All media recipes were made at pH 7 and autoclaved with the appropriate starting ingredients. NZY media for 1 L was made with 5 g yeast extract, 10 g casein hydrolysate, 5 g NaCL, and 1 g MgSO4. NZYB consisted of NZY plus phosphate buffer. TB media for 1 L was made containing 12 g yeast extract, 24 g casein hydrolysate, 4 mL glycerol, and phosphate buffer. FL media for 1 L was comprised of 10 g yeast extract, 20 g casein hydrolysate, 5 g NaCL, 1 g MgSO4, 20 mL glycerol, and phosphate buffer. Supplementation with glycerol and phosphate buffer occurred at the time of induction.
All transformations were carried out using E. coli strain C41 Overexpress (Lucigen), an optimal strain for terpene synthase expression (Prisic and Peters 2007 (link)). Transformations consisting of three or more plasmids required 0.5 µg plasmid DNA in 50 µL aliquots of chemically competent E. coli C41 cells. The resulting recombinant strains were grown under selective conditions using the appropriate antibiotics at concentrations of 25 µg/mL for carbenicillin, 20 µg/mL for chloramphenicol, 15 µg/mL tetracycline, and 15 µg/mL spectinomycin. For all concentrations listed, negative controls lacking resistance to one marker confirmed the ability of the lower antibiotic concentrations to fully inhibit growth. Expression growths were initiated from several colonies inoculated together into liquid media. For all shake flasks cultures, initial growth to log phase was carried out at 37 °C (A600 ∼0.6), with the temperature then dropped to 16 °C, whereupon the pH was adjusted to 7.0 and phosphate buffer and glycerol, where described, were added. Shaking was held at 200 rpm, and the cultures induced 1 h after dropping the temperature to 16 °C by the addition of IPTG to 1 mM. Baffled flasks were tested under the same conditions as regular Erlenmeyer flasks.
During growth, cultures were intermittently monitored for pH, cell density, and where applicable, product output (as indicated in the various figures). Cell density was monitored by absorbance of aliquots (one in ten dilutions) at 600 nm in a Varian spectrophotometer. Culture pH was monitored throughout growth and adjusted, if necessary, to ensure that the culture pH remained in the 6.5 to 7.5 range, although the cultures rarely required adjustments after the shift to 16 °C. Supplementation with pyruvate or mevalonolactone was administered via pulse feeding with 1 M solutions at regular intervals of 12 h for the first 36 h, up to the desired maximum concentration. Investigation of the role of cell density on product formation (Fig.3 ) was conducted in 48 h parallel aliquots for each density, from the time of IPTG induction, whereby the cell densities were controlled by moving the culture in aliquots from the initial incubation temperature of 37 to 16 °C, where it was held for the remainder of the time course. The values recorded for the cell density are the average of the two aliquots; the percent variance between the duplicate cell densities was smaller than the percent variance between the determined abietadiene yield, therefore error in abietadiene yield is displayed. Note that the data reported in Tables 1 and 2 , as well as Figs. 2 , 3 , 4 , 5 , 7 , and 8 , were derived from parallel fermentation runs (i.e., all the data reported in each of these came from cultures grown together in the same incubator, each in duplicate).
Bioreactor growths were conducted in a New Brunswick BioFlo110 fermentor, set up according the manufacturer's instructions. Under our settings, the cultures were stirred at 300 rpm, their temperature maintained at 20 °C, pH held at 7.2 (using 5 M KOH and 5 M HCL reservoirs connected to the A and B pumps), chemically resistant tubing was used, and air flow was maintained at 4 lpm using triple micron filtered air. Pulse feeding supplements were introduced through the feeding septa, and cell density and product output monitored, as well as pH verified, by intermittent sample removal for subsequent measurement.
All transformations were carried out using E. coli strain C41 Overexpress (Lucigen), an optimal strain for terpene synthase expression (Prisic and Peters 2007 (link)). Transformations consisting of three or more plasmids required 0.5 µg plasmid DNA in 50 µL aliquots of chemically competent E. coli C41 cells. The resulting recombinant strains were grown under selective conditions using the appropriate antibiotics at concentrations of 25 µg/mL for carbenicillin, 20 µg/mL for chloramphenicol, 15 µg/mL tetracycline, and 15 µg/mL spectinomycin. For all concentrations listed, negative controls lacking resistance to one marker confirmed the ability of the lower antibiotic concentrations to fully inhibit growth. Expression growths were initiated from several colonies inoculated together into liquid media. For all shake flasks cultures, initial growth to log phase was carried out at 37 °C (A600 ∼0.6), with the temperature then dropped to 16 °C, whereupon the pH was adjusted to 7.0 and phosphate buffer and glycerol, where described, were added. Shaking was held at 200 rpm, and the cultures induced 1 h after dropping the temperature to 16 °C by the addition of IPTG to 1 mM. Baffled flasks were tested under the same conditions as regular Erlenmeyer flasks.
During growth, cultures were intermittently monitored for pH, cell density, and where applicable, product output (as indicated in the various figures). Cell density was monitored by absorbance of aliquots (one in ten dilutions) at 600 nm in a Varian spectrophotometer. Culture pH was monitored throughout growth and adjusted, if necessary, to ensure that the culture pH remained in the 6.5 to 7.5 range, although the cultures rarely required adjustments after the shift to 16 °C. Supplementation with pyruvate or mevalonolactone was administered via pulse feeding with 1 M solutions at regular intervals of 12 h for the first 36 h, up to the desired maximum concentration. Investigation of the role of cell density on product formation (Fig.
Bioreactor growths were conducted in a New Brunswick BioFlo110 fermentor, set up according the manufacturer's instructions. Under our settings, the cultures were stirred at 300 rpm, their temperature maintained at 20 °C, pH held at 7.2 (using 5 M KOH and 5 M HCL reservoirs connected to the A and B pumps), chemically resistant tubing was used, and air flow was maintained at 4 lpm using triple micron filtered air. Pulse feeding supplements were introduced through the feeding septa, and cell density and product output monitored, as well as pH verified, by intermittent sample removal for subsequent measurement.
