Global changes in gene expression associated with phenotypic switching of wild yeast

BACKGROUND: Saccharomyces cerevisiae strains isolated from natural settings form structured biofilm colonies that are equipped with intricate protective mechanisms. These wild strains are able to reprogram themselves with a certain frequency during cultivation in plentiful laboratory conditions. The resulting domesticated strains switch off certain protective mechanisms and form smooth colonies that resemble those of common laboratory strains. RESULTS: Here, we show that domestication can be reversed when a domesticated strain is challenged by various adverse conditions; the resulting feral strain restores its ability to form structured biofilm colonies. Phenotypic, microscopic and transcriptomic analyses show that phenotypic transition is a complex process that affects various aspects of feral strain physiology; it leads to a phenotype that resembles the original wild strain in some aspects and the domesticated derivative in others. We specify the genetic determinants that are likely involved in the formation of a structured biofilm colonies. In addition to FLO11, these determinants include genes that affect the cell wall and membrane composition. We also identify changes occurring during phenotypic transitions that affect other properties of phenotypic strain-variants, such as resistance to the impact of environmental stress. Here we document the regulatory role of the histone deacetylase Hda1p in developing such a resistance. CONCLUSIONS: We provide detailed analysis of transcriptomic and phenotypic modulations of three related S. cerevisiae strains that arose by phenotypic switching under diverse environmental conditions. We identify changes specifically related to a strain’s ability to create complex structured colonies; we also show that other changes, such as genome rearrangement(s), are unrelated to this ability. Finally, we identify the importance of histone deacetylase Hda1p in strain resistance to stresses

CRISPR–Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains

There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR–Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories. Keywords: CRISPR–Cas9, Genome editing, Industrial yeast, Biorefineries, Chemical productio

EasyClone 2.0:expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains

Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10295-015-1684-8) contains supplementary material, which is available to authorized users