The Molecular Mechanism Of The Genes Responsible For The Inhibitors On The Xylonate Production By Whole Cell Catalysis Using Gluconobater Oxydans NL71

This paper was aiming to research on the problem of the reduced xylonic acid production the concentrated lignocellulosic hydrolysate by whole cell catalysis using Gluconobacter oxydans NL71. Characteristics of reaction kinetic on Gluconobacter oxydans NL71 biocatalysis of xylose to xylonate in the typical three lignocellulosic inhibitors of formate, furfural and 4-hydroxybenzaldehyde(PHB) were studied respectively. With the high throughput sequencing platform, the complete genome of G. oxydans NL71 was sequenced, and the high-throughput RNA sequencing technology(RNA-Seq) was utilized to interrogate G. oxydans NL71 transcriptome in order to address the above fundamental issues. Many crucial genes, constituted the major reasons for explaining the cellular responses related to inhibitory effects, were selected from the transcriptome results and then analyzed by Real-time Quantitative PCR(RQ-PCR).The results showed that formate, furfural and PHB were the critical three lignocellulosic inhibitors during G. oxydans NL71 biocatalysis of xylose to xylonate in lignocellulosic biomass. When the concentration of formic, furfrual and PHB increased to 5 g/L, 8 g/L and 3 g/L respectively, the whole-cell catalysis performance bagan to be inhibited with the decreased xylose metabolic rate and D-xylonate productivity. Based on the analysis of the xylonic acid production in the lignocellulosic hydrolysate by whole cell catalysis using Gluconobacter oxydans NL71, the inhibitory effect of formate was the most obvious, followed by the furfural and PHB.Based on Hiseq 2500 and Miseq sequencing platform, the draft genome sequence of G. oxydans NL71 was sequenced and deposited at NCBI under the accession LCTG00000000.. The results indicated that the total size of the complete genome of G. oxydans NL71 was 3.25 Mb with a G+C content of 55.71%, 3,226 predicted protein-coding sequences. There were rRNA(5), tRNAs(59), GIs(11), Prophage(3), GO(1124)?KEGG(1758)?COG(1600)?NR(3012)?Swiss-Prot(1161)?TrEMBL(2964) found in the genome.The transcriptome analyses during G. oxydans NL71 biocatalysis of xylose to xylonate in the critical three lignocellulosic inhibitors of formate, furfural and PHB were performed by RNA sequencing with Hiseq 2000. Accordingly, the digital gene expression profile screened 572, 714 and 408 DEGs in Blank vs Formic, Blank vs Furfural and Blank vs PHB between the two comparisons. 19 key genes, screened for explaining the cellular responses related to inhibitory effects, were as follows: glyceraldehyde 3-phosphate dehydrogenase(NL71GM001713) in glycolysis; 2-oxoglutarate dehydrogenase(NL71GM001355) in TCA cycle; robise 5-phosphate isomerase(NL71GM001290) in peotose phosphate pathway; ribose ABC transporter periplasmic-binding protein(NL71GM001291), ribose ABC transporter ATP-binding protein(NL71GM001292) in ABC transporters; preprotein translocase subunit A(NL71GM000616), signal recognition paticle protein(NL71GM002123) in bacterial secretion system; methyl-accepting chemotaxis protein(NL71GM000741), chemotaxis protein(NL71GM000736) in bacterial chemotaxis; NAD(P) transhydrogenase subunit alpha(NL71GM002001), thioredoxin(NL71GM000876), alcohol dehydrogenase(NL71GM001165), ferredoxin(NL71GM000337), monothiol glutaredoxin(NL71GM002512) in electron transport; peroxiredoxin(NL71GM001610), alkyl hydroperoxide reductase(NL71GM001611), glutathione peroxidase(NL71GM000375), superoxide dismutase(NL71GM002096), cytochrome c peroxidase(NL71GM000364) in detoxification. On the basis, the above genes were discussed their biological function combined with the cell physiology, biochemistry and the metabolic flux.