A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.

Deproteinized sweet and sour cheese whey concentrates were investigated for their suitability as substrates for the production of single-cell protein with Kluyveromyces marxianus CBS 6556 up to a 100-l scale. An important factor for gaining high cell concentrations was the use of the Crabtree-negative strain K. marxianus CBS 6556. Supplements such as trace elements, ammonium and calcium were required for the complete conversion of sweet whey concentrates into biomass, whereas sour whey concentrates had to be supplemented with ammonium, trace elements and vitamins. After improvement, biomass dry concentrations of up to 50 g l-1 could be reached with Yx/s values of 0.52 for sweet whey and of up to 65 g l-1 with Yx/s values of 0.48 for sour whey concentrates. The chemical oxygen demand of the whey concentrates were reduced by 80%. The cells were used for the analysis of amino acid and ash composition, showing a distinct increase of eight out of ten essential amino acids compared to sweet and sour whey protein and exceeding the World Health Organisation guidelines for valine, leucine, isoleucine, threonine, phenylalanine and tyrosine.

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