- Email: [email protected]
Small molecules: The lexicon of biodiversity
Journal of Biotechnology 129 (2007) 3–5
Small molecules: The lexicon of biodiversity Julian Davies ∗ Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Received 28 January 2005; accepted 20 July 2006
Abstract The extent of microbial diversity in the Biosphere is not known (and probably never will be!). One aspect of this diversity is the production of biologically active small molecules; within the Streptomycetes alone this may be millions of compounds with an extraordinary diversity and complexity of structure. First recognised as pigments and later, in the 1950s, as antibiotics, it is now clear that the small molecules produced by microbes have many different functions in nature. This huge collection of biologically active compounds with various properties has been used as pharmaceuticals and agriculturals. They also interact with proteins and RNA with high specificity and have been shown to be regulators and effectors of diverse biochemical reactions. The use of small molecules (other than as pharmaceuticals) deserves to be explored in order to exploit microbial biotechnology to the full. © 2006 Published by Elsevier B.V. Keywords: Antibiotics; Effectors; Regulation; Cell-signalling; Riboswiches; Microbial molecular diversity
The enormous diversity of the microbial world in terms of genera, species and products presents many opportunities, yet the potential for commercial development remains largely unexplored outside the pharmaceutical industry. Novel processes dependent on cell-based or enzyme-based conversions are frequently mentioned as prospects in the fields of “white” biotechnology or “green” chemistry. Surprisingly, investment in the industrial exploration of this enormous biocatalytic potential has been disappointingly small, even at a time when increasing and often ∗
Tel.: +1 604 822 5856; fax: +1 604 822 6041. E-mail address: [email protected].
0168-1656/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jbiotec.2006.11.023
uncontrolled industrial pollution is a worldwide threat to health. Hopes that the international Kyoto agreement for the protection of the environment (signed by the forward-looking countries of the world) would lead to a demand for the introduction of environmentally friendly processes on a large scale are still unrealized. By contrast, the modern pharmaceutical industry (the creation of which was entirely due to the development of bioactive microbial products) took advantage of microbial molecular diversity to revolutionize the treatment of infectious diseases and provide therapeutic options for many other common ailments. The discovery of pharmaceutically active small molecules was one of the most successful investments of all time, and
4
J. Davies / Journal of Biotechnology 129 (2007) 3–5
hundreds of billions of dollars have been earned by a relatively small number of specialized fermentation companies. The impact of microbial products has benefited human health on this planet immeasurably. Also, the modern biotechnology industry (post-recombinant DNA) arose in an unexpected way from the discovery of antibiotic resistance plasmids and the development of gene transfer methodology in the laboratory. However, the wealth of small molecules has been little exploited beyond therapeutics. It is difficult to give an estimate of the chemical diversity of the microbial world. The well-studied strain Streptomyces hygroscopicus has been demonstrated to produce more than 40 different small molecules. The nucleotide sequence of the genome of Streptomyces coelicolor, the first bacterium of its class to be sequenced, indicates the potential to produce some 24 distinct compounds (Bentley et al., 2002). Thus, the streptomycetes alone may produce upwards of a million different small molecules, all with distinct biological activities (Davies, 2004). Considering the many different bacteria that make small molecules, there may be billions of natural compounds of molecular mass less than 1000 Da! Fewer than 1% of all microbial strains can be grown under laboratory conditions; thus the potential of their small molecule products can barely be imagined. While many will have therapeutic value (and must be investigated further, which is good for the pharmaceutical industry), there are numerous other directions to explore.Given the vast chemical space defined by natural products (Dobson, 2004), what roles might these bioactive molecules play in nature? One can assume that each and every compound has a bioactive function. Because many of these compounds were discovered in the search for antibiotics, it has been generally assumed that cell-growth inhibition in microbial communities is their main natural function. This may prove to be only a minor role, however; it is more likely that most of the compounds are signaling or regulatory molecules, acting within cells or communities to maintain homeostatic conditions. They interact with proteins in a specific manner and their potential uses in biotechnology are many and varied. In general, the myriad processes involving enzymatic reactions are controlled by factors such as temperature, reactant concentration, ionic environment, pH, and other physical conditions. But we should not forget that many small molecules are enzyme effectors. This has been demonstrated for
some so-called antibiotics such as the aminoglycosides, which exert allosteric effects on the activity of certain phospholipases (Morris et al., 1996). They are also known to modulate RNA enzyme (ribozyme) activities (Schroeder and Wallis, 2001). In fact, a variety of “inhibitors” exert such modulation and could be used for fine control of cellular or cell-free reactions. For example, although aminoglycosides are generally presumed to be anti-bacterial agents, their ability to bind to nucleic acids and proteins in a site- (sequence-) specific manner is being exploited by several research groups to generate agents effective in the biological control of viruses (Werstuck and Green, 1998). Small molecules are extremely effective modulators of transcription in bacteria and possibly all types of cells (Goh et al., 2002). One regulatory process in bacteria (and all cells?) of increasing interest involves riboswitches (Mandal and Breaker, 2004; Grundy and Henkin, 2004); this is perhaps the first discovery of a general mechanism whereby small molecules modulate messenger RNA function. The binding of compounds such as guanine, alanine and other chemicals to specific RNA sequences (aptamers) permits activation or repression of gene expression and can be used to characterize metabolic networks in bacterial cells. It is likely that many so-called microbial secondary metabolites play similar roles in gene expression. Antibiotics possess dual modes of action. At low concentrations (between 1% and 10% of the minimal inhibitory concentration) they act as global regulators by modulating the transcription of some 10% of the promoters in a bacterial cell (Goh et al., 2002; Tsui et al., 2004). At concentrations above the MIC the growth of the microbe is blocked and in some cases the target organism is killed; there are relatively few antibiotics that have true bactericidal activity. Many antibiotics have activity on eukaryotic cells, even though there is no antibiosis. For example, it has been shown recently that -lactam antibiotics such as ceftriaxone exert neuro-protective effects by increasing transcription of the glutamate transporter gene in spinal cord cultures (Rothstein et al., 2005). We have found that antibiotics such as rifampicin and azithromycin modulate transcription of some 400 mammalian cell genes in tissue culture (unpublished results). Thus it is likely that, in addition to antibiotic activity, these small molecules have valuable uses in regulating many reactions by effects on cell transcription processes.
J. Davies / Journal of Biotechnology 129 (2007) 3–5
The possibility of regulating biosynthetic processes in vivo and in vitro with small molecule effectors may be of advantage for sequential reaction control in commercial bioprocesses; for example, specific biocatalytic steps could be controlled by the addition of low concentrations of the effectors. For such uses the compounds must have a highly specific activity. In addition, while compounds such as antibiotics have been employed extensively as selective agents in genetic engineering (a spinoff from the development of antibiotic resistance), they have been little considered as reagents for other purposes; pigmented compounds might be used for monitoring reactions or as the basis for diagnostic kits. Since many low-molecular-weight compounds have high affinity for cellular proteins, could they be developed as an alternative to antibodies? It is important to note that the cellular targets (receptors) of small molecules are phylogenetically conserved, which must have implications in their evolution (Briones et al., 2005). This has been demonstrated in many instances; receptors such as ribosomal proteins for small molecule modulation of protein synthesis or topoisomerases in DNA replication have protein domains that are conserved across many genera. Patterns of antibiotic resistance on the ribosome can be employed to confirm phylogenetic relationships. These considerations add support to the notion that a variety of small molecules could be employed to modulate many types of cell function in bacteria, archaea, and eukaryotes. For a specific example, could this be a natural function of the bacterial product phosphonomycin? It is used as an herbicide, but might it have another role to play in bacteria/plant interactions? It is reasonable to predict that a variety of enzymic reaction processes may be enhanced, repressed, or even revealed in the presence of low concentrations of naturally occurring small molecules. Microbial products have been used pharmaceutically for their suppressive activities; it is time they were employed in what is, perhaps, their natural role as modulators or effectors, which could be either up- or down-regulation of biocatalysis. The vast chemical space and biochemical wealth of small molecules hold many surprises and benefits for both research and industry.
5
References Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., Hopwood, D.A., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417 (6885), 141–147. Briones, C., Manrubia, S.C., Lazaro, E., Lazcano, A., Amils, R., 2005. Reconstructing evolutionary relationships from functional data: a consistent classification of organisms based on translation inhibition response. Mol. Phylogenet. Evol. 34 (2), 371–381. Davies, J.E., 2004. Streptomycetes and beyond. Microbiol. Aust. 25, 8–10. Dobson, C.M., 2004. Chemical space and biology. Nature 432 (7019), 824–828. Goh, E.-B., Yim, G., Tsui, W., McClure, J., Surette, M.G., Davies, J., 2002. Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc. Natl. Acad. Sci. 99, 17025–17030. Grundy, F.J., Henkin, T.M., 2004. Regulation of gene expression by effectors that bind to RNA. Curr. Opin. Microbiol. 7 (2), 126–131. Mandal, M., Breaker, R.R., 2004. Gene regulation by riboswitches. Nat. Rev. Mol. Cell Biol. 5 (6), 451–463. Morris, J.C., Ping-Sheng, L., Zhai, H.-X., Shen, T.-Y., MensaWilmot, K., 1996. Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418 2-deoxy2-fluoro-scyllo-inositol-1-0-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C. J. Biol. Chem. 271, 15468–15477. Rothstein, J.D., Patel, S., Regan, M.R., Haenggeli, C., Huang, Y.H., Bergles, D.E., Jin, L., Dykes Hoberg, M., Vidensky, S., Chung, D.S., Toan, S.V., Bruijn, L.I., Su, Z.Z., Gupta, P., Fisher, P.B., 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433 (7021), 73–77. Schroeder, R., Wallis, M.G., 2001. RNA-binding Antibiotics. Landes Biosciences, Georgetown, Texas. Tsui, W.H., Yim, G., Wang, H.H., McClure, J.E., Surette, M.G., Davies, J., 2004. Dual effects of MLS antibiotics: transcriptional modulation and interactions on the ribosome. Chem. Biol. 11 (9), 1307–1316. Werstuck, G., Green, M.R., 1998. Controlling gene expression in living cells through small molecule–RNA interactions. Science 282, 296–298.
Small molecules: The lexicon of biodiversity Julian Davies ∗ Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Received 28 January 2005; accepted 20 July 2006
Abstract The extent of microbial diversity in the Biosphere is not known (and probably never will be!). One aspect of this diversity is the production of biologically active small molecules; within the Streptomycetes alone this may be millions of compounds with an extraordinary diversity and complexity of structure. First recognised as pigments and later, in the 1950s, as antibiotics, it is now clear that the small molecules produced by microbes have many different functions in nature. This huge collection of biologically active compounds with various properties has been used as pharmaceuticals and agriculturals. They also interact with proteins and RNA with high specificity and have been shown to be regulators and effectors of diverse biochemical reactions. The use of small molecules (other than as pharmaceuticals) deserves to be explored in order to exploit microbial biotechnology to the full. © 2006 Published by Elsevier B.V. Keywords: Antibiotics; Effectors; Regulation; Cell-signalling; Riboswiches; Microbial molecular diversity
The enormous diversity of the microbial world in terms of genera, species and products presents many opportunities, yet the potential for commercial development remains largely unexplored outside the pharmaceutical industry. Novel processes dependent on cell-based or enzyme-based conversions are frequently mentioned as prospects in the fields of “white” biotechnology or “green” chemistry. Surprisingly, investment in the industrial exploration of this enormous biocatalytic potential has been disappointingly small, even at a time when increasing and often ∗
Tel.: +1 604 822 5856; fax: +1 604 822 6041. E-mail address: [email protected].
0168-1656/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jbiotec.2006.11.023
uncontrolled industrial pollution is a worldwide threat to health. Hopes that the international Kyoto agreement for the protection of the environment (signed by the forward-looking countries of the world) would lead to a demand for the introduction of environmentally friendly processes on a large scale are still unrealized. By contrast, the modern pharmaceutical industry (the creation of which was entirely due to the development of bioactive microbial products) took advantage of microbial molecular diversity to revolutionize the treatment of infectious diseases and provide therapeutic options for many other common ailments. The discovery of pharmaceutically active small molecules was one of the most successful investments of all time, and
4
J. Davies / Journal of Biotechnology 129 (2007) 3–5
hundreds of billions of dollars have been earned by a relatively small number of specialized fermentation companies. The impact of microbial products has benefited human health on this planet immeasurably. Also, the modern biotechnology industry (post-recombinant DNA) arose in an unexpected way from the discovery of antibiotic resistance plasmids and the development of gene transfer methodology in the laboratory. However, the wealth of small molecules has been little exploited beyond therapeutics. It is difficult to give an estimate of the chemical diversity of the microbial world. The well-studied strain Streptomyces hygroscopicus has been demonstrated to produce more than 40 different small molecules. The nucleotide sequence of the genome of Streptomyces coelicolor, the first bacterium of its class to be sequenced, indicates the potential to produce some 24 distinct compounds (Bentley et al., 2002). Thus, the streptomycetes alone may produce upwards of a million different small molecules, all with distinct biological activities (Davies, 2004). Considering the many different bacteria that make small molecules, there may be billions of natural compounds of molecular mass less than 1000 Da! Fewer than 1% of all microbial strains can be grown under laboratory conditions; thus the potential of their small molecule products can barely be imagined. While many will have therapeutic value (and must be investigated further, which is good for the pharmaceutical industry), there are numerous other directions to explore.Given the vast chemical space defined by natural products (Dobson, 2004), what roles might these bioactive molecules play in nature? One can assume that each and every compound has a bioactive function. Because many of these compounds were discovered in the search for antibiotics, it has been generally assumed that cell-growth inhibition in microbial communities is their main natural function. This may prove to be only a minor role, however; it is more likely that most of the compounds are signaling or regulatory molecules, acting within cells or communities to maintain homeostatic conditions. They interact with proteins in a specific manner and their potential uses in biotechnology are many and varied. In general, the myriad processes involving enzymatic reactions are controlled by factors such as temperature, reactant concentration, ionic environment, pH, and other physical conditions. But we should not forget that many small molecules are enzyme effectors. This has been demonstrated for
some so-called antibiotics such as the aminoglycosides, which exert allosteric effects on the activity of certain phospholipases (Morris et al., 1996). They are also known to modulate RNA enzyme (ribozyme) activities (Schroeder and Wallis, 2001). In fact, a variety of “inhibitors” exert such modulation and could be used for fine control of cellular or cell-free reactions. For example, although aminoglycosides are generally presumed to be anti-bacterial agents, their ability to bind to nucleic acids and proteins in a site- (sequence-) specific manner is being exploited by several research groups to generate agents effective in the biological control of viruses (Werstuck and Green, 1998). Small molecules are extremely effective modulators of transcription in bacteria and possibly all types of cells (Goh et al., 2002). One regulatory process in bacteria (and all cells?) of increasing interest involves riboswitches (Mandal and Breaker, 2004; Grundy and Henkin, 2004); this is perhaps the first discovery of a general mechanism whereby small molecules modulate messenger RNA function. The binding of compounds such as guanine, alanine and other chemicals to specific RNA sequences (aptamers) permits activation or repression of gene expression and can be used to characterize metabolic networks in bacterial cells. It is likely that many so-called microbial secondary metabolites play similar roles in gene expression. Antibiotics possess dual modes of action. At low concentrations (between 1% and 10% of the minimal inhibitory concentration) they act as global regulators by modulating the transcription of some 10% of the promoters in a bacterial cell (Goh et al., 2002; Tsui et al., 2004). At concentrations above the MIC the growth of the microbe is blocked and in some cases the target organism is killed; there are relatively few antibiotics that have true bactericidal activity. Many antibiotics have activity on eukaryotic cells, even though there is no antibiosis. For example, it has been shown recently that -lactam antibiotics such as ceftriaxone exert neuro-protective effects by increasing transcription of the glutamate transporter gene in spinal cord cultures (Rothstein et al., 2005). We have found that antibiotics such as rifampicin and azithromycin modulate transcription of some 400 mammalian cell genes in tissue culture (unpublished results). Thus it is likely that, in addition to antibiotic activity, these small molecules have valuable uses in regulating many reactions by effects on cell transcription processes.
J. Davies / Journal of Biotechnology 129 (2007) 3–5
The possibility of regulating biosynthetic processes in vivo and in vitro with small molecule effectors may be of advantage for sequential reaction control in commercial bioprocesses; for example, specific biocatalytic steps could be controlled by the addition of low concentrations of the effectors. For such uses the compounds must have a highly specific activity. In addition, while compounds such as antibiotics have been employed extensively as selective agents in genetic engineering (a spinoff from the development of antibiotic resistance), they have been little considered as reagents for other purposes; pigmented compounds might be used for monitoring reactions or as the basis for diagnostic kits. Since many low-molecular-weight compounds have high affinity for cellular proteins, could they be developed as an alternative to antibodies? It is important to note that the cellular targets (receptors) of small molecules are phylogenetically conserved, which must have implications in their evolution (Briones et al., 2005). This has been demonstrated in many instances; receptors such as ribosomal proteins for small molecule modulation of protein synthesis or topoisomerases in DNA replication have protein domains that are conserved across many genera. Patterns of antibiotic resistance on the ribosome can be employed to confirm phylogenetic relationships. These considerations add support to the notion that a variety of small molecules could be employed to modulate many types of cell function in bacteria, archaea, and eukaryotes. For a specific example, could this be a natural function of the bacterial product phosphonomycin? It is used as an herbicide, but might it have another role to play in bacteria/plant interactions? It is reasonable to predict that a variety of enzymic reaction processes may be enhanced, repressed, or even revealed in the presence of low concentrations of naturally occurring small molecules. Microbial products have been used pharmaceutically for their suppressive activities; it is time they were employed in what is, perhaps, their natural role as modulators or effectors, which could be either up- or down-regulation of biocatalysis. The vast chemical space and biochemical wealth of small molecules hold many surprises and benefits for both research and industry.
5
References Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., Hopwood, D.A., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417 (6885), 141–147. Briones, C., Manrubia, S.C., Lazaro, E., Lazcano, A., Amils, R., 2005. Reconstructing evolutionary relationships from functional data: a consistent classification of organisms based on translation inhibition response. Mol. Phylogenet. Evol. 34 (2), 371–381. Davies, J.E., 2004. Streptomycetes and beyond. Microbiol. Aust. 25, 8–10. Dobson, C.M., 2004. Chemical space and biology. Nature 432 (7019), 824–828. Goh, E.-B., Yim, G., Tsui, W., McClure, J., Surette, M.G., Davies, J., 2002. Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc. Natl. Acad. Sci. 99, 17025–17030. Grundy, F.J., Henkin, T.M., 2004. Regulation of gene expression by effectors that bind to RNA. Curr. Opin. Microbiol. 7 (2), 126–131. Mandal, M., Breaker, R.R., 2004. Gene regulation by riboswitches. Nat. Rev. Mol. Cell Biol. 5 (6), 451–463. Morris, J.C., Ping-Sheng, L., Zhai, H.-X., Shen, T.-Y., MensaWilmot, K., 1996. Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418 2-deoxy2-fluoro-scyllo-inositol-1-0-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C. J. Biol. Chem. 271, 15468–15477. Rothstein, J.D., Patel, S., Regan, M.R., Haenggeli, C., Huang, Y.H., Bergles, D.E., Jin, L., Dykes Hoberg, M., Vidensky, S., Chung, D.S., Toan, S.V., Bruijn, L.I., Su, Z.Z., Gupta, P., Fisher, P.B., 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433 (7021), 73–77. Schroeder, R., Wallis, M.G., 2001. RNA-binding Antibiotics. Landes Biosciences, Georgetown, Texas. Tsui, W.H., Yim, G., Wang, H.H., McClure, J.E., Surette, M.G., Davies, J., 2004. Dual effects of MLS antibiotics: transcriptional modulation and interactions on the ribosome. Chem. Biol. 11 (9), 1307–1316. Werstuck, G., Green, M.R., 1998. Controlling gene expression in living cells through small molecule–RNA interactions. Science 282, 296–298.