Enhancement of catalytic activity of recombinant aldolases by directed evolution and translational coupling

Abstract

More and more, aldolases are increasingly recognized as useful catalysts that carry out the reversible addition of a ketone donor to an aldehyde acceptor in achieving high stereoselectivity. Especially, threonine aldolase (TA) and 4-hydroxy-2-oxovalerate aldolase (HOA) were considered as potentially valuable biocatalysts in that both of them accept non-phosphorylated substrates unlike most of other aldolases that need phosphorylated substrates of which preparation is not trivial in organic synthesis. In spite of this advantage as biocatalysts, however, both of them have not been widely used in organic synthesis due to their low catalytic activity or low solubility. In this study, directed evolution and translational coupling have been employed to improve the catalytic activity of TA and HOA. The novel LTA gene was cloned from Pseudomonas aeruginosa and overexpressed in a soluble form in E. coli. 50-60 mg of recombinant aldolase was purified to homogeneity from 1 L culture by one-step affinity column chromatography. The recombinant aldolase was characterized as a low-specificity LTA and showed the best catalytic activity at 30℃ in phosphate buffer of pH 7.0. To improve its catalytic activity, the directed evolution approach was applied to the recombinant LTA. On the basis of the toxicity of acetaldehyde, which is a substrate of LTA, two high-throughput screening systems were proposed. According to the positive selection scheme, the 2.1-fold enhanced catalytic activity was obtained. MhpE aldolase (E. coli HOA) has been known as a member of 3-HPP catabolic pathway enzymes. When MhpE was overexpressed in E. coli, its solubility was largely dependent on the expression of MhpF. In vivo binding assay showed that MhpE was directly associated with MhpF. In addition, the translations of mhpF and mhpE genes were fully coupled by reinitiation mechanism although the mhpE gene has a Shine-Dalgarno sequence, which appears to be very efficient. When the mhpF and mhpE genes wer...