engleski

Process development for Escherichia coli based pyruvate production

The commercial demand for pyruvic acid has been 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. Compared with chemical method, biotechnological production of pyruvate is an alternative approach because of low cost. The main goal in this project is the development of a pyruvate production process from glucose using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate in acetyl-CoA or acetate, therefore, strain requires acetate for growth. Acetate concentration is a parameter critical for regulation of biomass growth and pyruvate production. By low activity of pentose-phosphate pathway CO2 will be mainly produced via TCA cycle. Because in mutant YYC202 Acetyl-CoA could be produced only from acetate, one can expect correlation between acetate consumption rate (ACR) and CO2 production rate (CTR). Interesting, we could show that the CTR is equal to the ACR. Therefore, CTR (calculated on-line by CO2 and O2 exhaust gas analysis) was used for on-line calculation and regulation of the acetate feed. This correlation was used to carry out a process under acetate limitation, saturation and accumulation just by changing parameter f in equation displayed bellow. For the on-line measurement and regulation of glucose concentration levels an &#8220 ; ; On-line general analyzer&#8221 ; ; (OLGA) was used. On such a way developed control loops were used in 7.5 l bioreactor to find optimums (molar yield pyruvate/glucose, space time yield and pyruvate final concentration) of the fed-batch process concerning acetate and glucose concentrations. Also, intra-cellular measurements of pyruvate concentrations by LC-MS were done. At optimal process conditions final pyruvate concentration of 500 mM (44 g l-1), space-time yield of 24.7 mmol l-1 h-1 and molar yield pyruvate/glucose of 0.80 mol mol-1 were achieved. Pyruvate export is probably controlled by active mechanism, because extra-cellular pyruvate concentration was 102 fold higher than intra-cellular through the process.

Escherichia coli; pyruvate; CTR; fed-batch process

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