Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor

Abstract

IntroductionActinomycetes are a source for discovery of important secondary metabolites containing a number of drugs and analogs that have been commercialized and are still used in human/animal health and crop protection [1,2]. Actinomycetes-produced natural products (NPs) are associated with diverse biosynthetic gene clusters (BGCs). BGCs are groups of genes responsible for producing specialized metabolites [3]. The majority of these actinomycetes NP BGCs belong to three types: polyketide synthase (PKS), non-ribosomal peptide synthase (NRPS), and a combination of PKS and NRPS [4]. Among them, some polyketides made by PKS complete the final structure after further modification by enzymes, such as P450 hydroxylase and glycosyltransferase [5,6]. The representative polyene macrolide antibiotics synthesized by type I PKS include amphotericin, nystatin, candicidin, and NPP, which are generally antifungal compounds with 3-8 conjugated bonds to the core macrolactone rings with 20-40 carbon atoms [7,8].A rare actinomycete, Pseudonocardia autotrophica, produces NPP A1. This compound has the same core macrolactone structure as nystatin A1, but unlike nystatin A1, it contains a unique disaccharide. NPP A1 has high solubility and low hemolytic activity compared to nystatin A1, but its antifungal activity is lower than that of nystatin A1 [9]. Therefore, NPP B1, which has a heptaene structure from the core macrolactone structure of tetraene, was developed by substituting two site-specific amino acids in module 5 of the NPP A1 biosynthetic gene nppC [10]. NPP B1 was developed by genetic manipulation and is less toxic but more powerful as an antifungal drug with similar efficacy in vivo compared to amphotericin B. Therefore, NPP B1 is a promising candidate for pharmacokinetically improved and less toxic polyene antifungal antibiotics [11,12]. On the other hand, the production level of NPP B1 was very low compared to the strain producing NPP A1 [10]. Various NPP B1 titer-As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-exp...

Generation of New Glycoanalogues of Polyene Antibiotics by Synthetic Biology—Testing Current Technical Boundaries

“…Disaccharides, such as lactose, that can make a free aldehyde group available may also be used. The Korean group, led by Eung-Soo Kim, has carried out extensive genetic engineering of NPP A1 biosynthesis [26][27][28][29][30]. Analogues were generated by inactivating the C10 hydroxylase P450, by engineering the PKS to make a heptaene rather than a tetraene, and by substituting NypY for NppY to replace GlcNAc with mannose.…”