טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIfraimov Ekaterina
SubjectTool Development for Genetic Improvement of S.Cerevisia
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yechezkel Kashi


Abstract

Today, yeast are the most important microorganisms in terms of commercial utilization. In addition to food industry, the biotechnology industries use yeasts for the production of important enzymes, chemicals, therapeutic proteins and recently for biofuel production. In industrial practice, optimal physiological conditions can never occur, and the yeast are exposed to a number of stress conditions reducing yeast’ performance and viability. Thus, the ability to withstand an applied stress is a crucial characteristic of commercially used yeast.

It is estimated that about 99% of the potential biodiversity of yeast is still unknown. For the genetic improvement of S. cerevisiae industrial strains, we wanted to take advantage of the great biodiversity existing in nature.

Strain improvement of yeast relies on classical breeding and genetic crossing of two strains, followed by screening for progeny exhibiting enhanced properties of interest. Sexual breeding resulting in re-shuffling parental lines’ alleles to be randomly distributed among offspring. This effect creates large phenotypic differences among offspring. The more genotypically diverse the parental lines are, the more variability we enter to the gene pool. However, the more distinct the parental lines are, the more complicated the crossbreeding will be. The offspring of an interspecific cross are very often sterile, this prevents the movement of genes from one species to the other, keeping both species distinct.

Therefore, there is a need for an efficient screening and isolation methods of the sole viable hybrids between Saccharomyces cerevisiae and the selected species for crossbreeding. We believed that we could overcome sexual reproductive isolation through EZ-Ascospore Isolation process using Saccharomyces sensu stricto genus as proof of concept first.

We have established a step by step protocol for efficient recovery of the viable crossbreeding offspring, allowing genetic improvement by random interspecific sexual breeding. This protocol uses double selection: G418 and EZ ascospore isolation procedure to select the offspring from the mix of parental and undesired crossbreeding culture.  The protocol was successfully tested and S288c X Saccharomyces kudriavzevii Advanced intercross line population was created.  Enrichment step revealed individuals in this population with potent ethanol resistance abilities.

The implementation of this method and approach enables improvement of the productivity and performance of industrial strains in different fermentations. Obtaining of new resistant strains will allow industrial and research achievements. The resultant strains obtained by these improvement and screening protocols are by no means considered genetically modified organisms, this makes them applicable in the food and beverage industry.