title: Emergence of terpene cyclization in Artemisia annua.
abstract: The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-β-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of ~27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism.
authors: John Innes Centre, Department of Metabolic Biology, Norwich Research Park, Norwich NR4 7UH, UK.
authors: Melissa
authors: M
authors: Salmon
authors: John Innes Centre, Department of Metabolic Biology, Norwich Research Park, Norwich NR4 7UH, UK.
authors: Caroline
authors: C
authors: Laurendon
authors: John Innes Centre, Department of Metabolic Biology, Norwich Research Park, Norwich NR4 7UH, UK.
authors: Maria
authors: M
authors: Vardakou
authors: John Innes Centre, Computational and Systems Biology, Norwich Research Park, Norwich NR4 7UH, UK.
authors: Jitender
authors: J
authors: Cheema
authors: Institute of Food Research, Analytical Sciences Unit, Norwich Research Park, Norwich NR4 7UA, UK.
authors: Marianne
authors: M
authors: Defernez
authors: Plant and Food Research, 120 Mt Albert Road, Sandringham, Auckland 1025, New Zealand.
authors: Sol
authors: S
authors: Green
authors: School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK.
authors: Juan A
authors: JA
authors: Faraldos
authors: 1] John Innes Centre, Department of Metabolic Biology, Norwich Research Park, Norwich NR4 7UH, UK [2] Institute of Food Research, Food &Health Programme, Norwich Research Park, Norwich NR4 7UA, UK.
authors: Paul E
authors: PE
authors: O'Maille
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D031172:Artemisia annua / Q000201:enzymology / Q000378:metabolism*; D003500:Cyclization; D011755:Pyrophosphatases / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism*
chemical_list: D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase; D011755:Pyrophosphatases; C109459:beta-farnesene synthase
references: PLoS One. 2011;6(8):e23608
references: 21912599
references: Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12305-10
references: 10535917
references: Nature. 2009 Sep 24;461(7263):515-9
references: 19779450
references: Nat Chem Biol. 2008 Apr;4(4):217-22
references: 18347585
references: Methods Enzymol. 2012;515:21-42
references: 22999168
references: Proc Natl Acad Sci U S A. 2010 Jan 26;107 Suppl 1:1747-51
references: 19805125
references: Science. 2012 Jun 29;336(6089):1667-70
references: 22745420
references: Proc Biol Sci. 2012 Jan 22;279(1727):247-56
references: 21697174
references: Fitoterapia. 2002 Oct;73(6):532-5
references: 12385883
references: MethodsX. 2014 Sep 08;1:187-96
references: 26150952
references: Science. 1972 Sep 22;177(4054):1121-2
references: 17840606
references: J Mol Biol. 2002 Aug 23;321(4):677-91
references: 12206782
references: PLoS Genet. 2013;9(7):e1003665
references: 23935519
references: Protein Eng. 2001 Sep;14(9):633-8
references: 11707608
references: ACS Chem Biol. 2010 Apr 16;5(4):377-92
references: 20175559
references: Nat Rev Genet. 2010 Aug;11(8):572-82
references: 20634811
references: Curr Opin Plant Biol. 2002 Jun;5(3):237-43
references: 11960742
references: BMC Biotechnol. 2008;8:91
references: 19055817
references: J Comput Chem. 2004 Oct;25(13):1605-12
references: 15264254
references: Anal Chem. 2006 Jul 1;78(13):4334-41
references: 16808440
references: Antimicrob Agents Chemother. 1997 Jul;41(7):1413-22
references: 9210658
references: Nat Protoc. 2010 Apr;5(4):725-38
references: 20360767
references: Comb Chem High Throughput Screen. 2010 Jan;13(1):27-38
references: 20201823
references: Bioinformatics. 2012 Dec 15;28(24):3332-3
references: 23097421
references: Science. 1997 Sep 19;277(5333):1820-4
references: 9295272
references: J Comput Chem. 2010 Jan 30;31(2):455-61
references: 19499576
references: Nature. 2011 May 12;473(7346):167-73
references: 21562557
references: Nat Chem Biol. 2008 Oct;4(10):617-23
references: 18776889
references: Nature. 2005 Jun 9;435(7043):824-7
references: 15944706
references: Nat Chem Biol. 2008 Aug;4(8):474-82
references: 18641628
references: Science. 1999 Oct 8;286(5438):295-9
references: 10514373
references: Biochemistry. 2003 Jul 1;42(25):7741-7
references: 12820883
references: Cell. 2009 Aug 21;138(4):774-86
references: 19703402
references: Anal Biochem. 2004 Dec 15;335(2):210-7
references: 15556559
references: Nature. 2006 Apr 20;440(7087):1078-82
references: 16495946
references: Science. 1997 Sep 19;277(5333):1815-20
references: 9295271
references: J Am Chem Soc. 2012 Dec 26;134(51):20844-8
references: 23214943
references: Biochemistry. 2010 Mar 2;49(8):1787-97
references: 20131801
references: Phytochemistry. 2009 Oct-Nov;70(15-16):1621-37
references: 19793600
references: BMC Bioinformatics. 2008;9:40
references: 18215316
references: Phytochemistry. 2008 May;69(8):1732-8
references: 18417176
references: Science. 2006 Apr 7;312(5770):111-4
references: 16601193