Isobutanol → (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal

Participants

Reaction Steps

Strains with integrated ALS genes expressed from the CUP1 promoter were transformed with pGV2082 (which carries the other 4 isobutanol pathway genes Ec_ilvC_coScQ110V (SEQ ID NO: 61), Ll_ilvD (SEQ ID NO: 65), Ll_kivd2_coEc (SEQ ID NO: 59), and Dm_ADH (SEQ ID NO: 60)). Strains were patched onto YPD plates containing 0.2 mg/mL G418. The following morning cells were removed from the plate with a sterile toothpick and resuspended in 4 mL of YPD with 0.2 mg/mL G418. The OD600 was determined for each culture. Cells were added to 50 mL YP with 5% dextrose and 0.2 mg/mL G418 such that a final OD600 of 0.1 was obtained. 1 mL of media was removed and the OD600 for this undiluted sample determined, leftover media was stored at 4° C. to act as media blank for the analytics submission, and to act as the t=0 sample for the fermentation. At t=24 h, 2 mL of media was removed and 25 μL used at a 1:40 dilution to determine OD600. The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 min and a 1:10 dilution read on the YSI. 50% glucose containing 0.2 mg/mL G418 was added to a final concentration of 100 g/L glucose. 1 mL of supernatant was analyzed by gas chromatography as described above. At t=48 h, 2 mL of media was removed and 25 μL used at a 1:40 dilution to determine OD600. The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 min and a 1:10 dilution read on the YSI. 50% glucose plus water (with 0.2 mg/mL G418) were added to give a final concentration of glucose of 100 g/L. 1 mL of supernatant was analyzed by gas chromatography. At t=72 h, 2 mL of media was removed and 25 μL used at a 1:40 dilution to determine OD600). The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 min and a 1:10 dilution read on the YSI. 1 mL of supernatant was analyzed by gas chromatography and high performance liquid chromatography.

Provenance & Evidence