Rapid identification of enzyme variants for reengineered alkaloid biosynthesis in periwinkle.
Monoterpene indole alkaloids from Catharanthus roseus (Madagascar periwinkle), such as the anticancer agents vinblastine and vincristine, have important pharmacological activities. Metabolic engineering of alkaloid biosynthesis can provide an efficient and environmentally friendly route to analogs of these synthetically challenging and pharmaceutically valuable natural products. However, the narrow substrate scope of strictosidine synthase, the enzyme at the entry point of the pathway, limits a pathway engineering approach. We demonstrate that with a different expression system and screening method it is possible to rapidly identify strictosidine synthase variants that accept tryptamine analogs not turned over by the wild-type enzyme. The variants are used in stereoselective synthesis of beta-carboline analogs and are assessed for biosynthetic competence within the terpene indole alkaloid pathway. These results present an opportunity to explore metabolic engineering of "unnatural" product production in the plant periwinkle.
Bernhardt
Peter
P
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
McCoy
Elizabeth
E
O'Connor
Sarah E
SE
D001483:Base Sequence; D030008:Catharanthus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D017931:DNA Primers; D026121:Indole Alkaloids / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016296:Mutagenesis
D017931:DNA Primers; D026121:Indole Alkaloids
Plant Cell. 2006 Apr;18(4):907-20
16531499
Protein Eng. 2003 Jun;16(6):451-7
12874379
Curr Pharm Des. 2001 Sep;7(13):1181-97
11472261
Bioorg Med Chem Lett. 2006 May 1;16(9):2475-8
16481164
Plant Cell Rep. 1997 Nov;17(1):50-54
30732419
Crit Rev Oncol Hematol. 2001 Nov;40(2):159-73
11682323
Tetrahedron. 1966 Jan;22(1):321-36
5926367
Phytochemistry. 1993 Feb;32(3):493-506
7763429
Biochem J. 1995 Mar 1;306 ( Pt 2):571-80
7887913
Curr Med Chem. 2004 Mar;11(5):607-28
15032608
Biotechniques. 1994 Oct;17(4):754-61
7530459
J Am Chem Soc. 2006 Nov 8;128(44):14276-7
17076499
Nat Prod Rep. 2006 Aug;23(4):532-47
16874388
Chem Biol. 2006 Nov;13(11):1137-41
17113995
Proc Natl Acad Sci U S A. 1984 Aug;81(15):4642-6
6087338
Biotechnol Prog. 2002 Nov-Dec;18(6):1183-6
12467449