The Genetic Basis of Variation in Clean Lineages of Saccharomyces cerevisiae in Response to Stresses Encountered during Bioethanol Fermentations

Abstract

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained.

Expression of RCK2 MAPKAP (MAPK-activated protein kinase) rescues yeast cells sensitivity to osmotic stress

“…Rck2p is a serine/threonine protein kinase homologous to mammalian calmodulin kinases, which requires phosphorylation for activation, phosphorylation is transiently increased during osmotic stress or in strains overexpressing the HOG pathway [ 18 ]. A QTL analysis for yeast revealed that on chromosome XII there were genes responsible for osmotolerance, upon further examination of the QTL, RCK2 was found to be up-regulated in yeast cells under osmotic stress [ 5 , 19 ].…”

Selection of yeast strains for bioethanol production from UK seaweeds

“…Natural strains may lack the relevant metabolic pathways for the conversion of certain sugars into ethanol (Roesijadi et al 2010 ), and therefore, the maximum conversion of the mixed array of monosaccharides found in seaweed hydrolysates into bioethanol cannot be fully achieved. However, phenotypic screening of yeast strains may be employed and can be a useful tool with the potential to identify strains with desirable traits for bioethanol production (Greetham et al 2014a , b ). This was recently highlighted in the work of Wimalasena et al ( 2014 ), where candidate strains for efficient second generation bioethanol production were identified by employing a phenotypic microarray (PM) analysis to measure tolerance to stresses on Saccharomyces spp yeast during fermentation.…”

“…This was recently highlighted in the work of Wimalasena et al ( 2014 ), where candidate strains for efficient second generation bioethanol production were identified by employing a phenotypic microarray (PM) analysis to measure tolerance to stresses on Saccharomyces spp yeast during fermentation. There are many advantages of utilising a PM approach, which include the high output of informative data in a short amount of time, and avoiding the need to use traditional yeast methodologies (Greetham et al 2014a , b ). The PM technique gives an insight into the yeast cell’s metabolic activity against various media (e.g.…”

Identification of Bio-oil Compound Utilizing Yeasts Through Phenotypic Microarray Screening

“…The application of a novel phenotypic microarray (PM) has proved to be an efficient, accurate and rapid screening tool to shortlist candidate yeast strains across a number of different biotechnological applications. The PM approach has previously been demonstrated to be successful in identifying strains for efficient second generation bioethanol production [25,26], to measure tolerance to stresses on Saccharomyces spp. yeast during fermentation [27], to determine the metabolic profiling of yeast on different pre-treatment hydrolysates [28] and to identify yeast strains with abilities to metabolise monosaccharides derived from macroalgae [29].…”