Identification Of Related Enzymes In PHA Synthesis Pathway And Their Influence On PHA Synthesis Of Massilia Sp. UMI-21

Polyhydroxyalkanoates(PHAs)are polymerized hydroxyalkanoates that can be used as energy storage substances that are accumulated in cells by a variety of microorganisms under the condition of excessive carbon sources and limited nutritional conditions.The chain-like polymer biopolyester material combines a variety of excellent properties,such as material diversity,biodegradability,gas barrier properties,thermoplasticity,biocompatibility,etc.,in the daily chemical,pharmaceutical industry,Biodegradable materials,food processing and packaging,and many other application fields have broad development and application prospects.However,the subsequent promotion of PHA faces problems such as high fermentation costs and limited types of successfully commercialized PHA,which hinder the further application and development of such biopolymers.In this study,Massilia sp.UMI-21,a microorganism with PHA production capacity isolated from the green algae Ulva,was used as the research objective,and the carbon source utilization of the strain and the structure of PHA production were identified;the genome sequencing of the strain was completed,Obtained its metabolic pathways of related enzymes that use starch as a carbon source to synthesize PHA;using gene knockout methods to identify the effect of amylolytic enzymes in its PHA metabolic pathways on PHA synthesis,and review the bacteria's PHA polymerization through in vitro gene recombination methods The substrate specificity of the enzyme PhaC.First,Massilia sp.UMI-21 can use soluble starch(cold water soluble),xylan,etc.as a carbon source to obtain a maximum of 1.214±0.078 g/L and 0.538±0.040 g/L PHAs,respectively.However,this strain cannot use monosaccharides such as glucose and fructose as a carbon source to accumulate PHAs.The GC and ~1H-NMR detection results of the product obtained with starch as the carbon source showed that it was poly-3-hydroxybutyrate(P(3HB)).DSC and GPC thermal analysis results of the product showed that the glass transition temperature of P(3HB)was-7.3?,the crystallization temperature was 31.9?,the melting temperature was 172.0?,and the molecular weight was about 66.4 k Da,The dispersion coefficient is about 2.49.Secondly,the sequencing analysis of the genome of the strain Massilia sp.UMI-21showed that UMI-21 contained no plasmids and only one chromosome,the genome size was5.3Mb,and the GC content was 67.06%.Gene function annotation results show that Massilia sp.UMI-21 uses starch to be hydrolyzed to glucose by?-amylase Amy A1 and Amy A2,and generates Acetyl Coenzyme A through the sugar metabolism pathway.Two Acetyl Coenzyme A molecules are?-ketone.Thiohydrolase(Pha A)condenses into an acetoacetyl-Co A molecule,and acetoacetyl-Co A reductase(Pha B)reduces Acetoacetyl-Co A to 3-hydroxybutyryl-Co A(3HB-Co A).Finally,3HB-Co A is polymerized into PHB by type I PHA synthase PhaC.The PHA synthesis pathway is a typical type I PHA synthesis pathway.Finally,the suicide plasmid knockout method was used to knock out the two?-amylase genes amy A1 and amy A2 in Massilia sp.UMI-21 to construct three gene knockout strains Massilia sp.UMI-21?amy A1,?amy A2 and?amy A1&?amy A2.The results of PHA starch fermentation showed that none of the three knockout bacteria accumulated PHA.Real-time fluorescent quantitative PCR showed that the knockout of amy A1 and amy A2 genes reduced their transcription levels,and the knockout of amy A2 gene reduced the transcription levels of amy A1.Using prokaryotic expression system to obtain Massilia sp.UMI-21 PHA polymerase PhaC(1.05 mg/L)substrate specificity analysis results show that the enzyme has the ability to 3HB-Co A,2HB-Co A and D-lactate-Co A and other substrates Polymerization capacity.The results of this study clarify the metabolic pathway of Massilia sp.UMI-21 using starch to synthesize PHA,and provide a reference for the transformation of the PHA synthesis pathway of this strain and the expansion of production.