Establishing Of Real-time Quantitative PCR System In Zymomonas Mobilis And Preliminary Analysis Of The Transcriptional Level Of The Carbon Metabolic Pathway

Bioethanol has been recognized as a potential alternative energy source. Amongvarious ethanol-producing microbes, Zymomonas mobilis has acquired specialattention due to its higher ethanol yield and tolerance. However, cellular metabolismin Z. mobilis remains unclear, hindering its practical application for bioethanolproduction. The complete genome sequence of Z. mobilis ZM4(ZM4) was reported in2005and2009. To elucidate the physiological characteristics, two of thegenome-scale metabolic network of Z. mobilis ATCC31821(ZM4) have beenreconstructed and validated based on its annotated gene, literature, physiological andbiochemical databases. The knowledge gained from the analysis could be applied toidentify potential cell engineering targets for enhancing carbon source utilization in Z.mobilis and improving the production of other desirable metabolites.Real-time quantitative PCR (qPCR) has become the benchmark technology fordetection and quantification of nucleic acids in a research, diagnostic, forensic andbiotechnology setting. The proposed mimimum standard for the provision ofinformation for qPCR ecperiments (“MIQE”) aims to restructure to day’s free-for-allqPCR methods into a more consistent format that will encourage detailed auditingexperimental detail, data analysis and reporting principles. General implementationof these guidelines is an important requisite for the maturing of qPCR into a robust,accurate and reliable nucleic acid quantification technology.There was little application of real-time qPCR in Z. mobilis. The main purpose ofthis paper is to establish the Real-time qPCR technology platform and the reliableanalysis of data standardization system in ZM4, including the selection of referencegenes and the gene expression at different growth stages. And then this method hasbeen used to the analysis of gene expression of ZM4and ZM4(gfo::FRT) in differentcarbon sources or sugar concentration.We have established the method of real-time qPCR for transcription analysis inZM4. Three softwares, geNorm, NormFinder and BestKeeper have been used toevaluate18candidate reference genes during the entire growth cycle in ZM4. Weselected7stablly expressed genes as reference gene. Then, three of genes were confirmed as internal control to analyze the expreesion of genes in alcohol metabolicpathway.Subsequently, transcriptomic and metabolomic profiles for the the main carbonmetabolic pathwayof ZM4and ZM4(gfo::FRT) fermentations in different carbonsources and sugar concentration have been elucidated by real-time qPCR andhigh-performance liquid chromatography (HLPC) analysis. It has been confirmed thestability of the7selected reference genes. And we also found that the deletion of gfogene effected the growth and the alcohol metabolic pathway, especially in the highsugar concentration. The two strains showed different growth states in differentcarbon sources.The results showed that it was very important to identify and validate theappropriate reference genes in different tissues and exprerimental conditions. And thetranscript abundance of genes in the the main carbon metabolic pathway weredifferent. The ZM4phenotypic change was the result of the roles of transcription andtranslation.