Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis.

Little is known about the mechanisms that couple regulation of secondary metabolic pathways to the synthesis of primary metabolic precursors. Camalexin, an indolic secondary metabolite, appears to be the major phytoalexin in Arabidopsis. It was previously shown that camalexin accumulation is caused by infection with plant pathogens, by abiotic elicitors, and in spontaneous lesions in the accelerated cell death mutant acd2. We demonstrate that the accumulation of this phytoalexin is accompanied by the induction of the mRNAs and proteins for all of the tryptophan biosynthetic enzymes tested. A strong correlation was observed between the magnitude of camalexin accumulation and the induction of tryptophan biosynthetic proteins, indicating coordinate regulation of these processes. Production of disease symptoms is not sufficient for the response because systemic infection with cauliflower mosaic virus or cucumber mosaic virus did not induce the tryptophan pathway enzymes or camalexin accumulation. Salicylic acid appears to be required, but unlike other documented pathogenesis-related proteins, it is not sufficient for the coordinate induction. Results with trp mutants suggest that the tryptophan pathway is not rate limiting for camalexin accumulation. Taken together, these results are consistent with the hypothesis that the regulation of the tryptophan pathway in plants responds to needs for biosynthesis of secondary metabolites.     

Regulation of leucine biosynthesis in Bacillus subtilis

The biosynthesis of alpha-isopropylmalate (alphaIPM) synthetase, IPM isomerase, and betaIPM dehydrogenase in Bacillus subtilis can be derepressed in leucine auxotrophs by limiting them for leucine. The derepression of the three enzymes is apparently coordinate. A class of mutants resistant to 4-azaleucine excretes leucine and has derepressed levels of all three enzymes. The azaleucine-resistance mutations may lie in a gene (azlA) encoding a repressor. Efforts to find mutations characteristic of a constitutive operator have been unsuccessful. No polar mutations have been found among nine leucine auxotrophs that have characteristics of frameshift mutations. The enzyme catalyzing the first step in leucine biosynthesis, alphaIPM synthetase, is sensitive to feedback inhibition by leucine. We conclude that leucine biosynthesis is controlled by the inhibition of the activity of the first biosynthetic enzyme by leucine, and by the repression of the synthesis of the first three biosynthetic enzymes by leucine. The repression of the three enzymes may be under the control of a single repressor and a single operator, or of a single repressor and a separate operator for each structural gene.     

Improving lycopene production in Escherichia coli by engineering metabolic control

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.