Optimization of Saccharomyces cerevisiae central carbon metabolism for biomaterials production
Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
Shifting our oil-based economy to a bio-based economy is a critical component in the fight against climate change and for ensuring energy security. Improving the scalability of bio-based products by reducing production costs is an important part of this objective. We report here an attempt to increase the yield of mevalonate production, as mevalonate is a chemical of industrial interest. It can indeed be transformed into several high-value chemicals traditionally obtained from fossil resources. Some of these chemicals can be used as fuel additives or polymerized into compounds such as rubber.We used a kit of standardized genetic parts to build a gene deletion system based on CRISPR-Cas9 to delete several genes in one single step. Four parameters of the method were tested for improvement: (1) the length of the donor DNA; (2) the number of double-strand breaks per target gene; (3) the transcription mechanism of the guide RNAs; and (4) the delivery method of the donor DNA.The said gene deletion system was then used to delete eight genes hypothesised to generate by-products of the central carbon metabolism (ethanol and glycerol) in a Saccharomyces cerevisiae strain already engineered to produce mevalonate. No more than three genes were successfully deleted in the same strain. However, we report significant increases in the titer of mevalonate production (13.3 g/L final concentration, a 54% increase) and in the molar yield (7.94% of theoretical mass yield, a 50% increase) by a adh1Δ gpd1Δ gpd2Δ triple mutant strain. Continued improvement in the same strain with further deletions of the other alcohol dehydrogenases is expected to lead to a bio-based production of mevalonate in S. cerevisiae that is competitive with current fossil-based methods.
Place, publisher, year, edition, pages
2016. , 87 p.
Mevalonate, biomaterials, biofuels, energy security, climate change, global warming, CRISPR, Cas9, synthetic biology, Saccharomyces cerevisiae, Golden Gate, ethanol, glycerol, ADH, GPD, metabolic engineering, gene deletion
Bio Materials Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-307934OAI: oai:DiVA.org:uu-307934DiVA: diva2:1049233
Visolis Inc; Lawrence Berkeley National Laboratory; University California Berkeley
Master Programme in Molecular Biotechnology
2016-10-25, C8:305, Husargatan 3, Uppsala, 13:00 (English)
Lee, Brian, PhDDugar, Deepak, PhD
Henriksson, Lena, PhD