A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors.

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Based on experimental results of eight fed-batch fermentations using a recombinant L-phenylalanine-producing Escherichia coli strain, the applicability of principal-component analysis (PCA) for fermentation analysis was studied. Three principal components were identi®ed, representing approximately 90% of total variance. Among them, concentrations of tyrosine and acetate were identi®ed as key fermentation parameters. Their signi®cance was also con®rmed when measurement errors were taken into consideration by Monte-Carlo estimations. The error estimation approach was also used to investigate PCA suitability for the time-speci®c analysis of different fermentation phases. Relatively large principal-component score errors were calculated that limit the applicability of PCA for detailed fermentation course analysis.

Two novel mutations of the human CLCN1 chloride channel gene, c.592C>G (p.L198V) and c.2255A>G (p.K752R), are described, occurring coincidentally in the one myotonic patient. These individual mutations and a construct with both mutations in the one cDNA were transcribed and expressed in Xenopus oocytes where channel gating parameters were extracted from chloride currents recorded under voltage clamp. We found that the p.L198V mutation has its major effects on the common (or slow) gate of the chloride channel, as do other dominant ClC‐1 mutations, and may therefore be causative of the patient's symptoms (when co‐expressed with wild‐type human ClC‐1, the p.L198V mutation exerts a dominant negative effect on common gating) but the p.K752R mutation appears to be innocuous and may be a benign polymorphism. A third mutant, the recently described c.2795C>T (p.P932L), was expressed in HEK 293 cells. Despite the severity of the disease associated with this mutation, chloride currents in cells expressing p.P932L were not significantly different from those of cells expressing wild‐type ClC‐1. © 2004 Wiley‐Liss, Inc.