Text S1. Procedures for fermentation and analytical method.
Fermentations
Seed cultures were grown in 250 ml shake flasks containing 50 ml YPD media for
24h at 30°C, 180 rpm, and inoculated into the fermentor at an initial OD (A600) of
0.01. All fermentations were performed in a DasGip 1.0-liter stirrer-pro vessels
(Drescher Amold Schneider, Germany) with a working volume of 500ml YPD media,
at 30°C, 600 rpm agitation, and an aeration set to 1 vvm (volume of air flow per
working volume per minute). One drop of antifoam was added to each fermentor.
Dissolved oxygen was controlled above 30% using a polarographic oxygen electrode
(Mettler Toledo, Switzerland) by automatic adjustment of the impeller speed. The pH
was maintained at 7.0 by the pH sensor (Mettler Toledo, Switzerland) using 2 M
KOH. All fermentations were done in biological duplicates.
Analytical methods
The dry cell weight (DCW) was acquired by filtering 5 ml of the cell culture through
a 0.45 µm filter (Sartorius Stedim, Germany) and measuring the increased weight of
the dried filter. Concentrations of glucose, glycerol, ethanol, and acetate were
analyzed by the Dionex Ultimate 3000 HPLC (Dionex Softron GmbH, Germany) with
an Aminex HPX-87H column (BIORAD, USA) at 65°C using 5 mM H2SO4 as the
mobile phase at a flow rate of 0.6 ml/min.
Relative transcript levels determination by qPCR
Total RNA was isolated from yeast cells cultured at 30°C in shake flasks containing
20 ml of YPD medium, at constant shaking (180 rpm). A total amount of cells
equivalent to 10 ml OD600 1, was harvested during exponential growth phase and
disrupted using glass beads (Lysing matrix C, MP-Biomedicals) in a cell sonicator
(Fast-Prep 24, MP-Biomedicals). Total RNA was purified using the RNeasy kit
(Quiagen), and cDNA was synthesized by adding 100 ng of total RNA to a final RT
reaction volume of 20 µl, and 2 µl of the cDNA were used as template with the
Brilliant III Ultra fast SYBRGreen QPCR Master mix (Stratagene) in a Mx3005P
QPCR System (Agilent Technologies).
Cycle thresholds (Ct) were normalized and gene expression calculated relative to S.
cerevisiae ACT1 expression levels.
Insulin precursor Measurements
1 ml of the culture broth was centrifuged at 4000g for 5 min, and 800 μl of the culture
supernatant was mixed with 100μl 0.1 M HCl and 5.5 mM NaN3 final concentrations,
and stored at 4°C until measurement. IP was measured by indirect ELISA method
using precursor with synthetic leader (Tyo KEJ, et al. Submitted) as a standard.
Western blots were performed according to the western blotting beginner’s guide
(Abcam, UK) to validate the specific binding of the antibody, goat polyclonal
antibody sc7839 (Santa cruz, USA) and donkey anti-goat horseradish peroxidase
(HRP) secondary antibody sc2033 (Santa cruz, USA) were used as primary antibody
and secondary antibody. The image was captured using Molecular Imager Gel Doc
XR System (BioRAD, USA). ELISA measurements were performed according to the
indirect ELISA protocol (Abcam, UK) using the same primary and secondary
antibody. The image was captured using Plate reader Flourstar (BMG LABTECH,
Germany).
Amylase Measurements
1 ml of the culture broth was centrifuged at 4000g for 5 min, and 800 μl of the culture
supernatant was mixed with 100μl 0.1 M HCl and 5.5 mM NaN3 final concentrations,
and stored at 4°C until measurement. The activity of α-amylase in the liquid culture
was measured using the Ceralpha kit (Megazyme, Ireland) using α-amylase from A.
oryzae (Sigma, USA) as standard.
The standard α-amylase (Sigma 82650) is not pure amylase but a fungal extract. We
only know the activity of the product, which is 1.79 U/mgcomplex, but not the purity of
the amylase. So in order to calculate how much mg/L amylase is produced from each
strain, the purity of the standard amylase was measured by Protein 80 reagent kit
(Agilent, Lithuania) using Agilent 2100 Bioanalyzer (Agilent, Austria) and human
insulin as internal standard (Fig. S1). The result showed that the purity of the standard
amylase is 2.57%, which means the actual activity of the standard amylase is 69.6
U/mgamylase.