Stereoselective Production of Dimethyl-Substituted Carbapenams via Engineered Carbapenem Biosynthesis Enzymes

Abstract

Stereoselective biocatalysis by crotonase superfamily enzymes is exemplified by use of engineered 5-carboxymethylproline synthases (CMPSs) for preparation of functionalized 5-carboxymethylproline (5-CMP) derivatives methylated at two positions (i.e. C2/C6, C3/C6 and C5/C6), including products with a quaternary centre, from appropriately-substituted-amino acid aldehydes and C-2 epimeric methylmalonyl-CoA. The enzymatically-produced disubstituted 5-CMPs were converted by carbapenam synthetase into methylated bicyclic β-lactams, which manifest improved hydrolytic stability compared to the unsubstituted carbapenams. The results highlight the use of modified carbapenem biosynthesis enzymes for production of new carbapenams with improved properties.

Crotonases: Nature’s Exceedingly Convertible Catalysts

“…47,49,52 In many cases, the β-amino acid products of CarB catalysis have been converted into bicyclic β-lactams by CarA, which catalyzes the step after CarB in carbapenem biosynthesis (Figure 7A). 47,52 Such dual-enzyme catalysis has enabled the efficient production of multiple bicyclic β-lactams which would otherwise be challenging to efficiently synthesize via a nonenzymatic methodology. 47 The standout feature revealed by the substrate analogue and engineering work with CarB/ThnE is the potential for In some cases, stereoselectivity can be enhanced by coupling (engineered) CarB/ThnE asymmetric catalysis with production of the C-2 alkylated malonyl-CoA derivatives using a malonyl-CoA synthetase or Ccr.…”

“…Di- or trialkylated CMP derivatives can also be synthesized using combinations of malonyl-CoA and P5C derivatives. ,, In many cases, the β-amino acid products of CarB catalysis have been converted into bicyclic β-lactams by CarA, which catalyzes the step after CarB in carbapenem biosynthesis (Figure A). , Such dual-enzyme catalysis has enabled the efficient production of multiple bicyclic β-lactams which would otherwise be challenging to efficiently synthesize via a nonenzymatic methodology …”

Biocatalytic production of bicyclic β-lactams with three contiguous chiral centres using engineered crotonases

“…There is a need to develop efficient asymmetric routes for antibiotic production, where cost of goods is important. With a view to enabling routes to functionalised bicycle β-lactams, in particular C-1/C-6-functionalised bicyclic β-lactams as in carbapenems, we are investigating engineering of carbapenem biosynthesis enzymes9–11.…”

“…We describe the use of engineered CMPSs9–12,23–25, solely, and in tandem with an alkylmalonyl-CoA-forming enzyme, to catalyse the formation of 4,6-disubstituted- t -CMP stereoisomers, i.e. products with three contiguous chiral centres.…”