In Vitro Analysis of d‑Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate-<i>co</i>-3-hydroxybutyrate) Syntheses

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1_PsSTQK) efficiently produces poly­(lactate-<i>co</i>-3-hydroxybutyrate) [P­(LA-<i>co</i>-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly­(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, <i>in vitro</i> analysis of PhaC1_PsSTQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1_PsSTQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of ∼3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P­(LA-<i>co</i>-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P­(LA-<i>co</i>-3HB) biosynthetic systems