PubMed · 8753216
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M Ikemoto. 1995. [Arginase].. https://pubmed.ncbi.nlm.nih.gov/8753216/
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Expression of the catabolic gene encoding arginase in Saccharomyces cerevisiae, CAR1, is controlled by multiple nitrogen signals, such as the presence of the inducer, arginine, and the nature and amount of the nitrogen source. The present study has determined or confirmed the identity of the proteins involved in these different controls, as well as their targets in the CAR1 promoter. We show that Gln3p activates CAR1 expression through the GATAA sequences in the absence of an optimal nitrogen source, such as ammonia, glutamine or asparagine. Ume6p, which also controls the expression of early meiotic genes, represses CAR1 expression through a sequence called URS, as a function of nitrogen availability. Thus, the responses to the quality of the nitrogen source and to nitrogen starvation are achieved through different cis- and trans-regulatory elements. At least one of the multiple Rap1p and Abf1p binding sites is required for the basal transcription of the gene. The UAS(arg), containing the previously defined "arginine boxes" is the region that responds to the inducer through the action of the ArgRp-Mcm1p proteins, and its deletion alone significantly affects growth on arginine as sole nitrogen source. The functional UAS(arg) is about 60 nucleotides long, and contains two sequences homologous to the binding site for MADS-box proteins, to which ArgRIp and Mcm1p belong. No obvious palindromic sequence similar to the binding site of Gal4p, Ppr1p or Put3p is present in the UAS(arg), although ArgRIIp contains a Zn(II)2Cys6 motif. Interestingly, we have found that induction of CAR1 expression by arginine in the presence of an optimal nitrogen source is counteracted by Gln3p, independently of its action at the GATAA sequences.
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Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.