Response Of Glucose Metabolism To High Temperature Stress Of Pseudomonas Plecoglossicida

Pseudomonas plecoglossicidia JUIM01 is currently the main industrial strain of2-ketogluconic acid production,which has relatively high productivity and conversion rate of sugar and acid.2KGA was an important organic acid mainly used in the production of food antioxidant D-sodium erythorbate.Aiming at the core problem that the glucose metabolism of P.plecoglossicida is shifted from extracellular oxidation pathway to intracellular phosphorylation pathway during high temperature fermentation,which led to a significant decrease of 2KGA production performance.In this study,P.plecoglossicida JUIM01 strain was utilized to investigate the effects of high temperature stress on its 2KGA biosynthesis.The response mechanism of glucose metabolism of P.plecoglossicida to high temperature stress was preliminarily revealed from transcriptomics and proteomics levels.The research will provide a theoretical basis for efficient production of 2KGA and subsequent breeding and the construction of high temperature resistant engineering strains..The obtained results are listed as follow:(1)The fermentation parameters under different fermentation temperatures on2 KGA biosynthesis of P.plecoglossicida was tested and defined.Compared to fermentation at 32°C,the yield of 2KGA at 36°C and 40°C decreased by 3.8% and10.1%,the fermentation period decreased by 5.8% and 11.7%,also the conversion rate of sugar and acid decreased by 3.9% and 10.2%,and the 2KGA productivity increased by 2.1% and 1.7%,respectively,while the trend on fermentation p H is remain consistent.The transcription levels of genes related to glucose metabolism were detected by real-time quantitative PCR,the expression level of genes related to 2KGA synthesis pathway(gcd,2kgd H,gndL,gndC,gndS)was down-regulated under high temperature stress;The genes of transcription regulatory factors related to glucose metabolism(rcc R,hex R,ptxS)was expressed significantly at 40°C;The expression level of genes related to 2KGA catabolism pathway(kguT,kguK,kguD)were down-regulated with the increase of temperature;The expression level of genes related to glucose phosphorylation pathway(glk,zwf)were up-regulated.Under high temperature stress,glucose metabolism shifts from extracellular oxidation pathway to intracellular phosphorylation pathway that clearly less benefit and unfavored to the synthesis of 2KGA from fermentation product.(2)The response mechanism of glucose metabolism in P.plecoglossicida under high temperature stress was preliminarily revealed from transcriptomics perspective.The DEGs at 32°C,36°C and 40°C fermentation temperatures were analyzed by Illumina Hiseq sequencing.High temperature stress had a whole impact on the transcriptomics level,and the related DEGs in pentose phosphate pathway,tricarboxylic acid(TCA)cycle pathway,oxidative phosphorylation pathway,ubiquinone and other terpene quinone biosynthetic pathways were significantly affected.Under high temperature stress,the expression lecvel of genes related to 2KGA synthesis in glucose extracellular oxidation pathway were down-regulated and the extracellular oxidation pathway was inhibited.In the intracellular phosphorylation pathway,the expression level of relevant genes to glucose phosphorylation pathway weres up-regulated,while the expression of relevant genes to glucose phosphorylation pathway and 2KGA phosphorylation pathway were down-regulated,which maked glucose metabolism shifted from extracellular oxidation pathway to intracellular phosphorylation pathway.Moreover,the electron transfer efficiency can be adjusted by the coordinated expression of different components in respiratory chain and five terminal oxidases to improve the high temperature adaptability of the strain.(3)The response mechanism of glucose metabolism of P.plecoglossicida under high temperature stress was preliminarily revealed from the proteomics analysis.The TMT quantitative proteomics technology was used to analyze DEPs at 32°C,36°C,40°C fermentation temperatures.High temperature stress also had a global impact on P.plecoglossicida at proteomic level.The expressions of Glk and Zwf-1 in glucose phosphorylation pathway,KguK and KguD in 2KGA phosphorylation pathway were up-regulated under high temperature stress.In the contrary,the expressions of Gnt P and Gnu K in gluconate phosphorylation pathway and Gcd,Pqq F,Pqq D,Rib H,Blu B,2KGDH in extracellular oxidation pathway were down-regulated.These led to the shifting in glucose metabolism from extracellular oxidation pathway to intracellular phosphorylation pathway thus resulting in the decrease of 2KGA yield.Furthermore,five terminal oxidases in respiratory chain also responded to high temperature stress at proteomic level.