title: 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 neor), 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.
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Dipesh
authors.initials: D
authors.lastname: Dhakal
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Chaudhary
authors.affiliation: School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering Institute, Seoul National University, Seoul, Republic of Korea.
authors.forename: Jeong Sang
authors.initials: JS
authors.lastname: Yi
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Anaya Raj
authors.initials: AR
authors.lastname: Pokhrel
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Biplav
authors.initials: B
authors.lastname: Shrestha
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Prakash
authors.initials: P
authors.lastname: Parajuli
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Anil
authors.initials: A
authors.lastname: Shrestha
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.|Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Tokutaro
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.|Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Jung
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.|Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
authors.forename: Seung-Young
authors.initials: SY
authors.lastname: Kim
authors.affiliation: School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering Institute, Seoul National University, Seoul, Republic of Korea.
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
authors.affiliation: Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea. sohng@sunmoon.ac.kr.|Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea. sohng@sunmoon.ac.kr.
authors.forename: Jae Kyung
authors.initials: JK
authors.lastname: Sohng
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors; D007783:Lactones / Q000378:metabolism; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009615:Nocardia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D007783:Lactones; D011994:Recombinant Proteins; C034624:nargenicin A1
keywords: Bioactivity; Multi-monocistronic vector; Nargenicin A1 acid; Nocardia; Synthetic biology