(10f) Transcriptional And Metabolic Responses In High-Cell Density Cultures During Temperature-Induced Synthesis Of Recombinant Protein In Escherichia Coli: A Scale-Down Study

Temperature-induced expression of heterologous genes in high-cell density cultures of Escherichia coli is an economical way for producing recombinant proteins. However, due to inherent limitations in the design and operation of large-scale bioreactors, heat-transfer operations become less efficient at large-scales. Accordingly, the fast rates in temperature increase that can be achieved in a laboratory setting are not attainable at large-scales. In this study we determined if the rate at which temperature is increased in recombinant Escherichia coli cultures affected the heat shock response and the production of human pre-proinsulin. In particular, we studied the bacterial response at the transcriptional level by quantifying gene expression through qRT-PCR and using a scale-down approach to experimentally simulate rates of temperature increase typical of large-scale fermentors. Induction of recombinant human pre-proinsulin in high-cell density fed-batch cultures of E. coli was performed by temperature up-shifts at rates of 6, 1.7, 0.8 and 0.4 °C/min, for mimicking heat-transfer characteristics of 0.1, 5, 20, and 100 m3 fermenters, respectively. Under such conditions, the yield of recombinant protein decreased as the temperature rising rate increased. This correlated with a decrease in stress-related responses for the cultures simulating the larger-scale processes (see below). Induction at faster temperature increases resulted in a larger waste of carbon skeletons, as observed by a higher accumulation of acetate, formate, and lactate. The relative mRNA levels of heat-shock genes, increased between 2- to over 42-fold when cultures were induced at 6, 1.7 and 0.8 °C/min, but no increase was observed at 0.4 °C/min. In addition, transcription levels of stress genes (relA and spoT) only increased between 1.5- to over 4-fold at 6 and 1.7 °C/min, suggesting that cells subjected to gradual changes in temperature can overcome stress. mRNA levels of the transcription-translation machinery genes (EF-Fu, rpoA and tig), decreased between 40 to 80 % at 6, 1.7 and 0.8 °C/min, and a transient increase occurred in cultures subjected to temperature increases of 0.4 °C/min. Knowledge of the effect of the way temperature is increased on bacterial physiology and product formation is useful for a rational design of scale-down and scale-up strategies, as well as for temperature induction schemes.