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Development and Validation of Yeast Biosensors for Signaling Pathways

Tueros Farfan, Felipe Gonzalo LU (2016) KMBM01 20152
Applied Microbiology
Biotechnology
Department of Physics
Abstract
Efficient bioconversion of the pentose xylose is vital for the development of competent lignocellulose biorefineries. Although many proper attempts of metabolic engineering for recombinant xylose utilization have been made in Saccharomyces cerevisiae, growth on xylose is still far from being optimal. Previous findings point out a more complex explanation to this feature involving interactions between xylose and signaling pathways. To assess this question we have developed and validated a panel of GFP yeast biosensor strains for the characterization of three signaling pathways (Snf3/Rgt2, Snf1/Mig1, cAMP/PKA) when subjected to different xylose conditions. Interestingly, the presence of 50 g/L xylose alone did not trigger any effect on these... (More)
Efficient bioconversion of the pentose xylose is vital for the development of competent lignocellulose biorefineries. Although many proper attempts of metabolic engineering for recombinant xylose utilization have been made in Saccharomyces cerevisiae, growth on xylose is still far from being optimal. Previous findings point out a more complex explanation to this feature involving interactions between xylose and signaling pathways. To assess this question we have developed and validated a panel of GFP yeast biosensor strains for the characterization of three signaling pathways (Snf3/Rgt2, Snf1/Mig1, cAMP/PKA) when subjected to different xylose conditions. Interestingly, the presence of 50 g/L xylose alone did not trigger any effect on these signaling pathways, however, when having a mixed condition of 50 g/L xylose and 5 g/L glucose higher induction patterns were observed than in glucose 5 g/L for the low affinity hexose transporters (HXT2 and HXT4) and the SUC2 gene. Together these results support the hypothesis that xylose alone produces a true starvation response in non-engineered W303 S. cerevisiae strains instead of a non-fermentable carbon response. Furthermore, sufficient information has been acquired to propose that mixed conditions of glucose and xylose produce a more profound effect in signaling that should be investigated further. (Less)
Popular Abstract
Baker’s yeast Saccharomyces cerevisiae has for years been the ideal species for expression and production of numerous chemicals, such as bioethanol, bioplastics and genetically engineered proteins. Nowadays, interest to implement this species in the lignocellulose (main component of plant matter) biorefinery industry is clear, due to its known robustness and capacity to grow in environments that are toxic to many other microorganisms. Importantly, lignocellulose is a complex carbohydrate matrix, commonly degraded by enzymes to produce a solution rich in sugars (monosaccharides), being the hexose glucose and the pentose xylose the two major components. Although great efforts have been made to genetically modify S. cerevisiae to uptake and... (More)
Baker’s yeast Saccharomyces cerevisiae has for years been the ideal species for expression and production of numerous chemicals, such as bioethanol, bioplastics and genetically engineered proteins. Nowadays, interest to implement this species in the lignocellulose (main component of plant matter) biorefinery industry is clear, due to its known robustness and capacity to grow in environments that are toxic to many other microorganisms. Importantly, lignocellulose is a complex carbohydrate matrix, commonly degraded by enzymes to produce a solution rich in sugars (monosaccharides), being the hexose glucose and the pentose xylose the two major components. Although great efforts have been made to genetically modify S. cerevisiae to uptake and metabolize xylose, its growth on this pentose is still less efficient than on glucose, and thus not economically feasible for the industrial production scale.

Many advanced technological techniques and tools have been used to produce strains with higher capabilities in terms of xylose transport and growth. However, knowledge about how this yeast senses the presence of xylose is still mostly unknown. Being able to understand the role that xylose has outside and inside the cells could open the door for the identification of new targets for genetic manipulation that would aid the development of a tailor-made strain for efficient growth on xylose.

This Master Thesis project is part of a current research project at Applied Microbiology, which aims to unveil the effect of xylose in yeast physiology. For this purpose a panel of biosensors strains have been developed and validated to massively screen the effect that different extracellular xylose conditions have on three different intracellular mechanisms. These mechanisms are involved in sugar sensing, metabolism and stress response. The biosensor strains were constructed by isolating regulatory regions (promoters) from genes affected by these mechanisms and the green fluorescent protein gene. By measuring the different levels of green light emitted by the cells exposed to different xylose conditions valuable data was acquired, much of which would have been a complex challenge with traditional techniques.

Our findings suggest that S. cerevisiae is unable to sense the presence of xylose in the surroundings. However, interesting patterns of induction were observed in the hexose-sensing pathway when xylose and glucose were both present in the growth medium. This could imply that hexose transporters induced by glucose could have a transient level of transportation for xylose, and therefore, the signaling mechanism could be affected by intracellular xylose concentrations. Further experiments are still necessary to be certain that intracellular xylose is able to affect any physiological traits in S. cerevisiae. (Less)
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author
Tueros Farfan, Felipe Gonzalo LU
supervisor
organization
course
KMBM01 20152
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Xylose, yeast, signaling, Snf3/Rgt2, Snf1/Mig1, cAMP/PKA, flow cytometry, high-throughput, GFP
language
English
id
8875515
date added to LUP
2016-06-07 13:01:20
date last changed
2016-12-07 04:07:57
@misc{8875515,
  abstract     = {Efficient bioconversion of the pentose xylose is vital for the development of competent lignocellulose biorefineries. Although many proper attempts of metabolic engineering for recombinant xylose utilization have been made in Saccharomyces cerevisiae, growth on xylose is still far from being optimal. Previous findings point out a more complex explanation to this feature involving interactions between xylose and signaling pathways. To assess this question we have developed and validated a panel of GFP yeast biosensor strains for the characterization of three signaling pathways (Snf3/Rgt2, Snf1/Mig1, cAMP/PKA) when subjected to different xylose conditions. Interestingly, the presence of 50 g/L xylose alone did not trigger any effect on these signaling pathways, however, when having a mixed condition of 50 g/L xylose and 5 g/L glucose higher induction patterns were observed than in glucose 5 g/L for the low affinity hexose transporters (HXT2 and HXT4) and the SUC2 gene. Together these results support the hypothesis that xylose alone produces a true starvation response in non-engineered W303 S. cerevisiae strains instead of a non-fermentable carbon response. Furthermore, sufficient information has been acquired to propose that mixed conditions of glucose and xylose produce a more profound effect in signaling that should be investigated further.},
  author       = {Tueros Farfan, Felipe Gonzalo},
  keyword      = {Xylose,yeast,signaling,Snf3/Rgt2,Snf1/Mig1,cAMP/PKA,flow cytometry,high-throughput,GFP},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development and Validation of Yeast Biosensors for Signaling Pathways},
  year         = {2016},
}