Technical Session 22: Yeast IV Session
Johnathon B Layfield, NC State University, Raleigh, NC, USA
Co-author(s): Lucas Vann and John Sheppard, NC State University, Raleigh, NC, USA
ABSTRACT: In conventional batch and continuous
fermentation, the cell cycles of individual yeast are randomized within
the population, and the observed metabolic performance is the result of
an averaging effect. Synchronous cellular growth is characterized by
cells in a population aligned with respect to their metabolic processes
traversing the cell cycle and dividing mostly in unison. Thus,
synchronized populations of cells can be used as a tool to reveal more
precisely how an individual cell reacts under different environmental
conditions (Sheppard et al, 1999). S. cerevisiae is a unique
organism in that it serves as a model eukaryote for academic and
industrial research. Thus, a method for inducing and storing a
synchronous yeast culture for rapid use in metabolic studies is
advantageous to both academia and industry. In this study, a novel
method for inducing and retaining cell cycle synchronization in yeast
cells (diploid- and polyploid-type cells) was developed. This technique
is derived from the continuous phased-culture induction method (Dawson,
1969). The original induction method was based on a cyclical process in
which one-half of the cell culture was harvested and a fresh nutrient
solution added to replace the harvested volume at a period corresponding
to cell doubling. This replenishment of sufficient nutrients only for
cell doubling resulted in the growth and division of a single division
of cells prior to the beginning of a new cycle. After about six such
cycles, the cells became aligned with respect to their cell cycles and
began dividing synchronously. Our new method begins with a small volume
and doubles it each cycle by periodically adding fresh nutrient
solution, without having to remove any cells. This adaptation is better
suited for industrial applications, such as seed expansion, due to its
relative simplicity and equivalent effectiveness in producing cell
synchrony. This was demonstrated by measuring the synchrony index of
both S. cerevisiae 288C (diploid) and the brewing strain London
ESB 1986 (polyploid), which matched that produced using the conventional
continuous phased method (71 and 83%, respectively). We have also shown
that synchronized cells can be stored for later use in glycerol at
–80°C for at least 2 weeks without significant loss in synchrony. Small
volumes (1.5 and 10 mL) of both S. cerevisiae 288C and London ESB
1986 showed no loss of synchrony from the original synchrony procedure.
However, as the volume of a synchronous stock increased to 50 mL,
certain aspects of synchrony (depending on the strain) seemed to
degrade. The extra time required for both freezing and thawing the
larger synchronous stocks is thought to be the cause. However, for most
metabolic studies, freezing at –80°C is a viable approach for retaining
cell synchronization in S. cerevisiae.
n JASBC,
where his work on desiccation tolerance in lager yeast was selected as
an “Editor’s Pick” (August 2011). He also gave an oral presentation at
the 2009 ASBC Annual Meeting in Tuscon, AZ.
VIEW PRESENTATION 228
