Harnessing the biosynthetic code: Combinations, permutations, and mutations

Science

Volumn 282, Issue 5386, 1998, Pages 63-68

  Cane, David E ^a   Walsh, Christopher T ^b   Khosla, Chaitan ^c,d  

^a BROWN UNIVERSITY   (United States)

^b HARVARD MEDICAL SCHOOL   (United States)

^c STANFORD UNIVERSITY   (United States)

^d KOSAN BIOSCIENCES INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AVERMECTIN; CARBOXYLIC ACID; CYCLOSPORIN; ERYTHROMYCIN; NATURAL PRODUCT; PENICILLIN DERIVATIVE; PEPTIDE SYNTHASE; POLYKETIDE;

BIOSYNTHESIS; CATALYSIS; CHEMICAL MODIFICATION; CHEMICAL REACTION; ENZYME ACTIVE SITE; PEPTIDE ANALYSIS; PRIORITY JOURNAL; REVIEW; STRUCTURE ACTIVITY RELATION; STRUCTURE ANALYSIS;

APOENZYMES; BINDING SITES; MULTIENZYME COMPLEXES; PEPTIDE BIOSYNTHESIS; PEPTIDE SYNTHASES; PEPTIDES; PROTEIN ENGINEERING;

EID: 0032475992     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5386.63     Document Type: Review

References (66)

1. For a thematic issue reviewing the recent literature on the molecular genetics and enzymology of polyketide and non-ribosomal peptide biosynthesis, see Chem. Rev. 97 (November 1997).
2. It has often been argued that the observed biological potency and selectivity of these microbial metabolites is the result of evolutionary pressures. While this simple argument may be true for some natural products, an increasing number of examples suggest that the complete picture is far more complex. For example, erythromycin A, an antibiotic produced by the soil organism Saccharopolyspora erythraea and which inhibits ribosomal protein biosynthesis in bacteria, readily undergoes an intramolecular cyclization under acidic conditions such as found in the stomach so as to generate a rearranged metabolite that is inactive as an antibacterial but is now a potent agonist of the motilin receptor [S. Omura et al., J. Med. Chem. 30, 1943 (1987); P. A. Lartey et al., ibid. 38, 1793 (1995)]. Clearly these two properties associated with the same fundamental natural product skeleton are mechanistically unrelated, and it is unlikely that evolutionary pressures led to the simultaneous optimization of both properties. What is undisputed about these families of natural products are their intrinsic pharmacological properties.
3. It has often been argued that the observed biological potency and selectivity of these microbial metabolites is the result of evolutionary pressures. While this simple argument may be true for some natural products, an increasing number of examples suggest that the complete picture is far more complex. For example, erythromycin A, an antibiotic produced by the soil organism Saccharopolyspora erythraea and which inhibits ribosomal protein biosynthesis in bacteria, readily undergoes an intramolecular cyclization under acidic conditions such as found in the stomach so as to generate a rearranged metabolite that is inactive as an antibacterial but is now a potent agonist of the motilin receptor [S. Omura et al., J. Med. Chem. 30, 1943 (1987); P. A. Lartey et al., ibid. 38, 1793 (1995)]. Clearly these two properties associated with the same fundamental natural product skeleton are mechanistically unrelated, and it is unlikely that evolutionary pressures led to the simultaneous optimization of both properties. What is undisputed about these families of natural products are their intrinsic pharmacological properties.
4. J. Cortes, S. F. Haydock, G. A. Roberts, D. J. Bevitt, P. F. Leadlay, Nature 348, 176 (1990); S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); P. Caffrey, D. J. Bevitt, J. Staunton, P. F. Leadlay, FEBS Lett. 304, 225 (1992).
5. J. Cortes, S. F. Haydock, G. A. Roberts, D. J. Bevitt, P. F. Leadlay, Nature 348, 176 (1990); S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); P. Caffrey, D. J. Bevitt, J. Staunton, P. F. Leadlay, FEBS Lett. 304, 225 (1992).
6. J. Cortes, S. F. Haydock, G. A. Roberts, D. J. Bevitt, P. F. Leadlay, Nature 348, 176 (1990); S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); P. Caffrey, D. J. Bevitt, J. Staunton, P. F. Leadlay, FEBS Lett. 304, 225 (1992).
7. Many polyketides, particularly aromatic polyketides such as tetracyline, are biosynthesized by structurally simpler polyketide synthases called iterative or type II synthases. Although these PKSs are also subject to combinatorial manipulation, they are not discussed in this review. For recent comprehensive reviews, see (1).
8. R. H. Lambalot et al., Chem. Biol. 3, 923 (1996).
9. G. Weber, K. Schorgendorfer, E. Schneider-Scherzer, E. Leitner, Curr. Genet. 26, 120 (1994).
10. M. A. Marahiel, T. Stachelhaus, H. D. Mootz, Chem. Rev. 97, 2651 (1997); H. von Döhren, U. Keller, J. Vater, R. Zocher, ibid., p. 2675.
11. M. A. Marahiel, T. Stachelhaus, H. D. Mootz, Chem. Rev. 97, 2651 (1997); H. von Döhren, U. Keller, J. Vater, R. Zocher, ibid., p. 2675.
12. D. J. Smith, J. E. Alison, G. Turner, EMBO J. 9, 2743 (1990); S. Guttierez, B. Diez, E. Montenegro, J. F. Martin, J. Bacteriol. 173, 2354 (1991); J. R. Coque, J. F. Martin, P. Liras, Mol. Microbiol. 5, 1125 (1991); A. P. MacCabe et al., J. Biol. Chem. 266, 12646 (1991).
13. D. J. Smith, J. E. Alison, G. Turner, EMBO J. 9, 2743 (1990); S. Guttierez, B. Diez, E. Montenegro, J. F. Martin, J. Bacteriol. 173, 2354 (1991); J. R. Coque, J. F. Martin, P. Liras, Mol. Microbiol. 5, 1125 (1991); A. P. MacCabe et al., J. Biol. Chem. 266, 12646 (1991).
14. D. J. Smith, J. E. Alison, G. Turner, EMBO J. 9, 2743 (1990); S. Guttierez, B. Diez, E. Montenegro, J. F. Martin, J. Bacteriol. 173, 2354 (1991); J. R. Coque, J. F. Martin, P. Liras, Mol. Microbiol. 5, 1125 (1991); A. P. MacCabe et al., J. Biol. Chem. 266, 12646 (1991).
15. D. J. Smith, J. E. Alison, G. Turner, EMBO J. 9, 2743 (1990); S. Guttierez, B. Diez, E. Montenegro, J. F. Martin, J. Bacteriol. 173, 2354 (1991); J. R. Coque, J. F. Martin, P. Liras, Mol. Microbiol. 5, 1125 (1991); A. P. MacCabe et al., J. Biol. Chem. 266, 12646 (1991).
16. D. E. Cane and C.-C. Yang, J. Am. Chem. Soc. 109, 1255 (1987); S. Yue, J. S. Duncan, Y. Yamamoto, C. R. Hutchinson, ibid., p. 1253.
17. D. E. Cane and C.-C. Yang, J. Am. Chem. Soc. 109, 1255 (1987); S. Yue, J. S. Duncan, Y. Yamamoto, C. R. Hutchinson, ibid., p. 1253.
18. Internal capture by the amino group of the mature polypeptide chain is the release mechanism in the formation of the characteristic macrolactam of such cyclic peptides as cyclosporin A, gramicidin S, and bacitracin.
19. K. J. Weissman et al., Angew. Chem. Int. Ed. Engl. 37, 1437 (1998).
20. J. Cortes et al., Science 268, 1487 (1995); C. M. Kao, G. Luo, L. Katz, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 117, 9105 (1995); ibid. 118, 9184 (1996); I. Böhm et al., Chem. Biol. 5, 407 (1998).
21. J. Cortes et al., Science 268, 1487 (1995); C. M. Kao, G. Luo, L. Katz, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 117, 9105 (1995); ibid. 118, 9184 (1996); I. Böhm et al., Chem. Biol. 5, 407 (1998).
22. J. Cortes et al., Science 268, 1487 (1995); C. M. Kao, G. Luo, L. Katz, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 117, 9105 (1995); ibid. 118, 9184 (1996); I. Böhm et al., Chem. Biol. 5, 407 (1998).
23. J. Cortes et al., Science 268, 1487 (1995); C. M. Kao, G. Luo, L. Katz, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 117, 9105 (1995); ibid. 118, 9184 (1996); I. Böhm et al., Chem. Biol. 5, 407 (1998).
24. A. M. Gehring, I. M. Mori, C. T. Walsh, Biochemistry 37, 2648 (1998).
25. R. G. Summers et al., Microbiology 143, 3251 (1997).
26. A. M. A. van Wageningen et al., Chem. Biol. 5, 155 (1998).
27. C. J. Tsoi and C. Khosla, ibid. 2, 355 (1995); J. Rohr, Angew. Chem. Int. Ed. Engl. 34, 881 (1995); P. F. Leadlay, Curr. Opin. Chem. Biol. 1, 162 (1997).
28. C. J. Tsoi and C. Khosla, ibid. 2, 355 (1995); J. Rohr, Angew. Chem. Int. Ed. Engl. 34, 881 (1995); P. F. Leadlay, Curr. Opin. Chem. Biol. 1, 162 (1997).
29. C. J. Tsoi and C. Khosla, ibid. 2, 355 (1995); J. Rohr, Angew. Chem. Int. Ed. Engl. 34, 881 (1995); P. F. Leadlay, Curr. Opin. Chem. Biol. 1, 162 (1997).
30. M. Oliynyk, M. J. B. Brown, J. Cortes, J. Staunton, P. F. Leadlay, Chem. Biol. 3, 833 (1996); L. Liu, A. Thamchaipenet, H. Fu, M. Betlach, G. Ashley, J. Am. Chem. Soc. 119, 10553 (1997); X. Ruan et al., J. Bacteriol. 170, 6416 (1997).
31. M. Oliynyk, M. J. B. Brown, J. Cortes, J. Staunton, P. F. Leadlay, Chem. Biol. 3, 833 (1996); L. Liu, A. Thamchaipenet, H. Fu, M. Betlach, G. Ashley, J. Am. Chem. Soc. 119, 10553 (1997); X. Ruan et al., J. Bacteriol. 170, 6416 (1997).
32. M. Oliynyk, M. J. B. Brown, J. Cortes, J. Staunton, P. F. Leadlay, Chem. Biol. 3, 833 (1996); L. Liu, A. Thamchaipenet, H. Fu, M. Betlach, G. Ashley, J. Am. Chem. Soc. 119, 10553 (1997); X. Ruan et al., J. Bacteriol. 170, 6416 (1997).
33. S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); S. Donadio, J. B. McAlpine, P. J. Sheldon, M. Jackson, L. Katz, Proc. Natl. Acad. Sci. U.S.A. 90, 7119 (1993); D. Bedford, J. R. Jacobsen, G. Luo, D. E. Cane, C. Khosla, Chem. Biol. 3, 827 (1996); R. McDaniel et al., J. Am. Chem. Soc. 119, 4309 (1997); C. M. Kao et al., ibid., p. 11339.
34. S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); S. Donadio, J. B. McAlpine, P. J. Sheldon, M. Jackson, L. Katz, Proc. Natl. Acad. Sci. U.S.A. 90, 7119 (1993); D. Bedford, J. R. Jacobsen, G. Luo, D. E. Cane, C. Khosla, Chem. Biol. 3, 827 (1996); R. McDaniel et al., J. Am. Chem. Soc. 119, 4309 (1997); C. M. Kao et al., ibid., p. 11339.
35. S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); S. Donadio, J. B. McAlpine, P. J. Sheldon, M. Jackson, L. Katz, Proc. Natl. Acad. Sci. U.S.A. 90, 7119 (1993); D. Bedford, J. R. Jacobsen, G. Luo, D. E. Cane, C. Khosla, Chem. Biol. 3, 827 (1996); R. McDaniel et al., J. Am. Chem. Soc. 119, 4309 (1997); C. M. Kao et al., ibid., p. 11339.
36. S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); S. Donadio, J. B. McAlpine, P. J. Sheldon, M. Jackson, L. Katz, Proc. Natl. Acad. Sci. U.S.A. 90, 7119 (1993); D. Bedford, J. R. Jacobsen, G. Luo, D. E. Cane, C. Khosla, Chem. Biol. 3, 827 (1996); R. McDaniel et al., J. Am. Chem. Soc. 119, 4309 (1997); C. M. Kao et al., ibid., p. 11339.
37. S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991); S. Donadio, J. B. McAlpine, P. J. Sheldon, M. Jackson, L. Katz, Proc. Natl. Acad. Sci. U.S.A. 90, 7119 (1993); D. Bedford, J. R. Jacobsen, G. Luo, D. E. Cane, C. Khosla, Chem. Biol. 3, 827 (1996); R. McDaniel et al., J. Am. Chem. Soc. 119, 4309 (1997); C. M. Kao et al., ibid., p. 11339.
38. C. M. Kao et al., J. Am. Chem. Soc. 120, 2478 (1998); K. J. Weissman et al., Biochemistry 36, 13849 (1997).
39. C. M. Kao et al., J. Am. Chem. Soc. 120, 2478 (1998); K. J. Weissman et al., Biochemistry 36, 13849 (1997).
40. In some cases, presumptive domains recognized on the basis of their characteristic consensus sequences may be functionally silent. For example, although various modules of the 14-module rapamycin synthase possess auxiliary domains with inferred ketoreductase, dehydratase, or enoylreductase properties, for reasons that are still obscure these latent activities are not always manifest in the structure of the eventually formed natural product [J. F. Aparicio et al., Gene 169, 9 (1996); T. Schwecke et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7839 (1995)].
41. In some cases, presumptive domains recognized on the basis of their characteristic consensus sequences may be functionally silent. For example, although various modules of the 14-module rapamycin synthase possess auxiliary domains with inferred ketoreductase, dehydratase, or enoylreductase properties, for reasons that are still obscure these latent activities are not always manifest in the structure of the eventually formed natural product [J. F. Aparicio et al., Gene 169, 9 (1996); T. Schwecke et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7839 (1995)].
42. J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, C. Khosla, Science 277, 367 (1997); J. R. Jacobsen, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 120, 9096 (1998); J. R. Jacobsen, A. Keatinge-Clay, D. E. Cane, C. Khosla, Bioorg. Med. Chem. 6, 117 (1998); A. F. A. Marsden, B. Wilkinson, J. Cortes, N. J. Dunster, J. Staunton, P. F. Leadlay, Science 279, 199 (1998).
43. J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, C. Khosla, Science 277, 367 (1997); J. R. Jacobsen, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 120, 9096 (1998); J. R. Jacobsen, A. Keatinge-Clay, D. E. Cane, C. Khosla, Bioorg. Med. Chem. 6, 117 (1998); A. F. A. Marsden, B. Wilkinson, J. Cortes, N. J. Dunster, J. Staunton, P. F. Leadlay, Science 279, 199 (1998).
44. J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, C. Khosla, Science 277, 367 (1997); J. R. Jacobsen, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 120, 9096 (1998); J. R. Jacobsen, A. Keatinge-Clay, D. E. Cane, C. Khosla, Bioorg. Med. Chem. 6, 117 (1998); A. F. A. Marsden, B. Wilkinson, J. Cortes, N. J. Dunster, J. Staunton, P. F. Leadlay, Science 279, 199 (1998).
45. J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, C. Khosla, Science 277, 367 (1997); J. R. Jacobsen, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 120, 9096 (1998); J. R. Jacobsen, A. Keatinge-Clay, D. E. Cane, C. Khosla, Bioorg. Med. Chem. 6, 117 (1998); A. F. A. Marsden, B. Wilkinson, J. Cortes, N. J. Dunster, J. Staunton, P. F. Leadlay, Science 279, 199 (1998).
46. Summarized in C. Khosla, R. Gokhale, J. R. Jacobsen, D. E. Cane, Annu. Rev. Biochem., in press.
47. In some cases transfer of the resultant structurally modified intermediate to downstream modules may limit the overall processivity of the recombinant multi-enzyme system. Although the exact magnitude and causes of these kinetic bottlenecks remain to be assessed, reductions in in vivo productivity of modified polyketides of up to three orders of magnitude compared with the titer of the natural product produced by the parent PKS have been observed. For example, replacement of the methylmalonyl-specific AT domain in module 2 of the erythromycin PKS by a suitable malonyl-CoA transferase from the rapamycin PKS results in biosynthesis of the expected normethyl-triketide by a truncated, two-module variant of DEBS in yields that approximate natural triketide production, implying that the activities of the native enzymes in the hybrid module are not significantly eroded by the introduction of a heterologous AT domain. When the same AT substitution is performed in the full, 6-module DEBS PKS, however, the expected normethyl-macrolide is produced in considerably reduced yields compared with those of the natural product 6-dEB (J. Lau, H. Fu, D. E. Cane, C. Khosla, unpublished results). The reasons for this reduction in yield are not clear. Developing strategies to overcome these deleterious effects on chain transfer represents a major challenge.
48. R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, C. Khosla, J. Am. Chem. Soc. 115, 11671 (1993); C. M. Kao, L. Katz, C. Khosla, Science 265, 509 (1994); D. J. Bedford, E. Schweizer, D. A. Hopwood, C. Khosla, J. Bacteriol. 177, 4544 (1995).
49. R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, C. Khosla, J. Am. Chem. Soc. 115, 11671 (1993); C. M. Kao, L. Katz, C. Khosla, Science 265, 509 (1994); D. J. Bedford, E. Schweizer, D. A. Hopwood, C. Khosla, J. Bacteriol. 177, 4544 (1995).
50. R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, C. Khosla, J. Am. Chem. Soc. 115, 11671 (1993); C. M. Kao, L. Katz, C. Khosla, Science 265, 509 (1994); D. J. Bedford, E. Schweizer, D. A. Hopwood, C. Khosla, J. Bacteriol. 177, 4544 (1995).
51. J. Kealey, L. Liu, D. V. Santi, M. Betlach, P. J. Barr, Proc. Natl. Acad. Sci. U.S.A. 95, 505 (1998); R. S. Gokhale and C. Khosla, unpublished results; A. Gehring, I. Mori, R. Perry, C. Walsh, Biochemistry 37, 11637 (1998).
52. J. Kealey, L. Liu, D. V. Santi, M. Betlach, P. J. Barr, Proc. Natl. Acad. Sci. U.S.A. 95, 505 (1998); R. S. Gokhale and C. Khosla, unpublished results; A. Gehring, I. Mori, R. Perry, C. Walsh, Biochemistry 37, 11637 (1998).
53. J. Kealey, L. Liu, D. V. Santi, M. Betlach, P. J. Barr, Proc. Natl. Acad. Sci. U.S.A. 95, 505 (1998); R. S. Gokhale and C. Khosla, unpublished results; A. Gehring, I. Mori, R. Perry, C. Walsh, Biochemistry 37, 11637 (1998).
54. For example, genetic engineering can be used to introduce novel functionality into or delete existing functional groups from a polyketide or non-ribosomal peptide backbone, thereby facilitating subsequent semisynthetic modification. In such cases genetics can be viewed as yet another tool in the medicinal chemist's armamentarium. Alternatively, chemical synthesis can precede engineered biosynthesis, whereby synthetic mimics of biosynthetic starting materials or chain elongation intermediates are fed to a recombinant strain carrying a lesion in an early step in the biosynthetic pathway. Processing of these synthetic analogs by the appropriate domains of the multi-enzyme synthase results in the formation of analogs of the natural product containing functional groups or other chemical properties that are ordinarily unavailable from primary metabolism. (21).
55. R. Pieper, G. Luo, D. E. Cane, C. Khosla, Nature 378, 263 (1995).
56. A. M. Gehring, K. A. Bradley, C. T. Walsh, Biochemistry, 36, 8495 (1997).
57. N. L. Pohl, R. S. Gokhale, D. E. Cane, C. Khosla, J. Am. Chem. Soc., in press.
58. Ketolides are a class of semisynthetic derivatives of erythromycin that possess excellent activity against a wide range of drug-resistant bacteria [C. Agouridas, Y. Benedetti, A. Denis, O. Le Martret, J. F. Chantot, 35th Interscience Conference On Antimicrobial Agents and Chemotherapy, San Francisco, 17 to 20 September 1995 (abstract F157); G. Griesgraber et al., J. Antibiot. 49, 465 (1996)]. Exploration of the pharmacological properties of ketolides has been limited by the difficulty of semisynthesis from naturally occurring macrolides. Combinatorial biosynthesis is ideally suited to the selective de novo construction of several of the required functional elements in the target ketolide molecule. Recent reports that FK506 can stimulate nerve regeneration [B. G. Gold, Mol. Neurobiol. 15, 285 (1997)] have stimulated interest in the identification of the relevant structural features responsible for the newly discovered activity of this polyketide. The epothilones are a class of polyketide natural products from Sorangium cellulosum, which are potent Taxol-like cytotoxic agents that are considerably more water-soluble than Taxol and active against Taxol-resistant tumors [K. C. Nicolaou et al., Angew. Chem. Int. Ed. Engl. 36, 2097 (1997); D.-S. Su et al., ibid., p. 2093.
59. Ketolides are a class of semisynthetic derivatives of erythromycin that possess excellent activity against a wide range of drug-resistant bacteria [C. Agouridas, Y. Benedetti, A. Denis, O. Le Martret, J. F. Chantot, 35th Interscience Conference On Antimicrobial Agents and Chemotherapy, San Francisco, 17 to 20 September 1995 (abstract F157); G. Griesgraber et al., J. Antibiot. 49, 465 (1996)]. Exploration of the pharmacological properties of ketolides has been limited by the difficulty of semisynthesis from naturally occurring macrolides. Combinatorial biosynthesis is ideally suited to the selective de novo construction of several of the required functional elements in the target ketolide molecule. Recent reports that FK506 can stimulate nerve regeneration [B. G. Gold, Mol. Neurobiol. 15, 285 (1997)] have stimulated interest in the identification of the relevant structural features responsible for the newly discovered activity of this polyketide. The epothilones are a class of polyketide natural products from Sorangium cellulosum, which are potent Taxol-like cytotoxic agents that are considerably more water-soluble than Taxol and active against Taxol-resistant tumors [K. C. Nicolaou et al., Angew. Chem. Int. Ed. Engl. 36, 2097 (1997); D.-S. Su et al., ibid., p. 2093.
60. Ketolides are a class of semisynthetic derivatives of erythromycin that possess excellent activity against a wide range of drug-resistant bacteria [C. Agouridas, Y. Benedetti, A. Denis, O. Le Martret, J. F. Chantot, 35th Interscience Conference On Antimicrobial Agents and Chemotherapy, San Francisco, 17 to 20 September 1995 (abstract F157); G. Griesgraber et al., J. Antibiot. 49, 465 (1996)]. Exploration of the pharmacological properties of ketolides has been limited by the difficulty of semisynthesis from naturally occurring macrolides. Combinatorial biosynthesis is ideally suited to the selective de novo construction of several of the required functional elements in the target ketolide molecule. Recent reports that FK506 can stimulate nerve regeneration [B. G. Gold, Mol. Neurobiol. 15, 285 (1997)] have stimulated interest in the identification of the relevant structural features responsible for the newly discovered activity of this polyketide. The epothilones are a class of polyketide natural products from Sorangium cellulosum, which are potent Taxol-like cytotoxic agents that are considerably more water-soluble than Taxol and active against Taxol-resistant tumors [K. C. Nicolaou et al., Angew. Chem. Int. Ed. Engl. 36, 2097 (1997); D.-S. Su et al., ibid., p. 2093.
61. Ketolides are a class of semisynthetic derivatives of erythromycin that possess excellent activity against a wide range of drug-resistant bacteria [C. Agouridas, Y. Benedetti, A. Denis, O. Le Martret, J. F. Chantot, 35th Interscience Conference On Antimicrobial Agents and Chemotherapy, San Francisco, 17 to 20 September 1995 (abstract F157); G. Griesgraber et al., J. Antibiot. 49, 465 (1996)]. Exploration of the pharmacological properties of ketolides has been limited by the difficulty of semisynthesis from naturally occurring macrolides. Combinatorial biosynthesis is ideally suited to the selective de novo construction of several of the required functional elements in the target ketolide molecule. Recent reports that FK506 can stimulate nerve regeneration [B. G. Gold, Mol. Neurobiol. 15, 285 (1997)] have stimulated interest in the identification of the relevant structural features responsible for the newly discovered activity of this polyketide. The epothilones are a class of polyketide natural products from Sorangium cellulosum, which are potent Taxol-like cytotoxic agents that are considerably more water-soluble than Taxol and active against Taxol-resistant tumors [K. C. Nicolaou et al., Angew. Chem. Int. Ed. Engl. 36, 2097 (1997); D.-S. Su et al., ibid., p. 2093.
62. Ketolides are a class of semisynthetic derivatives of erythromycin that possess excellent activity against a wide range of drug-resistant bacteria [C. Agouridas, Y. Benedetti, A. Denis, O. Le Martret, J. F. Chantot, 35th Interscience Conference On Antimicrobial Agents and Chemotherapy, San Francisco, 17 to 20 September 1995 (abstract F157); G. Griesgraber et al., J. Antibiot. 49, 465 (1996)]. Exploration of the pharmacological properties of ketolides has been limited by the difficulty of semisynthesis from naturally occurring macrolides. Combinatorial biosynthesis is ideally suited to the selective de novo construction of several of the required functional elements in the target ketolide molecule. Recent reports that FK506 can stimulate nerve regeneration [B. G. Gold, Mol. Neurobiol. 15, 285 (1997)] have stimulated interest in the identification of the relevant structural features responsible for the newly discovered activity of this polyketide. The epothilones are a class of polyketide natural products from Sorangium cellulosum, which are potent Taxol-like cytotoxic agents that are considerably more water-soluble than Taxol and active against Taxol-resistant tumors [K. C. Nicolaou et al., Angew. Chem. Int. Ed. Engl. 36, 2097 (1997); D.-S. Su et al., ibid., p. 2093.
63. For example, although the erythromycin PKS genes are expressed in the heterologous S. coelicolor host at levels that are considerably higher than are observed in the natural erythromycin producer, Sac. erythraea, actual production of the resultant polyketides is an order of magnitude higher in the native producing strain. This difference is most likely due to the limited supply of the requisite polyketide building blocks, propionyl-CoA and methylmalonyl-CoA, in S. coelicotor. Traditionally, problems of low metabolite production have been overcome by classical strain improvement based on routine but time-consuming empirical application of random chemical mutagenesis, coupled with iterative screening of producing microorganisms, eventually leading in some cases to improvement in metabolite titers of more than three to four orders of magnitude over those of the wild-type strains. The application of functional genomic technologies in synergism with emerging approaches for kinetic analysis of complex metabolic networks could provide powerful new tools for the rapid and efficient increases in metabolite production.
64. A. Crameri, S.-A. Raillard, E. Bermudez, W. P. C. Stemmer, Nature 391, 288 (1998); P. A. Patten, R. J. Howard, W. P. Stemmer, Curr. Opin. Biotechnol. 8, 724 (1997).
65. A. Crameri, S.-A. Raillard, E. Bermudez, W. P. C. Stemmer, Nature 391, 288 (1998); P. A. Patten, R. J. Howard, W. P. Stemmer, Curr. Opin. Biotechnol. 8, 724 (1997).
66. The structural determinants of chain extension unit specificity have been narrowed to a sequence of 20 to 30 amino acids (23), suggesting that it should be possible to modify the substrate specificity of AT domains. Likewise, the recent determination of the structure of the adenylation domain of the phenylalanine-activating module of the gramicidin NRPS opens the door to a structure-based approach toward the engineered biosynthetic incorporation of novel amino acids into non-ribosomal peptides (7). 34. Research on many of the topics covered in this review has been supported by grants from the NIH (GM22172 to D.E.C., CA66736 to C.K., CM20011 to C.T.W.) and by a David and Lucile Packard Foundation Grant (to C.K.). The authors are also members of the Scientific Advisory Board of Kosan Biosciences. We thank P. Belshaw for assistance in design of the figures.