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l-tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of the recombinant l-tryptophan producer Escherichia coli NT367 was studied in controlled fed-batch processes. The chromosome of the recombinant l-tryptophan producer was equipped with additional genes coding for enzymes of the aromatic amino acids biosynthetic pathway and l-serine biosynthesis, including genes for feedback-resistant enzyme variants ( trpE , aroFBL, and serA ), deletions of enzymatic steps for the degradation of precursors or the product l-tryptophan ( sdaB and tnaA), and alterations in the regulation of l-tryptophan metabolism (deletion of trpL and trpR). The impact of glycerol supply rates as well as the application of a multicopy plasmid (pF112- aroFBL -kan) were investigated in fully controlled stirred-tank bioreactors on a 15 L scale. The combination of E. coli NT367 carrying pF112- aroFBL -kan and an appropriate biomass-specific glycerol supply-rate resulted in the highest final product concentration of 12.5 g L l-tryptophan with the lowest concentrations of other aromatic amino acids. Fed-batch production of l-tryptophan from glycerol was shown for the first time with recombinant E. coli.

A fed-batch process was studied with lactate and glycerol supply in the growth phase and glycerol supply during L-phenylalanine production with recombinant E. coli K-12. Lactic acid feeding was necessary for growth because the genes encoding the PEP-consuming pyruvate kinase isoenzymes (pykA, pykF) have been deleted. An unexpected glucose efflux (67.6 ± 2.3 mgGlucose  gCDW (-1) ) was measured after the cells were harvested and resuspended in a mineral medium for metabolic perturbation experiments. As the efflux prohibited the application of these experiments, characterization of intracellular carbon storage was necessary. Therefore, two genetically engineered strains (one lacking glycogen metabolism and another additionally lacking trehalose synthesis) were applied in the fed-batch process. Trehalose synthesis and accumulation from lactate was clearly identified as the source for glucose efflux after cell harvest and resuspension. Cultivations of strains with active pyruvate kinase successfully identified lactate as the carbon source causing intracellular trehalose storage. The usage of glycerol as sole carbon source during the whole process enabled an improved process performance and inhibited trehalose accumulation. Overall, this setup allows the application of perturbation experiments.

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