2. Materials and Methods
2.1 Strains and experimental
overview
An isogenic culture of the recombinant S. cerevisiae strain,
C.U17 was cultured in prolonged chemostat cultivations (Seresht et al.,
2013). The strain contains a 2µm vector with an expression cassette
encoding the Triose Phosphate Isomerase 1 (TPI1) promoter and a gene
encoding a single-chain insulin precursor. Both HIS3 and URA3 were used
as auxotrophic selection markers. The C.U17 strain is referred to as theinitial cell clone throughout the rest of the manuscript.After 271 hours of glucose-limited growth, end sample cells from one of
the cultivations were collected and stored as glycerol stocks at -80°C.
These cells are referred to as end sample cells . One of the
glycerol stocks were thawed, washed three times in PBS buffer and used
for FACS sorting (Fluorescence Activated Cell Sorting) of three
individual populations separated based on particle size measured as
forward scatter light area (FSC-A) (Figure 1A). 10,000 sorted cells from
each population were propagated in individual shake flasks with minimal
medium at 30°C, harvested at an OD600 of 20 and stored
as glycerol stocks at -80°C (Figure 1B). The glycerol stocks were used
to reinitiate new chemostat cultures with each of the FACS sorted
populations (Figure 1C).
2.2 Chemostat cultivations
Aerobic chemostat cultivations were performed in 0.5 L fully
instrumented and automatically controlled BIOSTAT® reactors (Sartorius
Stedim Biotech S.A, Germany). The strains were cultured in duplicates as
previously described in Wright et al., (2020) at a temperature of 28oC, pH of 5.9, aeration rate of 2 vvm and dilution
rate of 0.1 h-1. A minimal medium with a glucose
concentration of 75 g/L was used. Each cultivation was initiated with 8
hours batch phase and 52 hours fed-batch phase. Cell dry weight and
extracellular heterologous insulin production were measured each day as
previously described in Wright et al., (2020).
2.3 Fluorescent activated cell sorting
(FACS)
Samples for flow cytometry analysis were collected every day and stored
in glycerol at -80 °C. Prior to analysis, the samples were thawed and
washed three times in PBS buffer. Flow cytometry analysis and cell
sorting with respect to particle size (FSC-A) were performed using a
Sony Cell Sorter SH800S. 100.000 cells were analyzed in each sample
using a 100 µm microfluidic sorting chip. The samples were diluted to
obtain an event rate below 1000 eps. The raw flow cytometry data (fcs
files) were analyzed in the software environment R version 3.6.1 using
the flowCore package (Ellis et al., 2020). Stacked density plots of the
log2(FSC-A) distribution at different time points were constructed by
application of the ggplot2 package in R (Wickham, 2016). Histogram bar
charts of log2(FSC-A) were constructed by sorting the cell count data
into 833 uniformly sized bins using the build-in hist function in
R.
2.4 Analysis of intracellular
proteins
A minimum of four samples were withdrawn for analysis of intracellular
proteins at different time points of the reinitiated chemostat
cultivations of the three FACS sorted populations (See Supplementary
materials Table S1 for an overview of the different samples). The
samples were stored at -80 °C before further processing. Intracellular
proteins were quantified by label‐free quantification as previously
described in Wright et al., (2020). For analysis of the samples, liquid
chromatography was performed on a CapLC system (Thermo Fisher
Scientific) coupled to an Exploris 480 mass spectrometer (Thermo Fisher
Scientific). The peptides were separated with a flow rate of 1.2 µl/min
on a 75‐µm × 15 cm 2 µm C18 easy spray column. A stepped gradient, going
from 4% to 40% acetonitrile in water over 50 minutes was applied. Mass
spectrometry (MS)‐level scans were performed with the following
settings: Orbitrap resolution: 60,000; AGC Target: 1.0e6; maximum
injection time: 50 ms; intensity threshold: 5.0e3; and dynamic
exclusion: 25 s. Data‐dependent MS2 selection was performed in Top 12
mode with HCD collision energy set to 30 % (AGC target: 1.0e4; maximum
injection time: 22 ms).
2.5 Data processing of proteome
data
For analysis of the thermos rawfiles, Proteome Discover 2.3 (Thermo
Fisher Scientific) was applied. The following settings were used for the
analysis: Fixed modifications: Carbamidomethyl (C) and Variable
modifications: oxidation of methionine residues. First search mass
tolerance of 20 ppm and an MS/MS tolerance of 20 ppm. Trypsin was
selected as an enzyme and allowing one missed cleavage. False discovery
rate was set at 0.1%. The data was searched against the S.
cerevisiae database retrieved from Uniprot with proteome ID
AUP000002311 and the sequence of the heterologous insulin. The data sets
can be found at data.dtu.dk (https: //doi.org/10.11583/DTU.13536179).
Batch variations between different proteome datasets were reduced by
scaling each protein such that the mean log2(abundance) was the same
between data sets. A differential expression analysis was performed
between Population 1, Population 2 and Population 3 for
samples taken in the beginning of the cultures (≤ 48 hours of chemostat
growth) and again in the end of the cultures (after 254 hours of
chemostat growth). Only proteins which were measured in all samples
between the compared populations were included in the analysis meaning
that 2635 proteins were compared between Population 1 andPopulation 3 , 2811 proteins were compared betweenPopulation 1 and Population 2 and 2679 proteins were
compared between Population 2 and Population 3 . The
analysis was performed using the EdgeR package (Robinson, McCarthy, &
Smyth, 2010) in R version 3.6.1. The proteome from the beginning of
chemostat cultures with the three populations were furthermore compared
to a previously published proteome of the initial cell clone(Wright et al., 2020). For an overview of the samples used for the
comparison, see Supplementary materials Table S2. 2716 proteins betweenPopulation 1 and the initial cell clone were compared,
2770 proteins between Population 2 and the initial cell
clone were compared and 2692 proteins were included in the comparison
of Population 3 and the initial cell clone .
For each comparison between two strains , proteins were grouped in
clusters depending on whether the level of the proteins were higher
(log2 fold-change > 0.5, q-value < 0.05) or lower
(log2 fold-change < 0.5, q-value < 0.05) in strain A
compared to strain B. Gene ontology (GO) process terms were obtained
from geneontology.org/annotations/sgd.gaf.gz on 16 November, 2020. A
one‐sided Fisher’s exact test was used to investigate whether the
protein clusters, were enriched with proteins annotated with certain GO
process terms (q-value <0.05). The test was performed using
the R package bc3net package (de Matos Simoes, Tripathi, &
Emmert-Streib, 2012).
2.6 Microscopy
The morphology of FACS sorted populations were visually inspected using
a LMI-005-Leica Microscope and a Confocal Microscope-SP8.
2.7 Determination of maximum growth rate in batch
cultures
Maximum growth rates were determined based on OD600measurements from exponentially growing cells in 100 ml shake flasks
with minimal medium and 3 % v/v glucose (Seresht et al., 2013). Three
biological replicates were performed for each strain.