Engineered D-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaCl(ps)STQK) efficiently produces poly(lactate-co-3-hydroxybutyrate) [P(LA-co-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, in vitro analysis of PhaCl(ps)STQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaCl(ps)STQK 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-co-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-co-3HB) biosynthetic systems.