Human interferon-gamma (hIFNG) production is limited by various gene-level bottlenecks including translation, protein folding, and secretion which depends upon the physiological state of the organism. In this study gene-level and substrate-level modulations have been used to control Bacillus subtilis physiology for >15 fold extracellular soluble hIFNG production. Two variants of the native human interferon-gamma gene (hifng) were designed and synthesized, namely, cohifnghis and cohifng having codon adaptation index 25.33 and 26.89% higher than the native gene, respectively. BScoIFNG and BScoIFNGhis with ΔG of -100.0 and -113.7 kcal mol-1 resulted in 30 and 6.5% higher hIFNG compared to the native gene in complex medium. BScoIFNG produced 1.53 fold higher hIFNG using glucose-based defined medium as compared to the complex medium by modulating the physiological parameter growth rate from 0.35 to 0.26 hr-1. Further modulatory effect of various phosphotransferase transport system (PTS) and no-PTS sugars, sugar alcohols, and organic acids was quantified on the physiology of B. subtilis WB800N for extracellular hIFNG production. Sorbitol and glycerol emerged as the best hIFNG producers with lowest growth and substrate consumption rates. BScoIFNG produced maximum 3.15 mg L-1 hIFNG at 50 g L-1 glycerol with highest hIFNG yield (Yp/x = 0.136) and lowest substrate uptake rate (qs = 0.26).
Keywords: Acetate; PTS/non-PTS carbon sources; acetoin; codon optimization; overflow metabolism; physiological performance; soluble human interferon gamma.