Fig. 3
From: Renewable and tuneable bio-LPG blends derived from amino acids
Hydrocarbons production of E. coli strain BL21(DE3)∆∆ containing engineered KdcA/CvFAPG462V-dependent routes. Gaseous alkane yields dependent on the a enzyme homologues and b amino acid supplementation for pTrIHTrKFG462I construct. Cultures (10–20 mL) were grown in LB medium containing 30 μg/mL kanamycin for 4–6 h at 37 °C and 180 rpm. Luria broth contains ca 7, 8.8 and 5.4 mM valine, isoleucine and leucine, respectively [21]. Protein induction (100 μM IPTG) was performed, followed by culture supplementation with valine, leucine or isoleucine (30 mg/mL) in part B) after 1 h at 30 °C. Triplicate samples (1 mL) each of 3 biological replicate cultures were sealed into glass vials (4 mL) and incubated at 30 °C for 16–18 h at 200 rpm, illuminated continuously with a blue LED (455 nm or 470 nm). Gaseous hydrocarbon levels were determined by manual headspace injection on an Agilent 490 Micro GC. Errors represent one standard deviation of the replicates (biological and/or technical triplicates). Data for LB + valine was obtained from previous studies [4]. Expression constructs (inset): αKGSDH/V = trc-ilvE-αKGSDH-trc-kdcA-CvFAPG462V; Hpad/V = trc-ilvE-Hpad-trc-kdcA-CvFAPG462V; PadA/V = trc-PadA-Hpad-trc-kdcA-CvFAPG462V; αKGSDH/I = trc-ilvE-αKGSDH-trc-kdcA-CvFAPG462I; Hpad/I = trc-ilvE-Hpad-trc-kdcA-CvFAPG462I and PadA/I = trc-PadA-Hpad-trc-kdcA-CvFAPG462I