Genetic modules for α-factor pheromone controlled growth regulation of Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a commonly used microorganism in the biotechnological industry. For the industrial heterologous production of compounds, it is of great advantage to work with growth-controllable yeast strains. In our work, we utilized the natural pheromone system of S. cerevisiae and generated a set of different strains possessing an α-pheromone controllable growth behavior. Naturally, the α-factor pheromone is involved in communication between haploid S. cerevisiae cells. Perception of the pheromone initiates several cellular changes, enabling the cells to prepare for an upcoming mating event. We exploited this natural pheromone response system and developed two different plasmid-based modules, in which the target genes, MET15 and FAR1, are under control of the α-factor sensitive FIG1 promoter for a controlled expression in S. cerevisiae. Whereas expression of MET15 led to a growth induction, FAR1 expression inhibited growth. The utilization of low copy number or high copy number plasmids for target gene expression and different concentrations of α-factor allow a finely adjustable control of yeast growth rate.

Keywords: Saccharomyces cerevisiae; growth regulation; pheromone response; synthetic biology; α‐factor.

FIGURE 1

Approach for pheromone induced growth restoration (A) or growth inhibition (B) of S. cerevisiae BY4741 Δbar1Δfar1. Expression of the target genes MET15 or FAR1 was under control of the α‐factor sensitive FIG1 promoter. Met15p complements the methionine auxotrophy in the expression strain (A), whereas Far1p induces cell cycle arrest by inhibiting the Cdc28p/Cln1/2p complex (B). The p416 low copy number and the p426 high copy number plasmids, which differ in their replication origins (p416: CEN/ARS; p426: 2μ origin), served as vectors [33].

FIGURE 2

Expression analysis of S. cerevisiae BY4741 Δbar1Δfar1 carrying plasmid p416FIG1‐MET15‐HA₃ (A), p426FIG1‐MET15‐HA₃ (B), p416FIG1‐FAR1‐HA₃ (C) or p426FIG1‐FAR1‐HA₃ (D). Strains were cultivated for 24 h without (‐ α) or with 0.25 µM synthetic α‐factor (+ α). Soluble protein fractions were separated in a 10% SDS‐PAGE and Western blot analysis was performed using HA₃‐specific antibodies. The arrow indicates the recombinant target proteins Met15p‐HA₃ (Mw calc. ∼ 53 kDa) and Far1p‐HA₃ (Mw calc. ∼ 99 kDa), respectively. Total protein amount was visualized in the protein gels by colloidal Coomassie staining (lower panels).

FIGURE 3

α‐Factor dependent growth rescue of methionine auxotroph S. cerevisiae FIG1‐MET15 expression strains. Yeasts carrying p416 low copy number plasmid (A, B) as well as the p426 high copy number plasmid (C, D) were analyzed. For the halo assay (A, C), the filter paper was either soaked with 5 µg of synthetic α‐factor (+ α) or with water (‐ α). Plates were incubated for 96 h before evaluation. For the nephelometric measurements (B, D), growth of the strains was monitored for 40 h after the addition of different α‐factor concentrations (0 µM to 1.0 µM). As positive control, methionine was added directly to the medium (MET). Curves represent mean values (n = 6) and error bars indicate standard deviations (± SD). Growth rates were compared with the values of the control (0 µM α‐factor) by unpaired t‐test and are marked if significant (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001).

FIGURE 4

α‐Factor‐dependent FAR1 induced growth inhibition of S. cerevisiae. Growth of the FIG1‐FAR1 expression strains carrying the p416 low copy number plasmid (A, B), or the p426 high copy number plasmid (C, D), as well as a control strain possessing a functional, natural FAR1 gene (E, F) was analyzed. Halo assays (A, C, E) were performed using either 5 µg of synthetic α‐factor (+ α) or water (‐ α). Plates were incubated for 96 h before evaluation. For the nephelometric measurements (B, D, F), growth of the strains was monitored for 40 h after the addition of different α‐factor concentrations (0 µM to 1.0 µM). Curves represent mean values (n = 6) and error bars indicate standard deviations (± SD). Growth rates were compared with the values of the control (0 µM α‐factor) by unpaired t‐test and are marked if significant (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001).