Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to 2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin. The vast majority of antibiotic and antitumor drugs belong either to the polyketide or the nonribosomal families of natural products. A related family comprises hybrid compounds containing polyketide and nonribosomal peptide moieties. Their biosynthesis involves the participation of a modular polyketide synthase (PKS) for the condensation of acyl coenzyme A (acylCoA) precursors and a nonribosomal peptide synthetase (NRPS) that condenses amino acids after their activation to an aminoacyl-AMP precursor. Both type I PKSs and NRPSs are multifunctional enzymes that are organized into modules and use a similar strategy for the assembly of these short carboxylic and amino acid building blocks. The minimal set of domains in a type I PKS includes ketosynthase (KS), acyltransferase, and acyl-carrier protein activities responsible for the catalysis of one cycle of polyketide chain elongation. These PKS modules can contain further domains such as ketoreductase (KR), dehydratase (DH), or enoylreductase to reduce the keto groups generated during the condensation process (9). In a similar