A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from L-proline. Nargenicin A(1), a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A(1) in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A(1) was also derived from L-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A(1) is initiated by NgnN4 that catalyzes ATP-dependent activation of L-proline into L-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert L-proline into pyrrole-2-carboxylic acid moiety.

A number of secondary metabolites having therapeutic importance have been reported from the genus Nocardia. One of the polyketide antibiotic compounds isolated from Nocardia is nargenicin A(1). Recently, nargenicin A(1) has been isolated from Nocardia sp. CS682, a new Nocardia strain isolated from soil in Jeonnam, South Korea. It possesses strong antibacterial activity against methicillin-resistant Staphylococcus aureus. In this study, we applied a metabolic engineering approach based on recombinant DNA technology in order to boost the production of nargenicin A(1) from Nocardia sp. CS682. Initially, we optimized the transformation of this new strain by electroporation method. Heterologous expression of S-adenosylmethionine synthetase (MetK1-sp) in Nocardia sp. CS682 enhanced the production of nargenicin A(1) by about 2.8 times due to transcriptional activation of biosynthetic genes as revealed by reverse transcription polymerase chain reaction analysis. Similarly, expression of acetyl-CoA carboxylase genes improved nargenicin A(1) production by about 3.8 times in Nocardia sp. ACC18 compared to that in Nocardia sp. CS682 and Nocardia sp. NV18 by increasing precursor pool. Thus, enhanced production of nargenicin A(1) from Nocardia sp. CS682 can be achieved by expression of transcriptional activator genes and precursor genes from Streptomyces strains.

Nargenicin A 1 is a 28-membered polyketide macrolide, with antibacterial activity against methicillin-resistant Staphylococcus aureus, produced by Nocardia sp. CS682. In this study, the production of nargenicin A 1 was improved by enhancing the supply of different biosynthetic precursors. In Nocardia sp. CS682 (KCTC11297BP), this improvement was ~4.62-fold with the supplementation of 30 mM methyl oleate, 4.25-fold with supplementation of 15 mM sodium propionate, and 2.81-fold with supplementation of 15 mM sodium acetate. In Nocardia sp. metK18 and Nocardia sp. CS682 expressing S-adenosylmethionine synthetase (MetK), the production of nargenicin A 1 was improved by ~5.57-fold by supplementation with 30 mM methyl oleate, 5.01-fold by supplementation with 15 mM sodium propionate, and 3.64-fold by supplementation with 15 mM sodium acetate. Furthermore, supplementing the culture broth of Nocardia sp. ACC18 and Nocardia sp. CS682 expressing the acetyl-CoA carboxylase complex (AccA2 and AccBE) with 30 mM methyl oleate, 15 mM sodium propionate, or 15 mM sodium acetate resulted in ~6.99-, 6.46-, and 5.58-fold increases, respectively, in nargenicin A 1 production. Our overall results showed that among the supplements, methyl oleate was the most effective precursor supporting the highest titers of nargenicin A 1 in Nocardia sp. CS682, Nocardia sp. metK18, and Nocardia sp. ACC18.