Construction Of Recombinant Strains Of Zymomonas Mobilis For Economic Bioethanol Production From Molasses

Taking non-food materials as the substrate for bioethanol synthesis can not only fundamentally solve the problem of"competing with human for food"and"competing with livestock for feedstuffs"in the bio-manufacturing industry,but also is an important strategy to achieve"carbon neutrality".Molasses is one of the by-products from sugar industry,which is applied to produce fodder,yeast,sodium glutamate,organic acid and other products.The large amount of sucrose makes molasses suitable for microbial fermentation.Optimization of strains and fermentation conditions are relatively common applied in the study of microbial molasses fermentation.Zymomonas mobilis is a facultative anaerobic ethanologenic bacterium.It can ferment sucrose,glucose and fructose to synthesize ethanol with its unique Entner-Doudoroff（ED）pathway.Its high sugar and ethanol tolerance makes it suitable for molasses fermentation.Although Z.mobilis can ferment molasses to produce ethanol,high concentration of sugar and potential inhibitors may inhibit Z.mobilis for the efficient utilization of molasses.In this study,the differences between different carbon sources（molasses,sucrose,fructose and glucose）utilization of Z.mobilis 8b has been evaluated.It showed that 8b was inhibited in molasses or fructose medium.Since the primary fermentable sugar sucrose in molasses will be hydrolyzed into glucose and fructose for utilization by Z.mobilis,adaptive laboratory evolution（ALE）strategy was applied to adapt Z.mobilis 8b under conditions of fructose,sucrose or molasses.A mutant strain named 8b-F74 with enhanced fructose utilization was selected,which is also capable of efficient utilization of molasses.To reveal the molecular mechanisms of efficiently utilization of fructose or molasses by8b-F74,Whole-Genome Resequencing（WGR）and RNA Sequencing（RNA-Seq）technologies were used to identify the genetic differences between mutant strain 8b-F74 and parent strain 8b in different carbon sources.WGR result indicated that the codon 99 of gene glf（ZMO0366）had a missense mutation.Result of molecular dynamics simulation and assessment of fructose utilization verified that the structure of mutant Glf^G99S becomes more stable with fructose as the ligand molecule,thus it might enhance fructose utilization ability of 8b-F74.RNA-seq result showed that the expression of genes,which involved in nitrogen fixation and phosphoric acid transport,was significantly up-regulated when 8b-F74 fermented in molasses.In addition,the expression of gene encoding carbohydrate porin was up-regulated while the expression of genes encoding phage shock proteins was down-regulated in 8b-F74 using all carbon sources.Differential expression of these genes might increase the membrane permeability to carbohydrate,which improved high concentration sugar utilization of 8b-F74.Sac B and Sac C were the main sucrose hydrolases in Z.mobilis.Sucrose was the main fermentable sugar in molasses,and levan will be synthesized by Sac B when Z.mobilis ferments in sucrose with ethanol production reduced.In this study,sac B was deleted and sac C was over-expressed to divert sucrose metabolic flux for ethanol production improvement from sucrose-rich molasses by 8b-F74.Ultimately,recombinant strain F74-B^-C⁺was obtained,which can utilize molasses efficiently to produce ethanol.Sugar consumption of F74-B^-C⁺increased to88.26 g/L from 71.71 g/L by 8b,and correspondingly ethanol titer increased to 40.85 g/L from31.11 g/L and ethanol productivity improved to 1.70 g/L/h from 0.97 g/L/h in 8b,respectively.In summary,this study obtained a fructose and molasses efficiently utilization strain 8b-F74 by adaptive laboratory evolution in fructose.Omics studies demonstrated that the mutation of glf（ZMO0366）and differential expression of genes related to nitrogen fixation,phosphoric acid transport,porin,phage shock proteins might be the reason of efficient molasses utilization by 8b-F74.The recombinant strain F74-B^-C⁺was then obtained by deleting sac B and over-expressing sac C to optimize the molasses utilization of 8b-F74.In addition,the omics data of Z.mobilis in molasses and other carbon sources obtaining from this study provided abundant information for further optimizing molasses utilization and exploring the potential mechanism of molasses fermentation in Z.mobilis.It laid the foundation for the construction of Z.mobilis as a molasses fermentation chassis cell.