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

ABSTRACT: 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.

SUBMITTER: Fischbach MA

PROVIDER: S-EPMC1924594 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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

Fischbach Michael A MA Lai Jonathan R JR Roche Eric D ED Walsh Christopher T CT Liu David R DR

Proceedings of the National Academy of Sciences of the United States of America 20070709 29

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 mod ...[more]

PMID: 17620609

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Project description:BACKGROUND:?-Mannanase randomly cleaves the ?-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of ?-mannanase have tremendously expanded in recent years, the wild-type ?-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 ?-mannanase (RmMan5A) from Rhizomucor miehei shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of RmMan5A to the biorefinery of mannan biomasses is severely limited. RESULTS:To overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated ?-mannanase (mRmMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (kcat/Km) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mRmMan5A. CONCLUSIONS:Through directed evolution of RmMan5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure-function relationship of GH family 5 ?-mannanase was acquired, which could be used for modifying ?-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a ?-mannanase from zygomycete engineered by directed evolution.

Dataset Information

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

Publications

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

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