Development of a growth-dependent selection system for identification of L-threonine aldolases

Threonine aldolases (TAs) are useful enzymes for the synthesis of β-hydroxy-α-amino acids due to their capability to catalyze asymmetric aldol reactions. Starting from two prochiral compounds, an aldehyde and glycine, two chiral stereocenters were formed in a single step via C-C bond formation. Owing to poor diastereoselectivity and low activity, the enzymatic synthesis of β-hydroxy-α-amino acids by TAs is still a challenge. For identification of new TAs, a growth-dependent selection system in Pseudomonas putida KT2440 has been developed. This bacterium is able to use aromatic compounds such as benzaldehyde, which is the cleavage product of the TA-mediated retro-aldol reaction of phenylserine, as sole carbon source via the β-ketoadipate pathway. With DL-threo-β-phenylserine as sole carbon source, this strain showed only slight growth in minimal medium. This growth deficiency can be restored by introducing and expressing genes encoding TAs. In order to develop a highly efficient selection system, the gene taPp of P. putida KT2440 encoding a TA was successfully deleted by replacement with an antibiotic resistance cassette. Different growth studies were carried out to prove the operability of the selection system. Genes encoding for L- and D-specific TAs (L-TA genes of Escherichia coli (ltaE) and Saccharomyces cerevisiae (gly1) and D-TA gene of Achromobacter xylosoxidans (dtaAX)) were introduced into the selection strain P. putida KT2440ΔtaPp, followed by cultivation on minimal medium supplemented with DL-threo-β-phenylserine. The results demonstrate that only the selection strains with plasmid-encoded L-TAs were able to grow on this racemic amino acid, whereas the corresponding strain harboring the gene coding for a D-specific TA showed no growth. In summary, it can be stated that a powerful screening tool was developed to identify easily by growth new L-specific threonine aldolases or other enzymes from genomic or metagenomic libraries liberating benzaldehyde.