Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Biosynthesis of the Antibiotic Bicyclomycin in Soil and Pathogenic Bacteria Jonathan R. Chekan† and Bradley S. Moore*,†,‡ †
Scripps Institution of Oceanography and ‡Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, United States
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the construction of a targeted natural product. For example, the diketopiperazine (DKP) family of natural products has a characteristic dipeptide core that can be installed by one of two enzymatic mechanisms involving either a nonribosomal peptide synthetase (NRPS) or a cyclodipeptide synthase (CDPS).1 Recently, two groups in the United States and China independently employed a genome mining strategy to elucidate the fascinating DKP biosynthetic pathway to bicyclomycin,2,3 a clinically promising antibiotic with a fascinating chemical structure (Figure 1A). While the biosynthetic logic for how this molecule is constructed by soil bacteria has now been unraveled to reveal fascinating oxidative biochemistry, the most unexpected finding is that related genes are also prevalent in a common human bacterial pathogen. The broad-spectrum DKP antibiotic bicyclomycin targets prokaryotic transcription by binding to and inhibiting the transcription factor Rho.4 Notably, Rho is found is a wide variety of bacteria, and bicyclomycin has been shown to be effective against Gram-negative pathogens.5 Bicyclomycin is used in veterinary medicine to treat gastrointestinal tract infections and has also been reinvestigated for human use. While bicyclomycin was first isolated in the 1970s,5 little was known about its biosynthesis. Isotope studies suggested that it utilizes leucine and isoleucine as precursors to form the characteristic DKP core. However, the details of its cyclization and extensive oxidation involving eight of 10 sp3 carbons remained enigmatic. Using a genome mining strategy to identify the bicyclomycin biosynthetic cluster, Bo Li’s lab at the University of North Carolina sequenced the genome of the confirmed producer Streptomyces cinnamoneus ATCC 21532 and searched for known DKP cyclization enzymes as biosynthetic hooks.2 A single putative CDPS was identified in the entire genome (Figure 1B). Closer examination of the genomic context revealed six neighboring genes predicted to encode five αketoglutarate (αKG)-dependent dioxygenases and one cytochrome P450, as well as a major facilitator superfamily (MFS) multidrug transporter. Together, the combination of the predicted CDPS, oxidative, and transporter gene products strongly suggested that this cluster was responsible for the construction, transport, and immunity of bicyclomycin. The high density of oxidative genes was perhaps most noteworthy as the structure of bicyclomycin is quite unusual among DKP molecules. The Li lab used a combination of in vivo and in vitro techniques to validate the bicyclomycin cluster. First, they heterologously expressed the MFS transporter (bcmH) in
he proliferation of rapid and high-throughput genome sequencing technologies has had dramatic impacts on many scientific disciplines, including human health, cell biology, evolution, and biotechnology. In the field of chemistry, these advances have revolutionized our fundamental understanding of nature’s chemicals and the biosynthetic enzymes that construct them. While the isolation and application of natural product compounds have a long and successful history in extending and enriching human life, the ability to link genes to molecules provides abundant new research opportunities in their discovery and engineering. Traditionally, the identification of genes responsible for the biosynthesis of a natural product involved time-consuming genetic experiments; however, current technologies have enabled any lab to affordably sequence and interrogate a microbial genome. Genomic information can thus be mined for a “biosynthetic hook” gene to help connect a cluster of biosynthetic genes to a molecular product (Figure 1A). Numerous bioinformatic tools now exist to facilitate this process by employing gene sequences associated with an enzymatic activity predicted to participate in
Figure 1. (A) Genome mining approach to finding a biosynthetic gene cluster. (B) Bicyclomycin biosynthetic cluster identified in Streptomyces cinnamoneus, Streptomyces sapporonensis, and Pseudomonas aeruginosa. © XXXX American Chemical Society
Received: November 30, 2017
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DOI: 10.1021/acs.biochem.7b01204 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry ORCID
Escherichia coli and showed that previously sensitive E. coli were now resistant to bicyclomycin. They then expressed the putative CDPS (bcmA) in E. coli and observed the production of the cyclo(Ile-Leu) DKP product that was originally proposed as the biosynthetic precursor to bicyclomycin (Figure 1A). The Li lab next explored two of the eight oxidation events by examining αKG-dependent dioxygenases that catalyze single site-specific hydroxylations. Together, the in vitro and in vivo results clearly established bcm as the bicyclomycin biosynthetic cluster. Gong-Li Tang and his colleagues at the Shanghai Institute of Organic Chemistry independently followed a similar path to discover the bicyclomycin genes in a different streptomycete bacterium.3 After establishing the reactivity order of the six oxidases, they took the bold step to reconstitute the entire biosynthetic pathway with recombinant enzymes to produce bicyclomycin in a two-stage synthetic strategy involving the one-pot conversion of cyclo(Ile-Leu) DKP. This elegant total biosynthesis approach achieves the construction of a highly oxidized molecule from a simple precursor involving a remarkable display of enzymatic proficiency. It will be fascinating to learn the extent of the biosynthetic proficiency of these enzymes and whether this approach can be used to produce bicyclomycin and its analogues in scale. The Li lab again exploited a genomics approach to understand the distribution of bcm-like clusters among sequenced bacterial genomes.2 Instead of only finding bcm homologues in other soil-dwelling Streptomyces Gram-positive bacteria, as one might anticipate, they found bcm genes in a very unlikely place, the Gram-negative opportunistic human pathogen Pseudomonas aeruginosa. Still more remarkably, more than one-fourth of >2500 publically available P. aeruginosa genomes contain this antibiotic gene cluster. To confirm these clusters do indeed produce bicyclomycin, the Li lab heterologously expressed the homologous cluster from P. aeruginosa ATCC 14886 and confirmed it as a new producer of bicyclomycin. This high frequency of an antibiotic-producing cluster in an important human pathogen suggests that it may offer the infecting pathogen a competitive advantage in disrupting the host microbiome and causing disease. Scientific literature is filled with an enormous number of fascinating bioactive natural products that have an unknown biosynthetic route. The bicyclomycin study by the Li and Tang laboratories exemplifies how effective a genome mining approach can be in identifying a biosynthetic cluster if an appropriate biosynthetic hook can be predicted.2,3 In this case, searching for enzymes that could produce the unusual DKP core quickly led to the discovery of the bicyclomycin genes, which in turn begs new scientific questions and opportunities. What are the mechanisms of the bicyclomycin oxidase enzymes, and can they be employed as novel biocatalysts in C−H activation biotechnology? What is the function of bicyclomycin in P. aeruginosa, and does it play a role in human infection? As more genomic information and biosynthetic knowledge becomes available, it is clear that linking genes to compounds can result in unexpected observations that can fuel future knowledge and applications across many scientific disciplines.
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Bradley S. Moore: 0000-0002-4652-1253 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Belin, P., Moutiez, M., Lautru, S., Seguin, J., Pernodet, J.-L., and Gondry, M. (2012) The nonribosomal synthesis of diketopiperazines in tRNA-dependent cyclodipeptide synthase pathways. Nat. Prod. Rep. 29, 961−79. (2) Patteson, J. B., Cai, W., Johnson, R. A., Santa Maria, K. C., and Li, B. (2017) Identification of the biosynthetic pathway for the antibiotic bicyclomycin. Biochemistry, DOI: 10.1021/acs.biochem.7b00943. (3) Meng, S., Han, W., Zhao, J., Jian, X.-H., Pan, H.-X., and Tang, G.L. (2017) A six-oxidase cascade for tandem C-H bond activation revealed by reconstitution of bicyclomycin biosynthesis. Angew. Chem., Int. Ed., DOI: 10.1002/anie.201710529. (4) Skordalakes, E., Brogan, A. P., Park, B. S., Kohn, H., and Berger, J. M. (2005) Structural mechanism of inhibition of the Rho transcription termination factor by the antibiotic bicyclomycin. Structure 13, 99− 109. (5) Miyoshi, T., Miyairi, N., Aoki, H., Kohsaka, M., Sakai, H., and Imanaka, H. (1972) Bicyclomycin, a new antibiotic. I. Taxonomy, isolation and characterization. J. Antibiot. 25, 569−575.
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DOI: 10.1021/acs.biochem.7b01204 Biochemistry XXXX, XXX, XXX−XXX
