Study On The Production Of Polyhydroxyalkanoate By Vibrio Alginolyticus

The large-scale use of petroleum-based plastics has brought about global "white pollution" issue,and biodegradable plastics are considered as the best alternative to traditional plastics.Polyhydroxyalkanoate(PHA)is a class of biodegradable green plastics,which has been widely studied,but high production costs and single production host have restricted large-scale development.In this study,we reported that Vibrio alginolyticus is a new PHAproducing strain.Based on this,experiments involved in characterization of strains,fermentation to examine PHA production with different carbon sources,genome sequencing analysis,and investigation of the effect of overexpression of PHA synthesis gene,amylase gene and glucosidase gene on PHA production.Finally,the transcriptome analysis was conducted in the presence of glycerol and soluble starch as carbon sources.The main conclusions are as follows:(1)Screening two new V.alginolyticus were screened and and compared for their ability to PHA production polyhydroxy fatty acid esters.Two strains with the ability to produce PHA were identified as V.alginolyticus by 16 S r RNA,and the growth rate was higher than E.coli.A strain is named V.alginolyticus LHF01,which can produce poly-3-hydroxybutyrate(PHB)using a variety of carbon sources,of which glycerol is the optimum carbon source.Further with the optimization of Na Cl concentration and culture temperature,PHB production was increased from 1.87 g/L to 5.08 g/L.After adding Propionate,the V.alginolyticus LHF01 also produce polyhydroxybutyrate hydroxybutyrate.(2)The genome sequencing analysis of V.alginolyticus LHF01 was carried out,and the genes related PHA synthesis were functionally verified.The genome of V.alginolyticus LHF01 contained two chromosomes and a large plasmid with a total of 5.2 MB,and the GC content was 44.69%.The genome contained PHA synthesis manipulator pha BAPC,and its expression vector was constructed and transformed into E.coli,and a small amount of PHB is generated when glucose was substrate.The genetic operation platform of V.alginolyticus had been established.When recombinant V.alginolyticus was obtained by conjugation,the expression of PHA synthesis manipulator pha BAPC had increased PHB production by double and 4.55 g/L with the soluble starch as substrate.(3)The utilization of soluble starch and the influence of the expression of amylase and glucosidase genes were analyzed.V.alginolyticus LHF01 had the total consumption of 20 g/L soluble starch for 5 hours,and the PHB production is highest for 18 h,reaching 2.06 g/L.A total of seven genes expressing amylase and glucosidase were found in its genome,and corresponding expression vectors were constructed and converted to V.alginolyticus.The overexpression of the amylase gene named 03713 was found to increase the rate of consumption of soluble starch of V.alginolyticus by 20%,and the overexpression of the glucosidase gene named 04552 was found to make PHB production a small amount.(4)Transcriptome sequencing analysis of V.alginolyticus LHF01 cultured with glycerol and soluble starch as carbon sources was carried out.Transcriptome sequencing of strains cultured with glycerol and soluble starch as substrates and without substrates was obtained.When using glycerol or soluble starch as substrates,1729 or 1643 m RNAs with different expression levels were obtained in the transcriptome,of which 867 or 918 were upregulated genes,respectively.In the process of PHA synthesis,the addition of glycerol or soluble starch significantly increased the expression of β-ketothiolase and acetoacetyl-Co A reductase by GO function significant enrichment analysis.Metabolic pathways with increased differential gene expression were obtained,and strong promoters were screened by KEGG Pathway analysis and used for metabolic engineering of V.alginolyticus,so it bacome an industrial chassis cell capable of synthesizing a variety of high valueadded products using low-cost substrates.