Application of metabolic engineering to improve both the production and use of biotech indigo.

A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli. This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO). In comparison to a tryptophan-over-producing strain, the first indigo-producing strain made less than half of the expected amount of indigo. Severe inactivation of the first enzyme of aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroGfbr gene product), was observed in cells collected from indigo fermentations. Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo. Indigo production was thereafter improved by increasing the gene dosage of aroGfbr or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase. By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved. Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application.

Effect of glucose on the cultivation of mammalian cells.

Most of the glucose consumed by mammalian cells cultivated in vitro is converted to lactate. The glucose consumption rate appears to be affected by glucose concentration. In a batch cultivation of cells the glucose concentration can be manipulated at a low level by programmed feeding of glucose. In such a culture the specific consumption rate of glucose and the fraction of glucose converted to lactate can be reduced. This reduced conversion rate of glucose to lactate appears to coincide with an increased oxygen uptake rate. A possible consequence of such programmed feeding of glucose is the increased oxidation of glutamine and the concurrent increased production of ammonium. For the cultivation of hybridoma cells high concentrations of ammonium and lactate can be growth inhibitory. It is suggested that the identification of the optimum cultivation conditions is necessary if such a programmed feeding is to be used to increase cell concentration and medium utilization efficiency.