Engineering Success

Engineering success of Saccharomyces cerevisiae

In the implementation and experimental design of MonChassis, we were driven by the general principle of synthetic biology - design, build, test, and learn (DBTL).

Design

We engineered the yeast’s mevalonate pathway to overcome its rate-limiting steps. By introducing additional heterologous genes and silencing endogenous genes, we modified the pathway to suit our needs. Our goal was to increase geranyl diphosphate (GPP) production in Saccharomyces cerevisiae, as it acts as an essential precursor for monoterpenoid synthesis such as α-pinene. Hence, we engineered the cytosolic pathway, but also targeted another cell compartment. Since physiological conditions in peroxisomes are favorable for GPP production, we hypothesize that an increased α-pinene concentration is observed if the corresponding synthesis pathway is relocated there.

Build

Since we decided to clone all genes using the RFC1000 modular cloning system (MoClo), we designed specific overhangs for our promoter, gene of interest and terminator. We added them to our genes via Polymerase Chain Reaction (PCR) to generate our level 0 plasmids. Those form the basis on which we build multiple parts and vectors that can be easily assembled in the correct order. To verify our design, we cloned in silico using SnapGene software to simulate composition models according to the actual process in the laboratory.

Test

GC-MS analysis was chosen as a suitable assay to evaluate our metabolically modified yeast strains. Corresponding yeasts were cultured to achieve high cell densities, followed by extraction of α-pinene to obtain a high concentration. Using previously assigned internal standards for GC-MS measurements, we quantified final α-pinene levels and thus tested our strains.

Learn

After obtaining our GC-MS results, we compared our yeast strains equipped with different heterologous genes for α-pinene production. When testing our previous hypothesis, we realized that this was not consistently met. To draw actual conclusions from strain comparison, we need to ensure uniform conditions in their generation. At the metabolic level, this implies that α-pinene synthesis proceeds via the same precursors. Based on this, we can use our self-developed software in future approaches to specifically identify additional metabolic targets for further optimization [model].

Fusion of afraGFP with PTS1 for localization in peroxisomes

For verification of the general functionality of the C-terminal SKL peroxisomal targeting signal-1 (PTS1) described by Dusséaux et al., (2020), Agaricia fragilis-derived afra green fluorescent protein (afraGFP) originated from the iGEM distribution (BBF10K_003348) was fused with a C-terminal PTS1 tag according to our general cloning strategy. The ScpTEF promoter was selected for strong gene expression. As a control, an afraGFP shuttle construct lacking a C-terminal PTS1 tag was designed to accumulate in the cytosol. Subsequent yeast transformation was performed utilizing the shuttle vectors that resulted from our partnership with the iGEM Team TU Dresden. The fluorescence microscopy of transformed yeast cells was carried out in cooperation with the research group of Dr. Maria Bohnert at the Institute of Cell Dynamics and Imaging.

Yeast cultures were inoculated with overnight cultures incubated shaking at 30 °C for two hours prior to microscopy. To adhere the cells, the wells of an 8-well glass bottom were primed with concanavalin A. 50 µl were added to each well and discarded after 10 minutes of incubation. Subsequently, 100 µl of cell suspension were added and removed after a 10-minute adhesion time. Cells were covered with 600 µl of fresh Sc-all medium. Imaging was performed using a Nikon N-SIM microscope with a 100x objective and a CMOS camera. A wavelength of 488 nm was used for excitation and emission spectra was captured at 510 nm with a bandwidth of 15 nm. Exposure time was set to 100 ms and laser intensity to 2%.

Control cells expressing the afraGFP without the PTS1 tag showed bright, diffuse fluorescence throughout the cell, indicating cytosolic GFP localization (Fig. 2, A). In contrast, the introduction of afraGFP_PTS1 resulted in localization of fluorescence signal in distinct bright spots, indicating functional targeting to peroxisomes (Fig. 1, B), consistent with Dusséaux et al., (2020). Moreover, microscopy of S. cerevisiae wild-type CEN.PK2-1C as a negative control displayed no fluorescence signal at all. Hence, we could demonstrate proper function of our shuttle vectors and the PST1 tag resulting in strong expression and targeted translocation. Therefore, we included the PTS1 tag accordingly into our constructs for targeted monoterpenoid production in yeast peroxisomes.

Engineering success of DNPH assay

We developed a colorimetric assay as a part of mutational screening for protein engineering of a cytochrome P450 monooxygenase (BM3). This rapid mutation screening method for the detection of verbenone is based on the reaction of 2,4-dinitrophenylhydrazine (DNPH) with enones like verbenone that leads to a red complex formation of a hydrazone (Fig. 3). These can be examined visually or quantified by photometric measurements in a 96-well plate (Liu et al., 2016). To avoid extraction of verbenone from the cells by lysis, we used the autodisplay system (Jose et al., 2012). As the BM3 is thus presented on the surface of Escherichia coli cells, catalysis of α-pinene to verbenone takes place in the medium. During the process, we encountered several challenges and constantly improved our assay to properly quantify our produced verbenone. Among others, we adapted the composition of the DNPH reagent to form a soluble mixture and avoid red precipitates that interfere with the measurement. In total, we went through three Design-build-test-learn (DBTL) cycles (Fig. 4) and further development should tackle the interference of nicotinamide adenine dinucleotide phosphate (NADPH) in photometric detection. In summary, we successfully showed that the assay is suitable for verbenone quantification and could be used as a tool for improving the BM3 through mutational screening if the conversion ratio of verbenone is exceeding 10%.