Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes.

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Research Abstract Details 

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Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Abstract Text:

Michael A Fischbach,Jonathan R Lai,Eric D Roche,Christopher T Walsh,David R Liu,

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with approximately 10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (10(3) to 10(4) clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts.

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Publishing Authors By Initials

MA Fischbach,JR Lai,ED Roche,CT Walsh,DR Liu,

For similar proteins: recombinant proteins research abstracts see: proteins: recombinant proteins research

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MEDLINE DATE:

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Proceedings of the National Academy of Sciences of

VOLUME: 104

Page Numbers: 11951-6

Journal Abbreviation: Proc. Natl. Acad. Sci. U.S.A.

ISSN: 0027-8424

DAY: 9

MONTH: 07

YEAR: 2007

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 7505876

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Keywords Mesh Terms:

KEYWORDS: Recombinant Proteins

MESH TERMS: metabolism

Chemical & Substance for Abstract: Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Information

Substance Name: non-ribosomal peptide synthase

Registry Number: EC 6.3.2.-

Grant and Affiliation Information for Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes.

AFFILIATION: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM20011

ACRONYM: GM

MEDLINETA: Proc Natl Acad Sci U S A

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