Dissecting and exploiting intermodular communication in polyketide synthases

Science

Volumn 284, Issue 5413, 1999, Pages 482-485

  Gokhale, Rajesh S ^a   Tsuji, Stuart Y ^a   Cane, David E ^c   Khosla, Chaitan ^a,b  

^a Stanford University   (United States)

^b STANFORD UNIVERSITY   (United States)

^c BROWN UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID; NATURAL PRODUCT; POLYKETIDE; SYNTHETASE;

ARTICLE; BIOENGINEERING; CATALYSIS; CATALYST; ENZYME ACTIVITY; ENZYME SPECIFICITY; KINETICS; PRIORITY JOURNAL;

AMINO ACID SEQUENCE; CATALYSIS; ESCHERICHIA COLI; EVOLUTION, MOLECULAR; GENES, BACTERIAL; LACTONES; MACROLIDES; MOLECULAR SEQUENCE DATA; MULTIENZYME COMPLEXES; NUCLEAR MAGNETIC RESONANCE, BIOMOLECULAR; PEPTIDES; PROTEIN ENGINEERING; RECOMBINANT FUSION PROTEINS; STREPTOMYCES;

EID: 0033574768     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5413.482     Document Type: Article

References (32)

1. D. A. Hopwood, Chem. Rev 97, 2465 (1997); L. Katz, ibid., p. 2557; J. Staunton and B. Wilkinson, ibid., p. 2611; C. Khosla, ibid., p. 2577.
2. D. A. Hopwood, Chem. Rev 97, 2465 (1997); L. Katz, ibid., p. 2557; J. Staunton and B. Wilkinson, ibid., p. 2611; C. Khosla, ibid., p. 2577.
3. D. A. Hopwood, Chem. Rev 97, 2465 (1997); L. Katz, ibid., p. 2557; J. Staunton and B. Wilkinson, ibid., p. 2611; C. Khosla, ibid., p. 2577.
4. D. A. Hopwood, Chem. Rev 97, 2465 (1997); L. Katz, ibid., p. 2557; J. Staunton and B. Wilkinson, ibid., p. 2611; C. Khosla, ibid., p. 2577.
5. D. E. Cane, C. T. Walsh, C. Khosla, Science 282, 63 (1998); R. McDaniel et al., Proc. Natl. Acad. Sci. U.S.A. 96, 1846 (1999).
6. D. E. Cane, C. T. Walsh, C. Khosla, Science 282, 63 (1998); R. McDaniel et al., Proc. Natl. Acad. Sci. U.S.A. 96, 1846 (1999).
7. J. Cortes et al., Nature 348, 176 (1990); S. Donadio, M. J. Staver, J. B. McAlpine, S. J. Swanson, L. Katz, Science 252, 675 (1991).
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12. R. H. Lambalot et al., Chem. Biol. 3, 923 (1996).
13. 6)-tagged fusion protein. Expression of M5+TE was achieved by combining the engineered Nde I site from pJRJ10 [J. R. Jacobsen et al., Biochemistry 37, 4928 (1998)] with Eco Eco RI site from pCK15 (4). The Nde I-Eco RI fragment was cloned in pET21c to obtain the expression plasmid pRSG46. These plasmids were introduced through transformation into E. coli BL21(DE3) cells for protein expression. Similar expression constructs were engineered for M2+TE and M6+TE using an engineered Nhe I site immediately upstream of the corresponding ketosynthases (at position 7570, 5'-GCTAGCGAGCCGATC-3'; at position 28710, 5'-GCTAGCGACCCGATC-3'). In order to coexpress the sfp gene, the Nde I-Hind III fragment derived from the pUC8-sfp [M. M. Nakano et al., Mol. Gen. Genet. 232, 313 (1992)] was cloned into pET, which has a kanamycin resistance gene. The resultant plasmid, pRSG56, was cotransformed into BL21(DE3) containing other single-module plasmids.
14. 6)-tagged fusion protein. Expression of M5+TE was achieved by combining the engineered Nde I site from pJRJ10 [J. R. Jacobsen et al., Biochemistry 37, 4928 (1998)] with Eco Eco RI site from pCK15 (4). The Nde I-Eco RI fragment was cloned in pET21c to obtain the expression plasmid pRSG46. These plasmids were introduced through transformation into E. coli BL21(DE3) cells for protein expression. Similar expression constructs were engineered for M2+TE and M6+TE using an engineered Nhe I site immediately upstream of the corresponding ketosynthases (at position 7570, 5'-GCTAGCGAGCCGATC-3'; at position 28710, 5'-GCTAGCGACCCGATC-3'). In order to coexpress the sfp gene, the Nde I-Hind III fragment derived from the pUC8-sfp [M. M. Nakano et al., Mol. Gen. Genet. 232, 313 (1992)] was cloned into pET, which has a kanamycin resistance gene. The resultant plasmid, pRSG56, was cotransformed into BL21(DE3) containing other single-module plasmids.
15. C. M. Kao, R. Pieper, D. E. Cane, C. Khosla, Biochemistry 35, 12363 (1996); N. L. Pohl, R. S. Gokhale, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 120, 11206 (1998).
16. C. M. Kao, R. Pieper, D. E. Cane, C. Khosla, Biochemistry 35, 12363 (1996); N. L. Pohl, R. S. Gokhale, D. E. Cane, C. Khosla, J. Am. Chem. Soc. 120, 11206 (1998).
17. R. S. Gokhale, S. Y. Tsuji, D. E. Cane, C. Khosla, data not shown.
18. S. Donadio and L. Katz, Gene 111, 51 (1992).
19. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
20. 6 peptide was lost during purification.
21. R. S. Gokhale, D. Hunziker, D. E. Cane, C Khosla, Chem. Biol. 6, 117 (1999).
22. Previous attempts to generate heterologous module fusions, such as M1-M6+TE, M1-M3-KTE, and M1-M2-M6+TE, have been unsuccessful [R. McDaniel et al., ibid. 4, 667 (1997); C. M. Kao, J. Lau, R. S. Gokhale, C. Khosla, unpublished results]. Although none of these recombinant PKSs, constructed by engineering restriction sites at arbitrarily chosen positions in the linkers between ACP and KS domains, yielded any polyketides in vivo, the fusion proteins were stable and could be purified as homodimers. In vitro assays carried out with radiolabeled propionyl-CoA and unlabeled methylmalonyl-CoA did not produce any radiolabeled triketide product. Instead, when diketide thioester 2 and radiolabeled methylmalonyl-CoA were used as substrates, these bimodular constructs produced radiolabeled triketide, probably by exogenously priming at the downstream module. It has been previously shown that 2 does not acylate at the KS domain of M1 [N. Tsukamoto et al., Biochemistry 35 15244 (1996)].
23. Previous attempts to generate heterologous module fusions, such as M1-M6+TE, M1-M3-KTE, and M1-M2-M6+TE, have been unsuccessful [R. McDaniel et al., ibid. 4, 667 (1997); C. M. Kao, J. Lau, R. S. Gokhale, C. Khosla, unpublished results]. Although none of these recombinant PKSs, constructed by engineering restriction sites at arbitrarily chosen positions in the linkers between ACP and KS domains, yielded any polyketides in vivo, the fusion proteins were stable and could be purified as homodimers. In vitro assays carried out with radiolabeled propionyl-CoA and unlabeled methylmalonyl-CoA did not produce any radiolabeled triketide product. Instead, when diketide thioester 2 and radiolabeled methylmalonyl-CoA were used as substrates, these bimodular constructs produced radiolabeled triketide, probably by exogenously priming at the downstream module. It has been previously shown that 2 does not acylate at the KS domain of M1 [N. Tsukamoto et al., Biochemistry 35 15244 (1996)].
24. Previous attempts to generate heterologous module fusions, such as M1-M6+TE, M1-M3-KTE, and M1-M2-M6+TE, have been unsuccessful [R. McDaniel et al., ibid. 4, 667 (1997); C. M. Kao, J. Lau, R. S. Gokhale, C. Khosla, unpublished results]. Although none of these recombinant PKSs, constructed by engineering restriction sites at arbitrarily chosen positions in the linkers between ACP and KS domains, yielded any polyketides in vivo, the fusion proteins were stable and could be purified as homodimers. In vitro assays carried out with radiolabeled propionyl-CoA and unlabeled methylmalonyl-CoA did not produce any radiolabeled triketide product. Instead, when diketide thioester 2 and radiolabeled methylmalonyl-CoA were used as substrates, these bimodular constructs produced radiolabeled triketide, probably by exogenously priming at the downstream module. It has been previously shown that 2 does not acylate at the KS domain of M1 [N. Tsukamoto et al., Biochemistry 35 15244 (1996)].
25. Bsa Bl-Eco RI fragments containing ery M3 and ery M6 were doned behind ery M1. The M1-M3+TE and M1-M64-TE genes were excised as Pac I-Eco RI fragments and inserted into pCK12 (4). The resultant plasmids pST97 and pST96, respectively, were transformed in S. coelicolor strain CH999.
26. The reason for the inability of KR6 to reduce the triketide keto ester is unclear, because incubation of M5-M6+TE with diketide 2 and NADPH can synthesize 4.
27. P. R. August et al., Chem. Biol. 4, 69 (1998); T. Schupp, C. Toupet, N. Engel, S. Goff, FEMS Microbiol. Lett. 159, 201 (1998).
28. P. R. August et al., Chem. Biol. 4, 69 (1998); T. Schupp, C. Toupet, N. Engel, S. Goff, FEMS Microbiol. Lett. 159, 201 (1998).
29. The construction of ery M1-rif M5+TE was done as follows: Based on an alignment with ery ACP sequences, the natural sequence at 28024 5'-CGCGAC-3' in rif ACP5 was replaced with the Spe I recognition sequence of 5'-ACTACT-3'. Standard polymerase chain reaction techniques were used to carry out mutagenesis. The Bsa Bl-Spe I fragment containing rif M5 was excised and used to replace the corresponding ery M2 fragment in pCK12. The resultant expression plasmid, pST110, was transformed into S. coelicolor strain CH999.
30. To graft the interpolypeptide linker from the COOH terminus of ery M2 onto the rif M5, the Pac I-Spe I fragment of pST110 was inserted into a derivative of pCK7 [C. M. Kao, et al. Science 265, 509 (1994)]. The derivative had a Spe I site engineered in the homologous region at the end of ACP2. The resulting pST113 construct contained ery M1 linked to rif M5 (through the natural cis linker between ery M1 and M2), rif M5 noncovalently linked to ery M3 (through the natural interpolypeptide linker between ery M2 and M3), and the subsequent natural links to ery M4, M5, and M6.
31. P. J. Belshaw, C. T. Walsh, T. Stachelhaus, Science 284, 486 (1999).
32. Supported by grants from NIH (CA66736 to C.K. and GM22172 to D.E.C.) and NSF (BES9806774 to C.K.). S.Y.T. is a recipient of a predoctoral fellowship from NSF and a NIH Biotechnology Graduate Training Fellowship. We thank N. Wu for his assistance with NMR.