In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria

Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures.