Optimization Of Synthetic Medium Of Xylose-fermenting Zymomonas Mobilis And Construction Of The Strain Overexpressing RecG Genes

Zymomonas mobilis is considered to have broad application prospects in ethanolfermentation industry because of its unique metabolism and ability to rapidly andefficiently produce ethanol. However, a synthetic medium suitable for the growth andethanol production from recombinant Z. mobilis has not been obtained, andrecombinant Z. mobilis have low tolerance to acetic acid and other inhabitors inhydrolysate. This study first optimized the synthetic medium of recombinant Z.mobilis using statistical experimental designs. In addition, the plasmids containingrecG from E.coli and Z.mobilis has been constructed and transformed into the CP4and recombinant Z. mobilis. Finally, the recombinant strains were evaluated using theobtained synthetic medium.A synthetic medium for ethanol production by recombinant xylose-fermentingZymomonas mobilis was optimized using statistical experimental designs. Firstly,eight components, including xylose,（NH₄）₂SO₄, trisodium citrate, succinic acid,choline chloride, folic acid, pyridoxine and thiamine, were determined to havesignificant effects on ethanol production by P-B design. Then, the steepest ascentmethod was used to check whether we were prospecting the best range of the fivevariables （xylose, trisodium citrate, choline chloride, pyridoxine and thiamine）identified by the PB design.The RSM was used to optimize the concentration of theabove five wariables and constructed a mathematical model. The ANOVA of themodel indicated that xylose and trisodium citrate were the key medium componentsinfluencing ethanol production. What’s more, the ethanol production and growthresponse were directly proportional to the logarithm of the concentration of trisodiumcitrate in the range of0.1–1.6g/L.The validity of the developed model was verified.The ethanol concentration in the optimized medium was19.1g/L, which was6.7%higher than in the RM medium. The optimized medium was then simplified to givethe final synthetic medium （S2）, containing58.5g/L xylose,2.65g/L trisodium citrate,1g/L （NH₄）₂SO₄,1g/L MgSO₄·7H₂O,2g/L KH₂PO₄and2.74mg/L calciumpantothenate, in which the ethanol concentration was21.7g/L and20.7%higher thanin the RM medium. Construction and evaluation of acetic acid-resisant Z. mobilis strains:overexpression plasmids pBBR1-MCS2-Pgap-ErecG-rrnB andpBBR1-MCS2-Pgap-ZmrecG-rrnB were constructed that contained Z. mobilis GAPpromoter （Pgap）, E.coli rrnB terminater and exclusively the recG genes of E.coli andZ. mobilis, respectively. The two plasmids then were transformed into CP4and CP4（pZB22）. The resultant transformants containing pBBR1-MCS2-Pgap-ErecG-rrnBwere obtained and were named CP4（ErecG） and CP4（X, ErecG）, and containingpBBR1-MCS2-Pgap-ZmrecG-rrnB were named CP4（ZmrecG）. Then thepreliminary assessments of fermentation of the three strains were done. This has lainthe foundation for the following study of the role of recG gene in improving thetolerance of inhibitors in recombinant strains.