Improving xylose utilization through investigation of sugar signaling in S. cerevisiae

Abstract

Lignocellulose is an important renewable feedstock for the sustainable production of bioethanol and other chemicals. Xylose is the second most abundant sugar in lignocellulose. However, xylose cannot be utilized by many industrially used microorganisms like S. cerevisiae. Therefore, large efforts have been made to express heterologous pathways for xylose utilization in S. cerevisiae and increase efficiency thereof. In this thesis, two attempts at improving xylose utilization were carried out focusing either on the XR-XDH pathway or the Weimberg pathway.
In yeast engineered with the XR-XDH pathway, sugar signaling constituents have previously been targeted by metabolic engineering with promising results. More specifically, modifications that increase protein kinase A (PKA) activity have been shown to increase the efficiency of xylose utilization. PKA activity is regulated by fructose-1,6-bisphosphate (F16bP) levels in the cell, which in turn is positively regulated by fructose-2,6-bisphosphate (F26bP) levels. Consequently, increased levels of F26bP have been hypothesized to allosterically activate the production of F16bP which in turn might activate PKA and thereby positively impact the sugar signaling for xylose utilization. This hypothesis was investigated in this thesis through the overexpression of PFK27, the gene responsible for the formation of F26bP. However, only minor effects on the sugar signaling were observed and the xylose utilization was not affected. Notably, the nourseothricin (clonNAT) resistance encoded on the overexpression plasmids negatively affected cell growth on xylose, possibly altering the results of PFK27 overexpression.
The functional Weimberg pathway, another metabolic route for xylose utilization, was recently established in S. cerevisiae. However, efficiency remains low and growth on xylose as the sole carbon source is not possible for these strains. In this thesis, the genes of the upper Weimberg pathway, xylB and xylC, were overexpressed and xylB was exchanged for the recently discovered M. thermophila Mtxyd1 to increase efficiency of the Weimberg pathway. While the overexpression of xylB together with xylC achieved the desired effect and improved growth of the Weimberg strain already, the overexpression of Mtxyd1 increased the efficiency of xylose utilization to an even higher extent. Besides the pathway engineering, the sugar signaling response through the SNF1/Mig1p pathway in a strain with a xylose oxidative pathway was investigated through a GFP-coupled biosensor for the first time.

Popular Abstract

Green chemicals from green yeast
Baker's yeast isn't just for baking anymore - it's now a key player in the fight against climate change, transforming agricultural waste into biofuels. With the help of genetic engineering, we can introduce completely new “production lines” in the yeast and optimize them.
Most people have used yeast, or more specifically the baker’s yeast Saccharomyces cerevisiae, to bake delicious bread or cinnamon buns. Some might even have brewed their own beer with it. What many people do not know is that we can also use yeast for the production of a whole array of chemicals, including biofuels. Not only that, but yeast can also produce chemicals from renewable feedstocks, helping us to produce more sustainably and ultimately, fight climate change. One such renewable feedstock is lignocellulose. Lignocellulose is the “wood-like” part of plants. It’s a common waste product from agriculture or the pulp and paper industry. Lignocellulose contains a lot of the sugar xylose, a monosaccharide like the more commonly known glucose or fructose. The problem with xylose is that natural yeast cannot eat it. Luckily, we’ve used genetic engineering to introduce enzymes to the yeast, basically give it new machines or tools. With these enzymes, we can create entire metabolic pathways, “production lines” that connect the new tools. This way we can help the yeast to utilize xylose.
One metabolic pathway for xylose utilization is the Weimberg pathway. Part of this project was to make this “production line” more efficient by overexpressing enzymes, adding more “machines”, and exchanging one of them. The efforts made were successful, especially by switching one enzyme the yeast was able to consume xylose more efficiently and grow faster.
Another problem for yeast when consuming xylose is how it senses nutrients. Yeast is used to eating glucose, it does not recognize the nutrients in xylose and still feels like it is starving even though xylose is being consumed. But while the yeast is starving it tries to save energy: it grows slower and therefore produces all enzymes and also our desired products slower and less efficient. Whether the yeast is starving or satiated depends, among other things, on the intermediates formed while the yeast metabolizes sugar. By producing more of one of these intermediates, I tried to alter the yeast’s response to xylose. Unfortunately, this was unsuccessful and other ways to change the xylose response must be explored in the future.

• author: Egeler, Nina
• supervisor: Ed van Niel; Viktor Persson
• organization: Applied Microbiology; Biotechnology; Biotechnology (MSc)
• alternative title: Förbättrad xylosanvändning genom studier av sockersignalering i S. cerevisiae
• course: KMBM01 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Xylose, Saccharomyces cerevisiae, Lignocellulose, PFK27, Weimberg pathway, Sugar signaling, applied microbiology
• language: English
• id: 9165154
• date added to LUP: 2024-06-20 14:42:32
• date last changed: 2024-06-20 14:42:32