engleski

Process development for Escherichia coli based pyruvate production

The commercial demand for pyruvic acid is expanding because of its use as an effective starting material for the synthesis of many drugs, agrochemicals and nowadays in food industry as a fat burner. It is also a valuable substrate for the enzymatic production of amino acids such as L-tryptophane, L-tyrosine and L-dihydroxyphenylalanine (L-DOPA). The main goal in this project is the development of a pyruvate production process from glucose with a high molar pyruvate/glucose yield (approaching 2 mol pyruvate/mol glucose) and space-time yield using a recombinant Escherichia coli YYC202 strain1. This strain is completely blocked in its ability to convert pyruvate in acetyl-CoA or acetate, resulting in acetate-auxotrophy during growth in glucose minimal medium. Due to the strain genotype, acetate availability was assumed to represent a key fermentation variable. Experimental studies identified a &#8216 ; ; ; ; simple&#8217 ; ; ; ; correlation between acetate consumption rate (ACR) and CO2 production rate (CTR), interestingly, with an optimum equal molar ratio. Therefore, CTR (calculated on-line by CO2 and O2 exhaust gas analysis) was used for on-line calculation and regulation of the acetate feed (acetate limiting, saturating and accumulating conditions could be established). Additionally, glucose concentrations were controlled on-line. By controlling acetate and glucose, a series of lab-scale experiments were performed to optimize the molar yield of pyruvate/glucose, the space time yield and the final pyruvate concentration. At optimal process conditions a final pyruvate concentration higher than 500 mM (44 g/L), a space-time yield of 24.7 mmol/L/h and a molar yield pyruvate/glucose of 0.80 mol/mol were achieved. Evidence was obtained that high extra-cellular pyruvate concentrations were identified as an inhibiting factor for further pyruvate production. To face this problem with process engineering means, continuous experiments with cells retention (by applying diafiltration unit) were done, thus reducing the final pyruvate titers. Current results will be presented. 1. Bott, M., Gerharz, T., Takors, R., Zelic, B. (2001) Verfahren zur fermentativen Herstellung von Pyruvat, Detsche Patentanmeldung 101 29 711.4

E. coli; pyruvate; process development; product inhibition

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