Biocatalytic synthesis of amino-alcohols using a de novo designed transketolase-/beta-alanine: Pyruvate transaminase pathway in Escherichia coli.

Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of a biocatalytic stage can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de novo to facilitate a required synthetic sequence. This will allow higher space-time yields and reduce the number of intermediate process steps required. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyse asymmetric carbon-carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino alcohols from achiral substrates. These compounds are synthetically very useful in the production of a range of compounds with pharmaceutical application. The p-alanine: pyruvate aminotransferase (p-A: P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR426, for over-expression in E.coli strain BL21gold(DE3). The aromatic transaminase (ArTAm) from E.coli was also cloned, to create plasmid pQR416. The substrate specificity of the transaminases has been explored in order to assess the feasibility of functioning downstream of TK and the potential to access a wider range of TK-TAm pathway products. Both directed evolution and site directed mutagenesis have also been applied in order to facilitate acceptance of transketolase products. Over-expression of the p-A: P TAm alongside the native transketolase (from plasmid pQR411), in a single E.coli host, has created a novel bacterial biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of 2-amino-l,3,4-butanetriol (ABT) product, in up to 21 % yield, by the p-alanine: pyruvate transaminase, via transamination of L- erythrulose synthesised by transketolase, from glycolaldehyde (GA) and p- hydroxypyruvate (p-HPA) substrates. The degradation of the amino alcohol product and the low activity of the transaminase, initial rate 0.1 mM.hr1, relative to TK, initial rate 2.1 mM.min"1, are found to be limiting the process requiring further investigation