Study On Fermentation Conditions And Synthesis Characteristics Of PHA Synthetic Pseudomonas Sp.SG4502

Polyhydroxyalkanoates(PHAs)are a class of natural polymers that are biocompatible and biodegradable and can replace petroleum-based plastics.In addition to being used as additives for degradable bioplastics,they are used in food and regenerative medicine.It has broad application prospects in the fields of pharmaceuticals and pharmaceuticals.However,the large-scale production of PHAs has high fermentation costs and limited types of commercialized PHAs,which hinders the further application and development of such biopolymers.In this study,the culture conditions of Pseudomonas sp.SG4502 with PHA synthesizing ability were optimized,the possibility of PHA-producing strains as PHAproducing strains was analyzed,and the PHA-synthesis-related genes of this strain were analyzed at the molecular level.Effects of PHA synthesis.First of all,Pseudomonas sp.SG4502 the optimization results showed that the viscous solid product was about 0.26 g/L when the strain was cultured at 45?and pH 8.0 using 1%(w/v)sodium octanoate and 1%(w/v)glycerol as carbon source and 1%(w/v)urea as nitrogen source.GC-MC and ~1H-NMR results showed that the product synthesized using sodium caprylate as the carbon source is 3-hydroxycaprylic acid(3HO)?3-hydroxydecanoic acid(3HD),and 3-hydroxydodecanoic acid(3HDD)medium-long-chain mixed polyester;glycerol as a carbon source synthesis product is 3-hydroxydecanoic acid(3HD)and 3-hydroxydecano medium-long chain mixed polyester of dialkanoic acid(3HDD).Further GPC analysis results show that the molecular weight of the product synthesized using sodium caprylate as the carbon source is 3298 Da and the dispersion is 4.56;the molecular weight of the product synthesized using glycerol as the carbon source is 3105 Da and the a dispersion of 4.61.Secondly,at the molecular level,a strong promoter tac was inserted into the PHA synthetase genes phaC1 and phaC2 upstream of the strain using genetic engineering techniques to construct recombinant strains Pseudomonas sp.SG4502+tac-phaC1 and Pseudomonas sp.SG4502+tac-phaC2.By means of Nile Red staining fluorescence detection,real-time quantitative fluorescent PCR,~1H-NMR,GC-MS and other methods,the transcription level of the strong promoter tac gene to the phaC gene and the production,structure and properties of PHA were discussed.The results showed that using 1%sodium caprylate(w/v)as the carbon source,compared with wild strains,the recombinant strains+tac-phaC1 and+tac-phaC2 increased the transcription level of the phaC gene;the yield of PHAs synthesis of the three strains They are 0.26 g/L(wild type)?0.13 g/L(+tac-phaC1)and 0.40 g/L(+tac-pha C2).The composition of PHAs synthesized by the three strains are all containing medium and long chains 3HO?3HD and 3HDD mixed polyester with good thermal properties.The molecular weight and dispersion of the synthetic polyester are 3298Da?4.56(wild type),4965 Da?2.67(+tac-pha C1),5480 Da?2.518(+tac-phaC2).DSC and TG results show that the melting temperature of PHAs synthesized by the three strains is in the range of 60?to 65?,and in the range of the glass transition temperature T_g is-38.4?-40.965?,all have the low melting properties of MCL-PHA.Compared with the decomposition temperature of wild type(356.7?),the decomposition temperature of recombinant strains has increased,which are 365.1?and 371.4?.These results indicate that the recombinant strains The thermal stability of synthetic PHA is better than that of wild-type synthetic PHA.Finally,the knockout strain phaZ gene of Pseudomonas sp.SG4502 was knockedout by resistance insertion to construct a knockout strain Pseudomonas sp.SG4502?phaZ.Using Nile red fluorescence staining,real-time quantitative PCR,~1H-NMR,GC-MS and other methods,the transcription level of synthase-related genes and the production and structure of synthetic PHA by knocking out the degradation enzyme phaZ gene were discussed.The results showed that using 1%(w/v)sodium caprylate as the carbon source,compared with the wild type strain,the knockout strain Pseudomonas sp.SG4502?phaZ,which reduces the transcription level of the phaZ gene and increases the transcription level of the synthase phaC gene;knockout strain Pseudomonas sp.SG4502?phaZ.The amount of PHA synthesized by phaZ was 0.32 g/L,which was 0.0563 g/L higher than that of wild type PHA,and the composition monomers of the synthesized PHA were consistent with the results of wild-type strains.Further GPC analysis results showed that the knockout strain Pseudomonas sp.SG4502?phaZ,the molecular weight of the phaZ product was 7675 Da and the dispersion was 2.60.The DSC and TG results showed that the PHA synthesized by the knockout strain had a melting temperature of 63.94°C,a glass transition temperature of-36.69°C,and a decomposition temperature of 366.68°C,compared with the wild type product,the molecular weight increased obviously,the polydispersity decreased obviously,the T_g and T_m decreased,and the decomposition temperature increased obviously.The results show that the synthesized PHA has a low melting temperature and good thermal properties.The results of this study provide basic data for Pseudomonas sp.SG4502 to use low-cost carbon sources to reduce the cost of PHA synthesis and improve the ability of PHA synthesis at the molecular level,and provide a reference for other related research on PHA synthesizing bacteria.