Ben Shen

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). LTM, isomigrastatin and analogs were found to have a potent antiproliferative effect on tumor cell lines and selectively inhibit protein translation. A systematic comparative study of the effects of CHX and LTM on protein translation revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, defining a common binding pocket for both inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms.

Recent literature on polyketide biosynthesis suggests that polyketide synthases have much greater diversity in both mechanism and structure than the current type I, II and III paradigms. These examples serve as an inspiration for searching novel polyketide synthases to give new insights into polyketide biosynthesis and to provide new opportunities for combinatorial biosynthesis.

Decoding Human Cytomegalovirus

Dissecting HCMV Gene Expression Most of us are infected with human cytomegalovirus (HCMV), but severe disease is almost always limited to immunocompromised individuals or newborn infants. The virus has a relatively large (∼240 kb) DNA genome and shows a complex pattern of gene transcription, hinting at a complex regulatory and coding capacity. Stern-Ginossar et al. (p. 1088) mapped ribosome positions on HCMV transcripts during the course of viral infection of human fibroblast cells. The data suggest the presence of novel open reading frames (ORFs) lying within existing ORFs; very short ORFs upstream of canonical ORFs; ORFs antisense to canonical ORFs; and short, conserved ORFs encoded by long RNAs. Select ORFs were translated, dramatically expanding the coding capacity of the HCMV genome. A closer look at the human cytomegalovirus genome uncovers many new open reading frames. The human cytomegalovirus (HCMV) genome was sequenced 20 years ago. However, like those of other complex viruses, our understanding of its protein coding potential is far from complete. We used ribosome profiling and transcript analysis to experimentally define the HCMV translation products and follow their temporal expression. We identified hundreds of previously unidentified open reading frames and confirmed a fraction by means of mass spectrometry. We found that regulated use of alternative transcript start sites plays a broad role in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus. Our results reveal an unanticipated complexity to the HCMV coding capacity and illustrate the role of regulated changes in transcript start sites in generating this complexity.

A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or “warhead”) found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution

Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

BACKGROUND The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a β-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to “AT-less” multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.

Synthetic biology to access and expand nature's chemical diversity

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products.

Thiopeptide Biosynthesis Featuring Ribosomally Synthesized Precursor Peptides and Conserved Posttranslational Modifications

Thiopeptides, with potent activity against various drug-resistant pathogens, contain a characteristic macrocyclic core consisting of multiple thiazoles, dehydroamino acids, and a 6-membered nitrogen heterocycle. Their biosynthetic pathways remain elusive, in spite of great efforts by in vivo feeding experiments. Here, cloning, sequencing, and characterization of the thiostrepton and siomycin A gene clusters unveiled a biosynthetic paradigm for the thiopeptide specific core formation, featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. The paradigm generality for thiopeptide biosynthesis was supported by genome mining and ultimate confirmation of the thiocillin I production in Bacillus cereus ATCC 14579, a strain that was previously unknown as a thiopeptide producer. These findings set the stage to accelerate the discovery of thiopeptides by prediction at the genetic level and to generate structural diversity by applying combinatorial biosynthesis methods.