Directed Evolution of Transketolase , a Carbon - Carbon Bond - Forming Enzyme

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 (1,2dihydroxyethyl) 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 m uch 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) transform ation of a plasmid library into E. coli XL10-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 m easuring donor substrate (pyruvate) depletion. The first four steps were optimised to ensure the highest possible concentration of holotransketolase in each screening reaction. The HPLC m ethod utilised a 50mm guard column as the separation matrix and was capable of processing one sample every 1.2 minutes.