title: Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo.
abstract: Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress in this area. Here, we use directed evolution and a multiplexed assay to screen a library of >14,000 members to generate three derivatives of the antibacterial compound, andrimid. Another limiting factor in reengineering these mega-enzymes of secondary metabolism has been that commonly used hosts such as Escherichia coli often give lower product titers, so our reengineering was performed in the native producer, Pantoea agglomerans. This integrated in vivo approach can be extended to larger enzymes to create analogs of natural products for bioactivity testing.
authors: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Bradley S
authors: BS
authors: Evans
authors: Yunqiu
authors: Y
authors: Chen
authors: William W
authors: WW
authors: Metcalf
authors: Huimin
authors: H
authors: Zhao
authors: Neil L
authors: NL
authors: Kelleher
references: J Antimicrob Chemother. 1997 Nov;40(5):611-4
references: 9421307
references: J Am Chem Soc. 2006 Aug 23;128(33):10660-1
references: 16910643
references: Chem Biol. 2000 Jun;7(6):373-84
references: 10873839
references: Chem Biol. 1995 Jun;2(6):355-62
references: 9383437
references: Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17462-7
references: 17090667
references: FEMS Microbiol Lett. 1995 Jan 1;125(1):3-14
references: 7867917
references: Science. 2009 Jul 10;325(5937):161-5
references: 19589993
references: Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):11951-6
references: 17620609
references: Nature. 1998 Jan 15;391(6664):288-91
references: 9440693
references: Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
references: 9396791
references: Trends Biotechnol. 1996 Sep;14(9):335-41
references: 8818287
references: J Bacteriol. 1989 May;171(5):2614-8
references: 2651409
references: Protein Eng. 2003 Jun;16(6):451-7
references: 12874379
references: Chembiochem. 2002 Mar 1;3(2-3):125-34
references: 11921390
references: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5848-53
references: 10811885
references: Mol Gen Genet. 1998 Feb;257(3):308-18
references: 9520265
references: Antimicrob Agents Chemother. 2006 Aug;50(8):2707-12
references: 16870762
references: Chembiochem. 2002 Jun 3;3(6):490-504
references: 12325005
references: J Biol Chem. 2004 Jun 18;279(25):26066-73
references: 15066985
references: J Mol Evol. 1999 Dec;49(6):716-20
references: 10594172
references: Nucleic Acids Res. 2005 Oct 12;33(18):5799-808
references: 16221976
references: Chem Biol. 1999 Aug;6(8):493-505
references: 10421756
references: Chem Biol. 2000 Aug;7(8):623-42
references: 11048953
references: J Ind Microbiol Biotechnol. 2006 Feb;33(2):66-74
references: 16193281
references: Methods Mol Biol. 1997;67:141-9
references: 9031138
references: Chem Biol. 2000 Mar;7(3):211-24
references: 10712928
references: Science. 1999 Apr 16;284(5413):486-9
references: 10205056
references: Microbiology (Reading). 2002 Apr;148(Pt 4):1105-1118
references: 11932455
references: J Ind Microbiol Biotechnol. 2003 Aug;30(8):448-55
references: 12707798
references: J Antibiot (Tokyo). 1997 Mar;50(3):270-3
references: 9439702
references: J Biol Chem. 1995 Mar 17;270(11):6163-9
references: 7534306
references: Biotechniques. 1990 Apr;8(4):404-7
references: 2340178
references: Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5691-6
references: 15811944
references: Annu Rev Biochem. 1999;68:219-53
references: 10872449