Enhanced production of nargenicin A1 and creation of a novel derivative using a synthetic biology platform

Abstract

Nargenicin A1, an antibacterial produced by Nocardia sp. CS682 (KCTC 11297BP), demonstrates effective activity against various Gram-positive bacteria. Hence, we attempted to enhance nargenicin A1 production by utilizing the cumulative effect of synthetic biology, metabolic engineering and statistical media optimization strategies. To facilitate the modular assembly of multiple genes for genetic engineering in Nocardia sp. CS682, we constructed a set of multi-monocistronic vectors, pNV18L1 and pNV18L2 containing hybrid promoter (derived from ermE* and promoter region of neo ), ribosome binding sites (RBS), and restriction sites for cloning, so that each cloned gene was under its own promoter and RBS. The multi-monocistronic vector, pNV18L2 containing transcriptional terminator showed better efficiency in reporter gene assay. Thus, multiple genes involved in the biogenesis of pyrrole moiety (ngnN2, ngnN3, ngnN4, and ngnN5 from Nocardia sp. CS682), glucose utilization (glf and glk from Zymomonas mobilis), and malonyl-CoA synthesis (accA2 and accBE from Streptomyces coelicolor A3 (2)), were cloned in pNV18L2. Further statistical optimization of specific precursors (proline and glucose) and their feeding time led to ~84.9 mg/L nargenicin from Nocardia sp. GAP, which is ~24-fold higher than Nocardia sp. CS682 (without feeding). Furthermore, pikC from Streptomyces venezuelae was expressed to generate Nocardia sp. PikC. Nargenicin A1 acid was characterized as novel derivative of nargenicin A1 produced from Nocardia sp. PikC by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. We also performed comparative analysis of the anticancer and antibacterial activities of nargenicin A1 and nargenicin A1 acid, which showed a reduction in antibacterial potential for nargenicin A1 acid. Thus, the development of an efficient synthetic biological platform provided new avenues for enhancing or structurally diversifying nargenicin A1 by means of pathway designing and engineering.

Marine Rare Actinobacteria: Isolation, Characterization, and Strategies for Harnessing Bioactive Compounds

“…Fundamentally, engineering or modulating the precursor pathways can lead to enhancement or diversification of natural products (Dhakal et al, 2016). Combinatorial biosynthesis exploits the shuffling of anabolic pathways by precursor directed biosynthesis, enzyme level modulations, and pathway level recombination, leading to novel natural products (Sun et al, 2015; Winn et al, 2016).…”

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms

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“…However, silicon-based materials can only be produced chemically because of the absence of organosilicon molecules in living organisms ( Apeloig et al, 2001 ). Likewise, important secondary metabolites, such as polyketides and alkaloids, isolated from organisms (e.g., plants and marine microorganisms) can provide alternative therapeutics against health threats such as multidrug resistance bacteria and intractable cancers ( Dhakal et al, 2016 ). Yet, while more potent analogs of these natural products may be discovered and obtained chemically ( DeChristopher et al, 2012 ), novel biosynthesis routes to these superior non-natural drugs are elusive.…”

Coalition of Biology and Chemistry for Ameliorating Antimicrobial Drug Discovery

“…“Genome mining” approach has enabled the computation of mined genetic data and the connection to particular NPs, even if they are cryptic or produced in insignificant titers (Ziemert et al, 2016). Thus, native hosts, genetically tractable alternative hosts or suitable heterologous hosts, can be used as a platform for system-level metabolic engineering approaches (Luo et al, 2015; Dhakal et al, 2016). Some of the key foundations (Figure 1) are briefly summarized below.…”

“…While NPs exhibit a wide range of pharmacophores and a high degree of stereochemistry (Harvey et al, 2015), novel NPs with better activities still need to be developed. Versatile biological knowledge based synthetic-biology approaches, system-biology guided metabolic engineering techniques, enzymatic modifications, and synthetic chemistry methods can be utilized to maximize the benefit of NPs from the source organism (Dhakal et al, 2015, 2016). Thus, the optimum application of NPs can only be improved with considerable effort based on precise screening, higher production, and desirable structural diversification (Dhakal and Sohng, 2015).…”