title: Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design.
abstract: Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles.
authors.forename: Lemuel M J
authors.initials: LMJ
authors.lastname: Soh
authors.affiliation: Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California Davis , One Shields Avenue, Davis, California 95616, United States.
authors.forename: Wai Shun
authors.initials: WS
authors.lastname: Mak
authors.forename: Paul P
authors.initials: PP
authors.lastname: Lin
authors.forename: Luo
authors.initials: L
authors.lastname: Mi
authors.forename: Frederic Y-H
authors.initials: FY
authors.lastname: Chen
authors.affiliation: California NanoSystems Institute , 420 Westwood Plaza, Los Angeles, California 90095, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Damoiseaux
authors.affiliation: Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California Davis , One Shields Avenue, Davis, California 95616, United States.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Siegel
authors.affiliation: UCLA-DOE Institute of Genomics and Proteomics , 420 Westwood Plaza, Los Angeles, California 90095, United States.|Academia Sinica , 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan, R.O.C.
authors.forename: James C
authors.identifier: 0000-0002-4580-7276
authors.initials: JC
authors.lastname: Liao
mesh_terms: D000440:Butanols / Q000737:chemistry / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006207:Half-Life; D057166:High-Throughput Screening Assays; D007651:Keto Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology; D016296:Mutagenesis; D000072417:Protein Domains; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D044366:Transition Temperature
chemical_list: D000440:Butanols; D007651:Keto Acids; D011994:Recombinant Proteins; C040507:isobutyl alcohol; D002262:Carboxy-Lyases
keywords: directed evolution; high-throughput screening; isobutyl alcohol; keto acid decarboxylase; protein design; thermostability