Metabolic Engineering and Synthetic Biological Approach for Enhancing and Structurally Modulating Nargenicin A1 from Nocardia Sp. CS682

The infectious diseases caused by bacteria, particularly those gaining the multi-drug resistance, are major causes of mortality. Hence, the discovery of new antimicrobials or expanding the utility of existing antibiotics by overproduction and targeted modification resulting in better activities or pharmacological properties is crucial to combat such havoc [1, 2]. Recently, nargenicin A1 was rediscovered as potential antimicrobial candidate through whole-cell anti-sense assay and established as the first natural product having unique capability to inhibit DnaE, which is protein actively involved in DNA replication [3].Hence, in this study we attempted to enhance the production of nargenicin A1 by rational metabolic engineering approach and synthetic biological tools coupled with sensible precursor supplementation, assisted by response surface methodology to attain ~24 fold higher production in recombinant strain than the wild type strain, Nocardia sp. CS682. For this purpose a userdefined multimonocistronic vector was developed, evaluated and utilized for cloning genes responsible for precursor refactoring viz. ngnN2, ngnN3, ngnN4 and ngnN5 for pyrrole biogenesis, and glf,glk and ACCase complex genes for aglycon synthesis. Furthermore, by incorporating pikC, a substrate flexible cytochrome P450 [4,5] in the nargenicin A1 biosynthetic pathway, novel derivative of nargenicin was isolated from the recombinant strain. To the other end, the nargenicin A1 was structurally diversified utilizing efficient single vessel reaction system in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined flexible glycosyltransferase (GT) from Bacillus licheniformisDSM 13[2]. Novel nargenicin derivatives  were characterized by UPLC-PDA, HR-QTOF ESI-MS/MS analyses and NMR analyses as par needed. The overall conversion studies indicated both the in vivo and in vitro approaches were suitable approach for structural modification of nargenicin. Furthermore, assessment of solubility indicated that there was enhanced water solubility by almost ~1.8 fold in the case nargenicin A1 11-O-β-D-glucopyranoside than nargenicin, which is beneficial with pharmacokinetics and pharmacodynamic point of view. But, the evaluation of antibacterial potency of the derivatives of nargenicin showed that there was substantial reduction in biological potency of the novel derivatives in comparision to parental compound.

1. Dhakal, D., Le, T. T., Pandey, R. P., Jha, A. K., Gurung, R., Parajuli, P., Pokhrel, A.R., Yoo, J.C. & Sohng, J. K. (2015). Enhanced Production of Nargenicin A1 and Generation of Novel Glycosylated Derivatives. Applied biochemistry and biotechnology, 175: 1-16.
2. Dhakal, D. & Sohng, J. K.(2015) Commentary: Toward a new focus in antibiotic and drug discovery from Streptomyces arsenal. Fronteirs in Microbiology, 2015:6.
3. Painter, R.E., Adam, G.C., Arocho, M., DiNunzio, E., Donald, R.G., Dorso, K., Genilloud, O., Gill, C., Goetz, M., Hairston, N.N., Murgolo, N.(2015) Elucidation of DnaE as the antibacterial target of the natural product, Nargenicin. Chemistry & biology. 22:1362-73.
4. Jha, A.K., Dhakal, D., Van, P.T.T., Pokhrel, A.R., Yamaguchi, T., Jung, H.J., Yoon, Y.J. & Sohng, J.K. (2015). Structural modification of herboxidiene by substrate-flexible cytochrome P450 and glycosyltransferase. Applied microbiology and biotechnology, 99:3421-3431.
5. Pokhrel, A.R., Dhakal, D., Jha, A.K. and Sohng, J.K. (2015). Herboxidiene biosynthesis, production, and structural modifications: prospect for hybrids with related polyketide. Applied microbiology and biotechnology, 99:8351-8362.