Development of an Unnatural Amino Acid Incorporation System in the Actinobacterial Natural Product Producer Streptomyces venezuelae ATCC 15439

Abstract

Many Actinobacteria, most notably Streptomyces, produce structurally diverse bioactive natural products, including ribosomally synthesized peptides, by multistep enzymatic pathways. The use of site-specific genetic incorporation of unnatural amino acids to investigate and manipulate the functions of natural product biosynthetic enzymes, enzyme complexes, and ribosomally-derived peptides in these organisms would have important implications for drug discovery and development efforts. Here, we have designed, constructed, and optimized unnatural amino acid systems capable of incorporating p-iodo-l-phenylalanine and p-azido-l-phenylalanine site-specifically into proteins in the model natural product producer Streptomyces venezuelae ATCC 15439. We observed notable differences in the fidelity and efficiency of these systems between S. venezuelae and previously used hosts. Our findings serve as a foundation for using an expanded genetic code in Streptomyces to address questions related to natural product biosynthesis and mechanism of action that are relevant to drug discovery and development.

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

“…The process of cell-extract preparation can be divided into five stages in the order of (1) cell-growth, (2) washing, (3) sonication, (4) run-off and (5) dialysis. We merged the Streptomyces method for stages (1-2) with the E. coli TX-TL methodology for stages (3)(4)(5). This new protocol provided a significant baseline level of sfGFP fluorescence (154 nM), whereas by following the separate protocols on their own, only trace levels of sfGFP fluorescence (≈5 nM) were observed (data not shown).…”

“…Recently, Streptomyces venezuelae, the chloramphenicol producer, has been adopted by synthetic biology for its use in metabolic engineering [2], since it is relatively well characterized, has strong promoter tools and genome engineering plasmids for integration [3][4][5]. Whilst it is not as characterized as Streptomyces coelicolor A3(2), in contrast, S. venezuelae provides significant advantages such as a fast growth (≈T D = 40 min) and no aggregation during liquid culture [3].…”

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

“…These features simplify the engineering of PylRS and TyrRS for a desired ncAA by in vivo selection methods (Figure 1c) and facilitate the targeting of different codons for reassignment (Table 1). The M. jannaschii TyrRS•tRNA Tyr pair is used in bacteria such as E. coli [1 •• ], Salmonella [9], Streptomyces [10], and Mycobacterium [11] (Table 1); yet this pair is not suitable for eukaryotic applications because identity elements overlap with eukaryotic TyrRS•tRNA Tyr . However, the E. coli TyrRS•tRNA Tyr pair has been adapted for GCE in diverse eukaryotic species [12]; although, the repertoire of ncAA substrates is smaller (Table 1).…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…1B59) into histone H3 (Elsasser et al 2016). Methodological advances have also allowed the genetic incorporation of ncAAs into proteins in various multicellular organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Streptomyces venezuelae (Greiss and Chin 2011;Bianco et al 2012;Chin 2014;He et al 2015). Most recently, we have been able to genetically encode ncAAs in Bacillus cereus, allowing the synthesis of ribosomally derived natural products from unnatural building blocks (Luo et al 2016).…”