Metabolic model for diversity-generating biosynthesis.

A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.

Accessing the hidden majority of marine natural products through metagenomics.

Tiny marine animals represent an untapped reservoir for undiscovered, bioactive natural products. However, their small size and extreme chemical variability preclude traditional chemical approaches to discovering new bioactive compounds. Here, we use a metagenomic method to directly discover and rapidly access cyanobactin class natural products from these variable samples, and provide proof-of-concept for genome-based discovery and supply of marine natural products. We also address practical optimization of complex, multistep ribosomal peptide pathways in heterologous hosts, which is still very challenging. The resulting methods and concepts will be applicable to ribosomal peptide and other biosynthetic pathways.

Assessing the combinatorial potential of the RiPP cyanobactin tru pathway.

Ribosomally produced natural products, the RiPPs, exhibit features that are potentially useful in the creation of large chemical libraries using simple mutagenesis. RiPPs are encoded on ribosomal precursor peptides, but they are extensively posttranslationally modified, endowing them with properties that are useful in drug discovery and biotechnology. In order to determine which mutations are acceptable, strategies are required to determine sequence selectivity independently of the context of flanking amino acids. Here, we examined the absolute sequence selectivity of the trunkamide cyanobactin pathway, tru. A series of random double and quadruple simultaneous mutants were synthesized and produced in Escherichia coli. Out of a total of 763 mutated amino acids examined in 325 unique sequences, 323 amino acids were successfully incorporated in 159 sequences, leading to >300 new compounds. Rules for tru sequence selectivity were determined, which will be useful for the design and synthesis of combinatorial biosynthetic libraries. The results are also interpreted in comparison to the known natural products of tru and pat cyanobactin pathways.

Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo.

Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.