Strains and cultivation conditions
Media for evaluating biomass accumulation were developed using a
previously described strain expressing G-CSF under control of the pAOX1
promoter (Crowell et al., 2020). 24-well plate screens were conducted as
described previously, except cells were grown on a labForce shaker and
were only sampled 24 hours after inoculation (Matthews et al., 2017a).
BMGY, BMMY, and RDM media were formulated as described previously for
shake flasks (Matthews et al., 2017a). All cultivations were inoculated
from a working cell bank at an initial cell density of 0.1 OD/mL. For
each working cell bank, cells grown in 1 L shake flasks with a 200 mL
working volume of RDM were harvested during exponential growth (4-5.5
OD/mL) via centrifugation at 1500 rcf for 4 minutes at 23oC
and resuspended in an equal volume mixture of RDM and 50 v/v% glycerol.
This mixture was then distributed into 700 µL aliquots and stored at -80oC, resulting in a cell density of ~30
OD/mL for the cell bank.
Media for evaluating enhanced production were developed using a strain
expressing a rotavirus-derived subunit vaccine candidate, P[8],
under the control of the pAOX1 promoter described previously (Dalvie et
al., 2020). Biomass accumulation proceeded for 24 hours; cells reached
an initial induction density of ~20 OD/mL. Cultures were
then exchanged into production media and allowed to produce protein for
an additional 24 hours. Supernatant was harvested by centrifugation at
1500 rcf for 4 minutes at 23 oC and filtered using a
Captiva 96 well 0.2 µm filter plate (Agilent Technologies, Santa Clara,
CA) prior to titer measurement by RP-UHPLC.
Media components and supplements were purchased from Sigma-Aldrich, St.
Louis, MO, unless otherwise indicated in the supporting information. A
table of supplement and stock solutions with screening concentrations is
also included in the supporting information. During modular
optimization, all media were prepared in high throughput using an
Opentrons OT-2 liquid handler (Opentrons, Brooklyn, NY, software version\(\geq\)3.16.1) using Openblend. Modular media blending code and
instructions for setup and operation are provided in the Openblend
package (https://github.com/abiedermann/openblend_public). For
consistency, media used in final head-to-head comparisons were prepared
in bulk and filter sterilized through a 0.2 \(\mu\)m benchtop filter.