Analytical procedures
Biomass was measured by optical density at 600 nm as described
previously (Matthews et al., 2017a). An Agilent Bravo liquid handler was
used to dilute samples prior to measurements of OD into the Tecan
Infinite M200 Pro plate reader.
Reverse phase ultra-high performance liquid chromatography (UHPLC)
analysis was performed on Agilent 1290 Infinity II UHPLC system
controlled using OpenLab CDS software (Agilent Technologies, Santa
Clara, CA). The concentration of protein was determined using a
Poroshell 120 SB-Aq column (2.1 x 50 mm, 1.9µm) operated at 1.0 mL/min
and 70 oC (Agilent Technologies, Santa Clara, CA).
Buffer A was 0.1% (v/v) TFA in water and buffer B was 0.1% (v/v) TFA,
0.5% (v/v) water in ACN. A gradient was performed as follows: 30% B
for 1 min., 30-40% B over 3 min., 40-90% B over 0.5 min., 90% B for
0.5 min., 90-30%B over 0.5 min., and 30% for 1 min.; total method run
time was 6.5 minutes. Sample injection volumes were 50µL. A diode array
detector was set for absorbance detection at 214nm. Data analysis was
completed using OpenLab CDS Data Analysis (Agilent Technologies, Santa
Clara, CA).
Statistical analysis and DOE design was conducted using JMP (SAS
Institute, Cary NC). Quadratic models were fitted using effect screening
and non-significant terms (adjusted p-value > 0.01) were
eliminated sequentially in order of decreasing adjusted p-value to avoid
overfitting. Data was plotted using Prism 8.4.0 (GraphPad Software, San
Diego, CA).