Enzymes feedback-resistant

In the cases described below we will generally focus on metabolic pathway engineering rather than on classical strain improvement, for obvious reasons. One should be aware, however, that the classical approach has strengths that can make it a powerful partner of the rational approach. Thus, regulation problems have been addressed by the development of a feedback-resistant enzyme, using selective pressure and random mutagenesis as described above, in a research species, followed by introduction of the altered gene in the production species via recombinant techniques. 

C. glutamicum and E. coli, which share very similar biosynthetic pathways and control architectures, have been subjected to pathway engineering for the production of L-Trp. Modifications that have been reported include the, now familiar, feedback-resistant DAHP synthase and the enzymes in the Trp pathway were freed from regulation [72, 97] and overexpressed. 

As expected for a biosynthetic pathway [7], the activity of the first enzyme (PR-ATP synthetase ) is controlled by the end product of the pathway [8]. At the pH used in the standard assay, pH 8.5, feedback inhibition by histidine is noncompetitive with respect to both substrates (PRPP and ATP), having a Ki of 100 fiM. When the pH is reduced to one more likely representative of that in the cell, pH 7.5, the strength of the inhibition is about doubled [8]. Feedback-resistant mutants of the PR-ATP s)mthetase have been obtained by selecting for resistance to the histidine analog 2-thiazolealanine (for structure, see Fig. 2). This analog does not substitute for histidine in proteins, but inhibits the PR-ATP synthetase [8,11,12], and thereby produces bacteriostasis in the sensitive cell by cutting off its histidine supply. Feedback-resistant mutants excrete Itistidine into the medium, whereas... 

I + Slight repression enzyme L-1 feedback-resistant None leu A... 

This involves the engineering of endpoint or branch point enzymes in the amino acid biosynthetic pathway leading to the accumulation of amino acids of interest. Feedback resistance of enzymes also led to amino acid overproduction. Amplification of rate-limiting enzymes has led to the increase in phenylalanine production. Engineering the branch point enzymes has led to the conversion of tryptophan to tyrosine or phenylalanine. 

PRPP synthetase superactivity, diversity in the kinetic mechanisms underlying increased PRPP synthesis has been identified. This diversity has important implications for the design of methods for detection of abnormalities of the enzyme. The four categories of kinetic alteration thus far associated with PRPP synthetase superactivity in man are abnormal catalytic properties (increased maximal reaction veloc-city) 2) defective regulatory properties (purine nucleotide feedback resistance) 3) increased affinity for the substrate ribose-5-P and 4) combined alterations of catalytic and regulatory properties. 

PRPP synthetases. A. Enzyme activities in dialyzed hemolysates. Sigmoidal activation is seen for both enzymes. Activity of the mutant enzyme with an increased maximal reaction velocity is, however, increased by a constant proportion at all Pi concentrations. B. Enzyme activities in undialyzed (crude) hemolysates. The mutant enzyme, deficient in inhibitor responsiveness, shows hyperbolic activation with increased activity only at Pi concentrations below 2 mM. C. Enzyme activities in partially purified erythrocyte preparations from a normal individual and the patient with the feedback-resistant PRPP synthetase studied in B. Note hyperbolic activation of the purified normal enzyme and the resulting similarity of the Pi activation curves. D. Enzyme activities in dialyzed fibroblast extracts. The mutant enzyme, with combined increased maximal reaction velocity and diminished nucleotide responsiveness, shows both hyperbolic activation and increased enzyme activity at all Pi concentrations. 

FIGURE 3.3 L-Phenylalanine-mediated feedback inhibition of wild-type Escherichia coli K12 prephenate dehydratase (JN302) and four feedback inhibition-resistant enzyme variants (JN305-JN308). Activity is expressed as a percentage of normal wild-type enzyme activity. 

The 10 amino acids essential in the human diet (Arg, His, He, Leu, Lys, Met, Phe, Thr, Trp, Val) are synthesized by non-human organisms by multistep pathways starting from simple metabolic precursors. Amino acid biosynthesis is controlled by feedback inhibition and suppression of synthesis of biosynthetic enzymes. The ability of an amino acid analogue to block biosynthesis of the parent amino acid often contributes to the toxicity of the analogue. Mutants resistant to the toxic effects of the analogue can be valuable tools for studying various aspects of cellular mechanism (examples to be given below). 

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