Improving catalytic function by ProSAR-driven enzyme evolution

Richard J Fox, S Christopher Davis, Emily C Mundorff, Lisa M Newman, Vesna Gavrilovic, Steven K Ma, Loleta M Chung, Charlene Ching, Sarena Tam, Sheela Muley, John Grate, John Gruber (+3 others)
2007 Nature Biotechnology  
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
more » ... 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 B4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterollowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations. Although interest in the use of enzymes as biocatalysts for chemical applications is increasing 1,2 , the performance of natural enzymes is rarely adequate for commercially viable processes. Many enzyme properties, such as specific activity, stability, chemo-and enantioselectivity, susceptibility to substrate and/or product inhibition and sensitivity to rapidly changing conditions, are difficult to optimize using rational design. Despite advances in protein engineering, there is a continuing need for more efficient and effective methods to improve multiple biochemical characteristics of an enzyme simultaneously 3,4 . Directed evolution is a powerful tool for protein optimization. The most efficient methods combine multiple rounds of diversity generation and gene recombination with functional screening to identify improved variants. The iterative nature of this approach results in stepwise improvements in overall function, yielding substantial improvements in desired enzyme properties 5-9 . Directed evolution can be performed in a variety of ways 10 , which are distinguished, for example, by the approach to generating libraries from available diversity. The field of in vitro recombination-based directed evolution 9 has focused on alternative methods for generating gene libraries rather than fundamentally altering the efficiency of the evolutionary process 7,10 , and despite advances, there remains a need to make the process of increasing enzyme function more efficient 3,4 . Our approach involves focusing the selective pressure on the mutations themselves rather than on the mutated gene. Quantitative structure-activity relationships (QSAR) have been used extensively in small-molecule 11 and peptide optimization 12 and have been proposed to be useful in protein engineering 13,14 . The statistical modeling efforts are motivated by the desire to establish causal relationships between the structures of interacting molecules (e.g., small-molecule descriptors and amino acid sequences) and measurable properties of scientific or commercial interest (e.g., binding affinity and catalytic activity). Such models can then be interrogated to make decisions about how to modify a molecule's structure to achieve improvements in desired properties. By formalizing the decision making processes about which mutations to include in combinatorial libraries, the ProSAR algorithm is an extension of traditional SAR-based approaches to molecular optimization. This study applies the concepts of QSAR to the problem of enzyme engineering. A multivariate protein optimization strategy based on protein sequence activity relationships (ProSAR) 15,16 was used to develop a halohydrin dehalogenase (HHDH) with the potential for use in the manufacture of ethyl (R)-4-cyano-3-hydroxybutyrate (HN) (Fig. 1) , the regulated starting material for the production of the cholesterol-lowering drug atorvastatin (Lipitor). The specifications for the chemical and enantiopurity of HN are tightly controlled, since the hydroxyl present in HN is used to define the second stereocenter in atorvastatin and high chemical purity is essential for successful downstream chemistry. We projected that an economically viable process could be achieved if the following design criteria were met: complete conversion (100%) of at least 100 g per liter substrate, a volumetric productivity of 420 g product per liter per hour per gram of biocatalyst, a simple HN isolation procedure to recover high quality product in high yield, and a simple enzyme formulation process that obviates the need for extensive enzyme purification. We first expressed an Agrobacterium radiobacter HHDH in Escherichia coli and showed that it catalyzed the conversion of ethyl (S)-4-chloro-3-hydroxybutyrate (ECHB) to HN at neutral pH ( Fig. 1) , thereby avoiding the side reactions associated with chemical
doi:10.1038/nbt1286 pmid:17322872 fatcat:kdevragibzegvbor3vtill7ezy