Miller, O.J.; (2005) Directed evolution of transketolase, a carbon-carbon forming enzyme. Doctoral thesis , University of London.

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Abstract

The enzyme transketolase (Enzyme Commission number: 2.2.1.1) has significant potential as a biocatalyst in the production of pharmaceuticals and fine chemicals. The enzyme catalyses the irreversible transfer of a C2 (1,2-dihydroxyethyl) moiety from p-hydroxypyruvate (P-HPA) to a wide range of acceptor substrates in a stereospecific reaction. However, commercial application of transketolase is currently restricted by the limited availability and expense of p-HPA. This project describes efforts to generate and identify variants of E. coli transketolase that are capable of accepting pyruvate: a related, but much cheaper compound. The variants were prepared by a novel directed evolution technique, "focused" error-prone PCR (fepPCR), and then screened for the desired activity: pyruvate (donor) and glycolaldehyde (acceptor) to (S)-3,4-dihydroxybutan-2-one (and carbon dioxide). The high-throughput screen consisted of the following steps: (1) transformation of a plasmid library into E. coli XLIO-Gold competent cells (2) culture of individual colonies in 384-well plates (3) lysis of the cultures (4) incubation of the lysates with cofactors and the target substrates and finally (5) high-throughput HPLC analysis measuring donor substrate (pyruvate) depletion. The first four steps were optimised to ensure the highest possible concentration of holotransketolase in each screening reaction. The HPLC method utilised a 50mm guard column as the separation matrix and was capable of processing one sample every 1.2 minutes. Several libraries of transketolase variants were generated using a novel mutagenesis technique: "focused" error-prone PCR (fepPCR). FepPCR uses knowledge of an enzyme as a map for targeting mutation to the most beneficial regions of the gene - in this case, three stretches of residues in the active site of E. coli transketolase (Ser24-His26, Gly99-Prol01, and Asp469- His473). Primers were designed to flank these small target sites (9-15bp) and they were PCR-amplified from the tkt gene under conditions that drastically lowered the fidelity of Taq DNA polymerase (0.14 misincorporations per nucleotide). The three mutated fepPCR products (50-54bp) were then cloned into the tkt vector pQR711 singly and in combination to create four distinct libraries. The PCR-based cloning techniques QuikChange Site-Directed Mutagenesis (Stratagene Ltd.) and QuikChange Multi Site-Directed Mutagenesis (Stratagene Ltd.) were both found to be effective for this step. The four libraries were screened for the target activity and the following levels of coverage were achieved: 100% of all possible single point mutations in the three targets sites and 22% of all possible combinations of double point mutations in the target sites. Careful analysis of the HPLC chromatograms failed to identify any variants with the desired activity. It is proposed that larger libraries may yield positive results.

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