title: Metabolic engineering and adaption of Saccharomyces cerevisiae for biotechnological applications
Creators: name: Höler, Sebastian
Publisher: name: UNSPECIFIED
Year: 2021

description: Yeasts have been used for decades as cellular platforms for various biotechnological applications. They are “generally regarded as safe” (GRAS) unicellular eukaryotic microorganisms, which undergo rapid and robust growth allowing straightforward handling and production of many functional eukaryotic proteins. The pairing of simple genetic manipulation together with direct functional screening assays furthermore provides versatile applications of yeast in metabolic and protein engineering. The combination of selection and screening assays with randomly mutated libraries furthermore offer the possibility of altering and probing the function of proteins of interest. This dissertation reports in five chapters applications of metabolic engineering in S. cerevisiae with the goal of tailoring these yeast cells for biotechnological applications. Chapter I reports the engineering of retinal-synthesis in a K+ uptake deficient trk1Δ/trk2Δ yeast strain. This strain should be employed as a simple cellular platform for the functional expression of rhodopsin-based channel proteins. Growth rescue experiments with this strain are possible, since these yeasts cannot grow in standard low potassium medium, due to a deletion of the major potassium uptake systems (Trkp1p and Trk2p). The data show that an entire metabolic pathway for retinal synthesis can be introduced in this yeast strain. Growth assays in liquid medium reveal the functionality of a channelrhodopsin-2 variant, a cation channel which requires retinal for its function. Yeast growth is observed in a blue light-dependent manner in standard low potassium medium, demonstrating that the cells express a blue light-dependent K+ conductance. In complementary experiments also blue light-dependent growth inhibition was observed in a medium with high NaCl concentrations, indicating that the blue light-activated channelrhodopsin allows Na+ influx, which results in high intracellular Na+ accumulation and eventually in growth inhibition. Chapter II describes a system in which a K+ uptake deficient trk1Δ/trk2Δ yeast strain is used as a platform to explore blue light-triggered expression of heterologous proteins. For this purpose a CRY2-CIB1-based light-activated transcription system was used in order to trigger expression of the small viral potassium channel KcvPBCV-1 in this K+ uptake deficient yeast. The data indicate that this system is able to generate light-triggered K+ uptake, resulting in growth restoration of this yeast strain in low potassium medium. In combination with a microfluidic system it was possible to monitor the expression kinetics of the channel after blue light stimulation. This kinetic analysis revealed that the channel protein production in cells occurred already within 15 20 minutes after stimulation. In Chapter III the same trk1Δ/trk2Δ yeast system and rescue experiments were used to test a putative channel function of the envelope protein of SARS-CoV-2 (EpSARS CoV 2). Growth assays show that expression of EpSARS-CoV-2 is able to restore yeast growth in a low potassium medium. These data, in combination with luminometric measurements, support the view that EpSARS-CoV-2 exhibits cation conductance, mainly for K+ and presumably also for Ca2+. The presented growth assay can be used to screen for inhibitors of EpSARS CoV 2. Chapter IV describes the development of novel auto-selection systems, based on the use of essential genes (OLE1 or CDC19) as selection markers. This strategy enables stable and robust selection of transformed yeasts in complex media, such as YPD. The host strains (ole1Δ or cdc19Δ) can be grown in complex medium supplemented with the low-cost compounds oleic acid (for ole1Δ) or lactic acid (for cdc19Δ), thus allowing easy culturing and handling prior to transformation. Upon transformation with DNA/plasmids containing the markers OLE1 or CDC19, transformants can be stably selected and propagated in complex medium without supplementation. These auto-selection systems might be useful for high volume applications, since this reduces expenses on medium and supplements such as antibiotics. A quantitative analysis indicated a near 100% retention of OLE1- and CDC19-based plasmids in complex YPD or SD medium. In the last chapter, Chapter V, a yeast-based screen was developed for the identification of potential inhibitors for Trypanosoma brucei and Trypanosoma cruzi stearoyl-CoA desaturases (SCDs), which provide a promising drug target for treating Trypanosoma infections. The ole1Δ yeast strain from the previous chapter cannot produce unsaturated fatty acids and can therefore not grow without supplementation of oleic acid. Plasmid based expression of either the endogenous S. cerevisiae SCD OLE1 or the Trypanosoma SCDs allows growth of the ole1Δ strain in standard yeast medium (YPD or SD). Reduction of the activity of Ole1p or either of the heterologously expressed SCDs, either by reduced expression or by inhibition of the proteins, results in a decreased content of unsaturated fatty acids, which leads to flocculation and growth inhibition of yeast. By testing three compounds, Cay 10566, SCD1 inhibitor A-939572 or PluriSIn 1, that showed inhibition of human SCDs, the present yeast-based screen identified strong differences in the potency of the potential inhibitors. Cay 10566, which has been shown to inhibit human and mouse SCD1, had no effect on either of the SCDs, while SCD1 inhibitor A-939572 and PluriSIn 1 showed strong inhibition of Ole1p and even stronger inhibition of the Trypanosoma SCDs. Therefore, the yeast system can be used to screen for and identify selective inhibitors of the Trypanosoma stearoyl-CoA desaturases, thus providing a basis for developing drugs for treatment of trypanosomiasis.