition to manganese ions, the increased cellular iron cation concentration resulting from ISU1 downregulation (0.34-fold, p < 0.05) could also increase xylose isomerase activity and other cellular processes beneficial for xylose metabolism [ 26 ]. Although iron cations are not preferred by xylose isomerase, they play essential roles as cofactors for several cellular processes and have been reported to boost xylA activity [ 26 , 27 ].…”