Using CRISPR/Cas9 to regulate enzyme expression levels in the Weimberg pathway for optimal xylose consumption in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a commonly used industrial organism which cannot consume xylose naturally. This is a problem since xylose is a major component in renewable resources, for instance lignocellulose. The Weimberg pathway is a pathway that converts xylose into α-ketoglutarate. It has been successfully integrated into S. cerevisiae, enabling it to utilize xylose. The successful strain had the genes xylB, xylD, xylX (from Caulobacter crescentus) and ksaD (from Corynebacterium glutamicum) integrated with one copy of xylB and four copies of each of the others. In addition, the strain had the iron regulon repressor gene, FRA2, deleted to enhance the activity of the XylD enzyme.

In the present master thesis project, the aim was to find the optimal number of copies of the genes xylB, xylD, xylX and ksaD as well as what enzyme activities that are needed for the pathway to be functional. This was done by creating a pallet of strains that contained different copy numbers of the genes, using CRISPR/Cas9. Enzyme activity was measured before further analysis of biomass formation and metabolite production.

It was found that TMB CB 016 (xylB, xylD, 4x(xylX, ksaD)) had an activity in the coupled assay, measuring the activity of XylD, XylX and KsaD together. However, due to the problems with the assay the results are unreliable and need to be further investigated. The constructed strains did not grow as well as the original strain with the functional Weimberg pathway, nor were any of them able to consume xylose.

Popular Abstract

Trying to optimize the consumption of xylose in baker’s yeast

Baker’s yeast can not consume xylose naturally, but the Weimberg pathway has made it possible. This master thesis focuses on optimization of the Weimberg pathway for an optimal xylose consumption.

The world is becoming more sustainable. This includes the industries that are interested in using sustainable and renewable carbon sources in the production of chemicals. One of the promising renewable sources is called lignocellulose and it contains a high percentage of the sugar xylose. Baker’s yeast or Saccharomyces cerevisiae is a commonly used industrial organism, but it cannot consume xylose and instead prefers the sugar glucose. This means that if for example lignocellulose is used as the substrate, a major part of this cannot be used by the yeast.

Therefore, it is of great interest to make baker’s yeast able to consume xylose. This has successfully been done by introducing the Weimberg pathway into the yeast. The pathway consists of four enzymes called XylB, XylD, XylX and KsaD. These enzymes convert xylose into a compound called α-ketoglutarate that can be converted into a variety of chemicals. Introducing the pathway into the yeast means that the yeast obtains genes that work as a template for producing the four enzymes. What is interesting now is to optimize the pathway to make the yeast consume xylose in the most effective way.

In the present master thesis project the goal was to optimize the Weimberg pathway. This was achieved by looking at the number of copies of the genes coding for the four enzymes in the Weimberg pathway. A pallet of strains was constructed with different copy numbers of the genes. The strains were constructed using the engineering tool CRISPR/Cas9, which can make specific cuts in the genome and paste the gene of interest.

Once the strains had been constructed enzyme activity was measured. The enzyme activity of the enzymes XylD, XylX and KsaD were measured together. It was shown that one strain had higher activity of these enzymes than the existing strain with a working Weimberg pathway. This was very promising since that would mean that the pathway most likely worked in this strain and possibly perform even better than the existing working strain.

Unfortunately, the constructed strains grew poorly and none of them were able to consume any xylose. It is difficult to say if this is due to their poor growth or due to the Weimberg pathway not working (wrong copy numbers of the genes).
More research is needed, starting with constructing strains with better growth. It is of special interest to investigate the strain that had higher enzyme activity than the existing working strain. Progress in this area may contribute to a more sustainable, efficient and profitable use of for example lignocellulose in the industry.

• author: Heiling, Anna
• supervisor: Marie-F Gorwa-Grauslund; Celina Borgström
• organization: Applied Microbiology
• alternative title: Reglering av enzymnivåer i Weimberg pathway med hjälp av CRISPR/Cas9 för att optimera förbrukningen av xylose i Saccharomyces cerevisiae
• course: KMBM05 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry; Technology and Engineering
• keywords: Yeast, Weimberg pathway, xylose consumption, Applied Microbiology, Teknisk mikrobiologi
• language: English
• id: 9015858
• date added to LUP: 2020-06-12 17:15:44
• date last changed: 2020-06-12 17:15:44