Construction Of In Vitro PHA Synthesis Pathway Based On New Enzymes And Study Of Synthesis Performance

Polyhydroxyalkanoates（PHAs）are produced by a variety of bacteria and archaea under unbalanced growth conditions that limit nutrients and excess carbon supply,and are used intracellularly as a store of carbon and energy.In addition to being completely biodegradable in nature,PHAs have mechanical and thermal properties almost comparable to petroleum-based plastics and have received much attention in the materials field.However,the synthesis cost of PHAs is higher than petroleum-based plastics,the types of commercializable PHAs are limited,and the use of new enzymes to synthesize novel PHAs through designed synthetic pathways is of great importance.In this study,（R）-3-hydroxybutyric acid[（R）-3HB],D-lactic acid（D-LA）,4-hydroxybenzoate acid（4HBZ）,（R）-Mandelic acid[（R）-MA]as substrates.Using the Acetyl-Co A synthase ACS_MU from Massilia sp.UMI-21 and ACS_Pt from Pelotomaculum thermopropionicum JCM 10971 as Co A donor synthases,respectively.And Co A transferase Had A_Cd from Clostridium difficile and PCT_Cp from Clostridium propionicum JCM 1430 as polymeric precursor HA-Co A synthase,respectively.The classⅠPHA synthases PhaC_Re and PhaC_MU from Ralstonia eutropha,Massilia sp.UMI-21,and classⅡPHA synthases PhaC1_Ps（STQK）and PhaC1_{Pa93（STQK）}from Pseudomonas sp.SG4502,Pseudomonas aeruginosa 93-3-1 as precursor polymerases,respectively.Different in vitro synthesis pathways were constructed to explore the synthesis capacity and product structure of new enzymes related to the synthesis of PHA.First,the related enzymes in the designed pathway were expressed recombinantly in vitro.Four existing recombinant strains,BL21-p Cold-acs _Pt,BL21-p QE-pct _Cp,BL21-p Cold-pha C_Re,BL21-p QE-80L-pha C1_Ps（STQK）,and four newly constructed recombinantstrainsBL21-p QE-80L-acs MU,BL21-p ColdⅡ-had ACd,BL21-p QE-80L-pha C_MU,BL21-p QE-80L-pha C1_{Pa93（STQK）}were overexpressed and purified in vitro.The obtained protein concentrations were（mg/m L）:ACS_MU（6.2）,ACS_Pt（14.0）,Had A_Cd（15.0）,PCT_Cp（12.0）,PhaC_Re（16.0）,PhaC_MU（6.4）,PhaC1_Ps（STQK）（5.4）,PhaC1_{Pa93（STQK）}（6.8）,respectively.The substrate specificity of the three types of proteins was then analyzed and the specific activity of the ACS_MU protein for Co A was lower than that of ACS_Pt.Had A_Cdshowed enzymatic activity against（R）-3HB,D-LA,4HBZ,（R）-MA,（R）-HPBA with the highest activity against 4HBZ,while PCT_Cp has enzymatic activity only against（R）-3HB,D-LA.The PHA synthase PhaC_Re had enzymatic activity only for（R）-3HBCo A,PhaC_MUhad enzymatic activity for（R）-3HBCo A,D-LACo A,and PhaC1_{Pa93（STQK）}had higher enzymatic activity for（R）-3HBCo A,D-LACo A,4HBZCo A,（R）-MACo A,（R）-HPBACo A than PhaC1Ps（STQK）.Finally,three PHA synthesis pathways were constructed,the first using the enzyme composition ACS_MU/ACS_Pt,PCT_Cp,PhaC_Re,all of which were able to synthesize polyhydroxybutyrate（PHB）with（R）-3HB as substrate,demonstrating that the new enzyme ACS_MU can be used for the in vitro synthesis of PHB.The second one uses enzyme composition ACS_MU,PCT_Cp,PhaC_MU/PhaC1_Ps（STQK）/PhaC1_{Pa93（STQK）}with molar ratios of100:0,75:25,50:50,25:75,0:100 of（R）-3HB,D-LA as substrates,all three polymerases can synthesized PHB,and PhaC_MU,PhaC1_{Pa93（STQK）}synthesized PHB in higher yield than PhaC1_Ps（STQK）.The third one uses enzyme composition ACS_MU,Had A_Cd,PhaC1_{Pa93（STQK）}with molar ratios of 100:0,75:25,50:50,25:75,0:100 of（R）-3HB:4HBZ and（R）-3HB:（R）-MA as substrates,and the products synthesized from the two different ratios of substrates were PHB,and no 4HBZ,（R）-MA components were incorporated,which proved that the three new enzyme combinations could synthesize PHB.