Single active-site mutants are sufficient to enhance serine:pyruvate α-transaminase activity in an ω-transaminase.

We have analyzed the natural evolution of transaminase structure and sequence between an α-transaminase serine-pyruvate aminotransferase and an ω-transaminase from Chromobacterium violaceum with < 20% sequence identity, and identified the active-site regions that are least conserved structurally. We also show that these structural changes correlate strongly with transaminase substrate specificity during evolution and therefore might normally be presumed to be essential determinants of substrate specificity. However, key residues are often conserved spatially during evolution and yet originate from within a different region of the sequence via structural reorganizations. In the present study, we also show that α-transaminase-type serine-pyruvate aminotransferase activity can be engineered into the CV2025 ω-transaminase scaffold with any one of many possible single-point mutations at three key positions, without the requirement for significant backbone remodeling, or repositioning of the residue from a different region of sequence. This finding has significant implications for enzyme redesign in which solutions to substrate specificity changes may be found more efficiently than is achieved by engineering in all sequence and structure determinants identified by correlation to substrate specificity.

Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes.

Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 µmol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C10-C16 alkanes and represents the highest reported gain in product yields resulting from its use.