PHB体外合成体系的构建及产物性能OA

The chain-like polyhydroxyalkanoates(PHAs)accumulated in microorganisms as carbon and energy reserves have garnered significant attention due to their exceptional properties,such as biodegradability and biocompatibility.An in vitro synthesis system independent of metabolic pathways presents notable advantages for the development of novel PHAs.In this study,based on a self-constructed in vitro PHA synthesis system,(R)-2-hydroxybutyric acid and(R)-3-hydroxybutyric acid[(R)-2HB/(R)-3HB]with varying molar ratios were utilized as substrates to explore the structure and properties of the products polyhydroxyalkanoate(PHB)synthesized via three pathways.These pathways were composed of one acetyl CoA synthetase(ACSPt),two CoA transferases(ACTCp and PCTTt),and two PHA synthetases(PhaCRe,PhaC1Ps(STQK))that can be overexpressed in vitro at different temperatures.The results indicated that the mass concentrations of ACSPt,ACTCp,PCTTt,PhaCRe,and PhaC1Ps(STQK)were 8.0,20.0,12.0,16.0,and 6.0 mg/mL,respectively.Their specific activities were 0.058,0.460,0.520,0.105,and 0.026 U/mg,respectively.When the molar ratio of(R)-2HB and(R)-3HB was 0∶100 as the substrate,at 30℃using ACSPt/ACTCp/PhaCRe,and at 37℃using ACSPt/PCTTt/PhaCRe and ACSPt/PCTTt/PhaC1Ps(STQK),the synthesis yields of the PHB products were 0.66,0.80,and 0.66 g/L,respectively.The highest yield was achieved by ACSPt/PCTTt/PhaCRe at 37℃.1 H-NMR analysis confirmed that the products from all three pathways were PHB.GPC results demonstrated that the polydispersity indices of the PHB synthesized via the three pathways were below 2.0.The methodology employed in this study provides a reference for the synthesis of novel PHAs.