Bacillus subtilis: a microbial cell factory for human interferon gamma production and process development

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The B. subtilis WB800N microbial cell factory was tackled at three levels for enhanced extracellular human interferon gamma production namely: 1) genetic level 2) substrate, medium components and extracellular cultivation environment level and 3) process operation level. The genetic level modulation of the B. subtilis WB800N physiology was performed by designing three human interferon gamma genes (IFNγ) with different optimum codon usage, RNA free energy and secondary structures. At substrate level complex and completely defined medium were assessed for modulation of the B. subtilis physiology along with various carbon and nitrogen sources by applying metabolic modelling and the stoichiometric modelling approaches for higher IFNγ production. Various amino acids were screened for their positive enhancing effects on IFNγ production using stoichiometric modelling and demand. The concentration level of carbon and nitrogen sources along with other medium components were optimized using statistical Design of Experiments (DoE) approach and machine learning based artificial neural network (ANN) based evolutionary programing using genetic (GA) and simulated annealing (SA) algorithms. At process operation level, the batch process was established with physical and environmental condition optimization of the B. subtilis WB800N culture for an antifoaming agent, optimum dissolved oxygen mass transfer coefficient and agitation rate. Fed-batch mode of operation was established with organic acid and amino acid feeding for positive stimulatory effect on IFNγ production. The high cell density cultivation of the B. subtilis WB800N with glycerol-amino acid-organic acid feeding was established which resulted in further enhancement in IFNγ production.
Supervisor: V. V. Dasu