Recombinant collagen production optimization in Escherichia coli

Abstract

An Escherichia coli-based collagen-production process was used to investigate several process optimization objectives for use at the industrial scale. The effect of cooling on fermentation growth kinetics was studied, with preliminary results indicating that cooling an early to mid-batch phase fermentation (OD₆₀₀ = 2-4) to 10⁰C for up to 40 hours was not detrimental to the recovery of fermentation growth or the culture's ability to reach high cell density (OD₆₀₀> 50). A cost-effective assay of collagen-like polymer was examined under high-cell density conditions (as opposed to previously studied low density conditions) and an experimental design for tailoring the assay to high cell density fermentation samples is presented. In addition, a dual-plasmid strain of E. coli was designed for use in a novel process for the mass production of collagen-like polymers: one plasmid contains a thermally inducible recombinant collagen gene (CLP3.1-his), and the other contains an arabinose-inducible lytic gene (bacteriophage T4 t-holin) along with the basally expressed T7 lysozyme gene from the pLysS plasmid. A methodology for the optimization of the sequential induction of CLP3.1-his followed by induction of the t-holin is presented; a review of the literature suggests that decreased growth rate is detrimental to lytic efficiency (both the time required for lysis and the degree of lysis). The resulting process will enable lysis to take place in the bioreactor, thus avoiding the extra time and monetary cost of a separate cell homogenization step for E. coli disruption and endogenous protein release..