MOESM1 of Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae
Creators: name: Wehrs, Maren
name: Prahl, Jan-Philip
name: Moon, Jadie
name: Li, Yuchen
name: Tanjore, Deepti
name: Keasling, Jay
name: Pray, Todd
name: Mukhopadhyay, Aindrila
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology

description: Additional file 1: Figure S1. Phenotype of BJ5465.sfp.bpsA producing Indigoidine streaked on YPD plates. Overnight cultures of BJ5465.sfp.bpsA grown in 3 mL YPD media were streaked onto YPD plates and incubated at 30 °C. Indigoidine production was monitored over the course of 10 days. Red boxes mark spontaneous white mutants forming small colonies. Because these mutants did not grow on glycerol as sole carbon source (data not shown), we conclude that these mutants lack functional mitochondria and can be characterized as petites. Figure S2. Phenotype of BJ5465.sfp.bpsA spotted on solid media containing either glucose (YPD or glycerol (YPG). Overnight cultures of BJ5465.sfp.bpsA grown in 3 mL YPD media were spotted in twofold serial dilution onto YPD and YPG plates and incubated at 30 °C. Indigoidine production was monitored over the course of 7 days. Figure S3. Phenotype and Titer of BJ5465.sfp.bpsA grown in different carbon sources. BJ5465.sfp.bpsA was grown in rich media containing either glycerol, galactose, sucrose or glucose ranging in concentrations from 1 to 5% as the sole carbon source for 5 days. Indigoidine production was quantified after 3 days (dark blue bars) and 5 days (light blue bars). The carbon sources are utilized via different metabolic pathways in S. cerevisiae, namely respiratory for glycerol, mixed respiro-fermentative for galactose and fermentative for glucose. Error bars represent standard deviation (n = 3–4). Figure S4. Time profiles of dissolved Oxygen and Oxygen Uptake Rate during glucose starvation conditions at 2 L bioreactor scale. Glucose starvation conditions were realized using a DO signal-based pulse feeding strategy adding 0.4 g glucose per liter on demand upon carbon source depletion. Feed start after 24 h. Spikes in dissolved Oxygen were used to trigger feed pulses. Dissolved Oxygen (pO2) is shown in blue and Oxygen Uptake Rate (OUR) is shown in red. Figure S5. Time profiles of dissolved Oxygen and Oxygen Uptake during excess glucose conditions at 2 L bioreactor scale. Excess glucose was achieved via a semi-continuous feeding strategy with fixed glucose delivery of 4 g glucose per liter per hour. Dissolved Oxygen (pO2) is shown in blue and Oxygen Uptake Rate (OUR) is shown in red. Figure S6. DNA sequences and constructs used in this study. A Sequence of Streptomyces lavendulae bpsA codon optimized for expression in S. cerevisiae. B The codon-optimized version was cloned into an E5C plasmid backbone under the control of an IPTG inducible lacUV5 promoter for production of Indigoidine in E. coli. Figure S7. Standard curve of Indigoidine absorbance at 612 nm in DMSO. Absorbance values were obtained for serial dilutions of purified Indigoidine in DMSO. The equation for the trendline is y = 0.739x − 0.0721, R2 = 0.9948.