Dormancy-to-death transition in yeast spores occurs due to gradual loss of gene-expressing ability.

Dormancy is colloquially considered as extending lifespan by being still. Starved yeasts form dormant spores that wake-up (germinate) when nutrients reappear but cannot germinate (die) after some time. What sets their lifespans and how they age are open questions because what processes occur-and by how much-within each dormant spore remains unclear. With single-cell-level measurements, we discovered how dormant yeast spores age and die: spores have a quantifiable gene-expressing ability during dormancy that decreases over days to months until it vanishes, causing death. Specifically, each spore has a different probability of germinating that decreases because its ability to-without nutrients-express genes decreases, as revealed by a synthetic circuit that forces GFP expression during dormancy. Decreasing amounts of molecules required for gene expression-including RNA polymerases-decreases gene-expressing ability which then decreases chances of germinating. Spores gradually lose these molecules because they are produced too slowly compared with their degradations, causing gene-expressing ability to eventually vanish and, thus, death. Our work provides a systems-level view of dormancy-to-death transition.

Establishment of a Novel Cell Surface Display Platform Based on Natural "Chitosan Beads" of Yeast Spores.

Cell surface display technology, which expresses and anchors proteins on the surface of microbial cells, has broad application prospects in many fields, such as protein library screening, biocatalysis, and biosensor development. However, traditional cell surface display systems have disadvantages: the molecular weight of phage display proteins cannot be too large; bacterial display lacks the post-translational modification process for eukaryotic proteins; yeast display is prone to excessive protein glycosylation and misfolding of multisubunit proteins; and the compatibility of Bacillus subtilis spore display needs to be further improved. Therefore, it is extremely valuable to develop an efficient surface display platform with strong universality and stress resistance properties. Although yeast surface display systems have been extensively investigated, the establishment of a surface display platform using yeast spores has rarely been reported. In this study, a novel cell surface display platform based on natural "chitosan beads" of yeast spores was developed. The target protein in fusion with the chitosan affinity protein (CAP) exhibited strong binding capability with "chitosan beads" of yeast spores in vitro and in vivo. Moreover, this protein display system showed highly preferable enzymatic properties and stability. As an example, the displayed LXYL-P1-2-CAP demonstrated high thermostability and reusability (60% of the initial activity after seven cycles of reuse), high storage stability (75% of original activity after 8 weeks), and excellent tolerance to a concentration up to 75% (v/v) organic reagents. To prove the practicability of this surface display system, the semisynthesis of paclitaxel intermediate was demonstrated and its highest conversion rate was 92% using 0.25 mM substrate. This study provides a novel and useful platform for the surface display of proteins, especially for multimeric macromolecular proteins of eukaryotic origin.

Purification of Yeast Spores to Investigate Their Dynamics of Activation.

Germination is an important developmental process that supports resumption of growth in dormant spores. The study of the mechanisms underlying germination requires a pure spore population devoid of other cell types. This article describes the sporulation of wild Saccharomyces cerevisiae and Saccharomyces paradoxus strains, and the isolation and purification of ascospores. We also describe a method to synchronously induce germination in a spore population as well as to measure spore activation. This procedure can be applied, for example, to the study of environmental conditions that trigger germination. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Sporulation Basic Protocol 2: Spore purification Basic Protocol 3: Germination induction Support Protocol 1: Flow cytometry analysis Support Protocol 2: Heat-shock resistance measurement.