Engineering the Stereospecificity and Regioselectivity of Flavoenzymes Público
Teadt, Leann (Spring 2018)
Abstract
Enzymes are biocatalysts which accelerate biological reactions while typically maintaining high regio-, stereo-, and chemo- selectivity. However, most enzymes have evolved over thousands of years for specificity towards their native substrates. As a result, their usefulness as tools for biocatalysis is limited beyond these native substrates. Therefore, researchers have turned to protein engineering to adapt enzymes to work more efficiently towards desired compounds. This dissertation focuses specifically on engineering flavoenzymes, which are enzymes that contain a flavin cofactor, to alter their stereospecificity or regioselectivity. The specific flavoenzymes this work will focus on are Old Yellow Enzyme 1 (OYE1) from Saccharomyces pastorianus and cyclododecanone monooxygenase (CDMO) from Rhodococcus sp. HI-31.
Initially, the RApid Parallel Protein EvaluatoR (RAPPER) system was developed to rapidly and semi-quantitatively assess OYE1 variants. Preliminary work with RAPPER also reports OYE1 variants with enhanced activity as a result of increased flexibility in the β6 loop. These studies were followed up by the expansion of the RAPPER system to assess a variety of OYE1 variants. These variant libraries included single amino acid substitutions, deletions, and alanine scanning studies. The importance of substrate profiling was demonstrated as each variant was tested with multiple substrates to more accurately assess the impact of each substitution. As a result of this substrate profiling, several promising variants proved to be highly substrate dependent with respect to their catalytic activity.
Finally, efforts were made to engineer the regioselectivity of CDMO towards an N-protected β-amino ketone. In the course of these engineering efforts, two main regions of interest were identified as important towards determining product regioselectivity. Through a series of amino acid changes, both the chemically preferred (‘normal’) and the ‘abnormal’ products can be obtained with high selectivity. Taken together, these collective engineering efforts illustrate the capacity to specifically tailor biocatalysts for the production of industrially valuable products.
Table of Contents
Table of Contents
Chapter 1: General Introduction...1
General Introduction...2
Enzyme Engineering...2
Rational Design...4
Directed evolution...4
Library Selection and Screening...7
Semi-rational design...8
Cell-free translation systems...12
Flavoenzymes...14
Old Yellow Enzyme 1...18
Baeyer-Villiger Monooxygenases...22
Aim and scope of the dissertation...29
Chapter 2: RApid Parallel Protein EvaluatoR (RAPPER), from gene to enzyme function in one day...55
Abstract...56
Results and Discussion...56
Materials and Methods...62
Materials:...62
Preparation of linear DNA templates:...62
Mutagenesis by primer overlap extension PCR:...63
Cloning of oye1 variants:...64
PURExpress In Vitro Transcription-Translation:...64
Chapter 3: Cell-free protein engineering of Old Yellow Enzyme 1 from Saccharomyces pastorianus...67
Abstract...68
Introduction...68
Results and Discussion...70
Conclusion...76
Experimental...77
General information...78
Creating linear templates of OYE variants...78
In vitro transcription/translation of linear DNA...79
Large-scale protein expression and purification...79
Enzymatic activity assay for OYE1 variants...80
Supplemental information...82
Oligonucleotide sequences for construction of OYE1 variants...82
Chapter 4: Expanding the regioselectivity of Cyclododecanone Monooxygenase...97
Introduction...98
Results and Discussion...104
Initial characterization...104
Creation of a homology model...105
Selection of residues...106
Substitutions at positions 299 and 325...107
Substitutions at positions 190 and 191...108
Substitutions in positions 497-501...113
Combination of regional variants...114
Conclusion...115
Experimental...116
General Information...117
Chemical Baeyer-Villiger Oxidation of 1 and 4...117
Creation of the Mutant Library...118
Protein Expression and Purification...119
Enzymatic Activity Assay...119
Hydrolysis of Products...120
List of Primers Used...121
Chapter 5: Conclusions and Future Work...130
General Conclusions...131
The continuing use of in vitro transcription/translation systems...131
Engineering OYE1 using multiple substrates...132
Future studies on cyclododecanone monooxygenase...134
About this Dissertation
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