Exploring Insect Enzymes as Catalyst for Bioconversion of Value Added Products

Abstract

Demand for fine chemicals and value-added products has increased annually concomitantly with the rapid expansion of biocatalytic production of these chemicals. With an increased mandate for chemicals in the pharmaceutical, agricultural, scents and flavorants industry, it is essential to identify enzymes capable of performing specific, complex chemical reactions in an efficient, economical and environmentally friendly manner. Insects produce secondary metabolites that are potential value-added chemicals, therefore insect enzymes provide a rich source of catalysts that can be added to the collection of biocatalysts used to perform these intricate tasks. This thesis explores the biochemical role of three insect enzymes, CYP9T3, MPB-CPR, and CG4020, which are involved in lipid metabolism and could potentially be used as biocatalysts for value-added products. All three enzymes were expressed with a baculoviral system in Sf9 cells and used in functional assays. Eastern Ips pini CYP9T3 is a cytochrome P450 with 94% sequence identity to western Ips pini CYP9T2. CYP9T3 accepts myrcene, (+)- and (-)-α-pinene, (+)- and (-)- limonene, and (+)-3-carene as substrates, a pattern similar to CYP9T2. However, the enantiomeric ratio of (4R)-(-)-ipsdienol to (4S)-(+)-ipsdienol is significantly different between CYP9T3 and CYP9T2. Additionally, the product from (-)-α-pinene is transverbenol with CYP9T3 and myrtenol with CYP9T2. Assays with β-pinene, terpinolene, γ-terpinene, α-phellandrene did not yield detectable products by either CYP9T3 or CYP9T2. Cytochromes P450 require the transfer of electrons from a protein partner, commonly cytochrome P450 reductases. Mountain pine beetle cytochrome P450 ii reductase (MPB-CPR) is the first bark beetle cytochrome P450 reductase to be isolated and characterized. MPB-CPR is 69% identical to house fly-CPR (HF-CPR), the reductase used in previous bark beetle P450 functional assays. Recombinant MPB-CPR microsomes reduce cytochrome c with apparent Km and vmax of 85.04 µM and 8.42 nmol·min-1·µg total protein-1, respectively. Initial kinetic assays with CYP9T3 indicate that MPB-CPR reduces the P450 in the presence of myrcene with apparent Km and vmax values of 15.8 uM and 11.3 nmol·min-1·U enzyme-1, respectively. The final step in hydrocarbon biosynthesis in insects is the removal of one carbon from a fatty aldehyde through the action of an oxidative decarbonylase, CYP4G2. Experimental evidence from M. domestica microsomal preparations indicates that a fatty acyl-CoA is reduced to a fatty aldehyde prior to the oxidative decarbonylation reaction and the candidate enzyme is a fatty acyl-CoA reductase (FAR). CG4020, a Drosophila melanogaster FAR, has an expression pattern similar to CYP4G1, the D. melanogaster homologue of CYP4G2. Additionally, suppression of CG4020 produces flies with fifty percent less total hydrocarbon than wild type flies. Preliminary assays with expressed CG4020 did not confirm the biochemical function of this enzyme, however they did provide relevant background information from which future experiments can be designed.