Bioinformatics-based approaches to engineer the transmembrane Δ6 desaturase from Micromonas pusilla

Abstract

The increased awareness of the health benefits of ω3-long chain polyunsaturated fatty acids (ω3-LCPUFAs) has led to a drastic increase in the consumption of fish-oil supplements. This has resulted in environmental concerns and the identification of key membrane-bound desaturases involved in the biosynthesis of ω3-LCPUFAs in order to generate a sustainable source of ω3-LCPUFAs. The Micromonas pusilla Δ6 desaturase (MpΔ6des) is a membrane-bound desaturase that is specific for ω3-LCPUFA precursors and acyl-Coenzyme A substrates (acyl-CoAs). The incorporation of MpΔ6des into the ω3-LCPUFA biosynthesis pathway allows efficient ω3-LCPUFA production in transgenic plants. However, little is known of the molecular basis underlying its ω3-specificity, stability and acyl-CoAs specificity. MpΔ6des is relatively challenging in terms of protein engineering targets in that there is no molecular structure available, it cannot be expressed in easily manipulated prokaryotic systems such as Escherichia coli, and the activity cannot be rapidly screened via the conventional techniques. Thus, computational, structure-based, protein design and high-throughput directed evolution could not be used. To overcome the technical hurdles, we have applied bioinformatics-based techniques (consensus mutagenesis, ancestral protein reconstruction and sequence similarity networks) to engineer MpΔ6des and to better define the sequence-structure-function relationship of proteins within the desaturase superfamily. Consensus mutagenesis of MpΔ6des (Chapter 2) demonstrated that it is possible to modulate the ω3/ω6-specificity of MpΔ6des semi-independently. The geometry of the substrate-binding pocket of MpΔ6des was not only influenced by the residues located in the substrate-binding cavity, but also by distal residues, possibly through intramolecular interaction networks. An ancestral algal front-end Δ6 desaturase (ANC175) was inferred (Chapter 3), which resembles the properties of the progenitor of the algal Δ6 desaturases. The comparison between ANC175 and contemporary desaturases indicated that the divergence of the ω3/ω6-specificity of algal Δ6 desaturases is associated with the environmental differences seen in the habitats of the different algal species. Chapter 4 describes a bioinformatics analysis of the desaturase superfamily, showing that the four major desaturase subfamilies (the first desaturases, methyl-end desaturases, front-end desaturases and Δ4 sphingolipid desaturases) are structurally and functionally distinct. Conserved motif analysis of the front-end desaturases suggested that two cytosolic regions (a loop between AH1 and H2, and the cytosolic side of TM3) play crucial roles in determining the substrate head-group specificity of the front-end desaturase. Altogether, this thesis promotes a more detailed structural and functional understanding of the front-end desaturases, especially MpΔ6des. It validates the use of bioinformatics-based approaches such as consensus mutagenesis and ancestral protein reconstruction, showing that small libraries that are relatively “rich” in valuable mutations can be produced, even in the absence of detailed structural information or a high-throughput screen. We have successfully created novel variants of MpΔ6des with significantly enhanced ω3-specificity and with enhanced expression. These results also shed new light on the evolution of ω3/ω6-specificity in the front-end desaturase subfamily. Finally, the use of sequence similarity networks allowed us to propose a more detailed classification of the desaturase superfamily and identify specific sequence motifs that can be used to predict the substrate “head-group” specificity of these enzymes. Show more