Omics Study Of Efficient Fructose-utilizing Mutants Of Zymomonas Mobilis For Ethanol Production Using Molasses

Molasses is one of the by-products of sugar industry,which majorly contains sucrose,as well as small amount of glucose and fructose.Molasses is applied to many industries such as aquaculture,animal husbandry,special breeding and construction industry due to its rich sugars and sticky physical properties.Zymomonas mobilis can use fermentable sugars to produce ethanol through Entner-Doudoroff pathway.Meanwhile,it has strong tolerance to high concentrations of sugar and ethanol,which makes it an excellent ethanologenic bacterium.In this work,wild-type Zymomonas mobilis subsp.mobilis ZM4(ZM4)and fructose-utilizing mutants(ZMF~+-73 and ZMF~+-74)were used to evaluate their fermentation performance using different molasses.The ethanol titer of ZM4 was 25～35 g/L when culturing in diluted molasses,which contained 80～130 g/L sucrose,while the ethanol titer increased about 80～160%and the fermentation time reduced by 30～40 h for fructose-utilizing mutants.To explore the molecular mechanism of mutants’fermentative performance in molasses,next-generation sequencing(NGS)-based WGR(Whole Genome Resequencing)and RNA Sequencing(RNA-Seq)were applied to identify genomic and transcriptomic differences between mutants and wild-type in RMG5 and diluted beet-molasses.The WGR data indicated that the insertion mutation in ZMO0976(xyr A)and the deletion mutation in ZMO1237(ldh A)may cause the transcription to terminate prematurely.In addition,there were 44 SNP mutations for 43 genes,which are involved in DNA repair and replication,transportation,flagellar biosynthesis,glycolysis and pyruvate metabolism.Transcriptomic data demonstrated that four genes related to phosphate ABC transporter and three genes related to phosphate regulation were upregulated in mutant backgrounds.In addition,six genes related to flagella structure and two genes related to glucose transport and ethanol production were also differentially expressed in molasses.These changes may affect the capability of mutants on phosphate regulation,movement,sugar transport and ethanol production.In summary,this study demonstrated that Z.mobilis can utilize molasses directly for ethanol production.Two fructose-utilizing mutants can efficiently utilize molasses with better performance than wild type ZM4.In addition,the potential molecular mechanism related to the efficient fructose and molasses by mutant strains has been proposed based on genome resequencing and RNA-Seq results,which lay a foundation for future strain development.