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).