Improving catalytic function by ProSAR-driven enzyme evolution

Nat Biotechnol. 2007 Mar;25(3):338-44. doi: 10.1038/nbt1286. Epub 2007 Feb 18.

Abstract

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.

MeSH terms

  • Algorithms
  • Anticholesteremic Agents / chemical synthesis
  • Atorvastatin
  • Bacteria / enzymology
  • Catalysis
  • Directed Molecular Evolution / methods*
  • Heptanoic Acids / chemical synthesis
  • Hydrolases / genetics
  • Hydrolases / isolation & purification
  • Hydrolases / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Proteins / isolation & purification*
  • Proteins / metabolism
  • Pyrroles / chemical synthesis
  • Quantitative Structure-Activity Relationship*

Substances

  • Anticholesteremic Agents
  • Heptanoic Acids
  • Proteins
  • Pyrroles
  • Atorvastatin
  • Hydrolases
  • halohydrin dehalogenase

Associated data

  • GENBANK/AF397396