Camarasa, C. | bioRxiv Science

bioRxiv2024

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

systems biology↗

bioRxiv2022

Regulated use of alternative Transcription Start Sites controls the production of cytosolic or mitochondrial forms of branched-chain aminotransferase in Kluyveromyces marxianus

Following a whole genome duplication (WGD) event approximately 100 million years ago, the yeast lineage from which the model Saccharomyces cerevisiae derives maintained two copies of genes where it was necessary to synthesise proteoforms with different sub-cellular localisation. In contrast, yeasts that did not undergo the WGD event have a single gene that must encode both proteoforms. We adopted an integrated in silico and experimental approach to study how this is achieved with BAT1, a gene that encodes mitochondrial and cytosolic forms of a branched chain aminotransferase (BCAT) in pre-WGD yeast such as Kluyveromyces marxianus. We determined that condition-specific regulation of alternative transcription sites gives rise to mRNA isoforms that differ at the 5end and that, when decoded, generate a mitochondrial or cytosolic proteoform. Furthermore, targeted mutants lacking specific transcription factors were generated to establish how this differentiation was regulated. As in S. cerevisiae, Gcn4 and Leu3 activated expression of the mRNA encoding the mitochondrial proteoform under conditions when branched chain amino acid synthesis was required. Unlike S. cerevisiae, however, K. marxianus lacked tight regulation of the mRNA encoding the cytosolic proteoform supporting the hypothesis that maintaining paralogous genes in post-WGD yeasts facilitated development of more sophisticated expression control mechanisms.

genetics↗