Increasing production of 3-hydroxypropionic acid by modulating the activity of acetyl-CoA carboxylase 1 in Saccharomyces cerevisiae
Examensarbete för masterexamen
Koendjbiharie, Jeroen Girwar
3-Hydroxypropionic acid (3-HP) is an attractive chemical that can be produced from biomass, because it can be used as a precursor for a lot of commercially interesting products and thus contribute to a more sustainable, bio-based economy. The synthesis of 3-HP has already previously been shown to be possible in Saccharomyces cerevisiae via the heterologous expression of malonyl-CoA reductase (MCRCa). 3-HP production was further improved via the overexpression of an acetyl-CoA carboxylase 1 double mutant that is no longer phosphorylated by Snf1 (Acc1**). However, malonyl-CoA is also a precursor for fatty acids, and the increased fatty acid production associated with the overexpression of Acc1** is undesirable when aiming to produce 3-HP, and could, at least partly, originate from the presumed association of Acc1 with the cytosolic side of the ER membrane. It was hypothesized that by bringing the two enzymes, Acc1** and MCRCa, together with a scaffold, the 3-HP production could be increased by channeling the acetyl-CoA directly towards 3-HP and minimizing the leakage towards fatty acid synthesis. To investigate the use of a scaffold, the following two research questions were proposed: 1) Is it possible to abolish the association of Acc1 with the ER membrane by fusing Acc1 to a docking domain? 2) Is the 3-HP production improved when Acc1 is linked to MCRCa via a protein scaffold? Due to the limited time of the project it was not possible to adequately address the proposed questions. However, it was clear that it was not possible to effectively abolish the presumed association of Acc1** with the ER membrane by fusing dockerin a (Da) from C. thermocellum endoglucanase to the C-terminus of Acc1**, as it rendered to enzyme non-functional, most likely as the result of incorrect folding. The non-functionality of Acc1**-Da was not an inherent feature of Acc1** when fusing a protein domain to the C-terminus, as Acc1** was still functional with an enhanced green fluorescent protein (eGFP) fused to the C-terminus, as well as to the N-terminus, making the use of a scaffold still a viable strategy to improve the production of 3-HP and other malonyl-CoA derived products. In parallel, the extent in which Acc1** activity is regulated by the efficiency of its biotinylation by Bpl1 was also investigated. Cultivating S. cerevisiae with Acc1** integrated in the genome in the presence of different biotin concentrations did not lead to an increased production of 3-HP, both with and without overexpressing BPL1 via a multiple copy plasmid. This suggested that Acc1** activity is not significantly regulated by its biotinylation. However, to be conclusive about the ACCase activity, it should be directly measured, rather than indirectly, as the 3-HP production is affected by more than just the activity of Acc1**.
Livsvetenskaper , Biologiska vetenskaper , Bioinformatik och systembiologi , Life Science , Biological Sciences , Bioinformatics and Systems Biology