Further Improvement Of E.coli IS5-d For Xylitol Production Via CRP Mutation And Enhancement Of NADPH Regeneration

Xylitol is a pentose alcohol that is widely used in food,medicine,and chemical industries,and has important economic and social values.Biological production of xylitol has the advantages of mild conversion conditions,green technology,and the direct use of hemicellulose hydrolysate as a substrate,which promises alternative to the currently commonly used chemical hydrogenation process.As an ideal host strain for genetic engineering,Escherichia coli has many advantages including clear genetic pathway,simple culture conditions,fast growth rate,mature genetic manipulation methods,and the ability to use pentose,etc.At present,studies on xylitol production from hemicellulose hydrolysate using E.coli as a host have been reported,and a xylitol-producing strain E.coli W3110 IS5-d has also been constructed in our laboratory earlier.However,the slow transport rate under low xylose concentration conditions still adversely affects the volumetric productivity of xylitol and leads to relatively high residual xylose concentration.Additionally,the original strain can only use the ion-exchange detoxified hemicellulose hydrolysate efficiently,and the adaptability to lime-treated hemicellulose hydrolysate needs to be improved.To solve the above issues,the following work has been carried out in this thesis.First of all,the combination of CRP protein mutation and glucose phosphorylation transport pathway modification was used to further eliminate the inhibition of glucose on xylose transport.Based on the deletion of the glucose phosphorylation transport coding gene ptsG,and after the mutation of CRP into to cAMP-independent CRP*(I112L,T127I,A144T),the glucose uptake rate of the mutant IS5-dI decreased 17.4%,while the xylose uptake rate increased 10.5%,compared with the starting strain IS5-d,which only deleted ptsG,achieving a completely simultaneous absorption of xylose and glucose.Additionally,the xylose transport rate of the mutant increased 27.3%when the xylose concentration was lower than 5.7 g/L.Through the application of this combinatorial strategy,and using the ion-exchange detoxified corncob hydrolysate as a substrate,the 15-L bioreactor fermentation experiments revealed that xylitol concentration,volumetric productivity and xylitol yield per xylose for IS5-dI were 163.5 g/L,3.03 g/L/h and 1.02 g/g,increased by 14.3%,65.6%,and 9.7,respectively,compared with the starting strain IS5-d under the same fermentation conditions.Then,the process of the fermentation by IS5-dI to produce xylitol from iron-exchange treated hemicellulose hydrolysate was optimized.The optimal fermentation conditions were as follows:seed age 13 h,fermentation temperature 37? DO 35%in the early stage and 20%in the later stage,corn steep liquor initio concentration 10 g/L constant flow rate feeding(30 L/h),hydrolysate initio feeding after 10-h fermentation and keeping xylose concentration basically constant(60 g/L),continuous feeding and controlling glucose concentration not to exceed 5 g/L.Under the optimized conditions,52-h fermentation in a 15-L bioreactor yielded 165 g/L xylitol.The volumetric productivity was 3.17 g/L/h and the xylitol yield per xylose was 1.01 g/g,which was the highest level of xylitol production reported to date,by E.coli from hemicellulose hydrolysate.Moreover,no glucose,xylose,arabinose,or other residual sugars was detected in the fermentation broth.Next,a dual modulation strategy to redirect the carbon flux of the glucose central metabolic pathway in E.coli cells was adopted,that is,the PPP pathway gene was overexpressed while the EMP pathway gene was deleted,to strengthen the NADPH regeneration.Strain 2bzwf was constructed by inserting one copy of zwf gene into the genome of the IS5-d strain,resulting in a 26.3%increase in NADPH/NADP+.Strain 2bzwfgnd was constructed by inserting one copy of gnd gene into the genome of the 2bzwf strain,resulting in an 8.3%increase in NADPH/NADP+.Strain 2bpgi was constructed by deleting pgi gene of the 2bzwfgnd strain,resulting in a 46.2%increase in NADPH/NADP+.The engineered strain 2bpgi produced xylitol via 76 h fed-batch fermentation from ion-exchange-detoxified corncob hydrolysate in a 15-L bioreactor.The xylitol concentration,volumetric productivity,xylitol yield per xylose and xylitol yield per glucose were 161.6 g/L,2.13 g/L/h,1.02 g/g and 2.50 g/g,increased by 13.0%,16.4%,9.7%and 67.8%,respectively,compared with the starting strain IS5-d.Finally,with the crp*mutant IS5-dI as the starting strain,further semi-rational site-directed mutagenesis was carried out on I127 of its CRP,and a total of 9 mutants with the different polarity of amino acid of CRP from that of the original strain IS5-d were obtained.After screening with lime-treated corncob hydrolysate as fermentation substrate,strain IS5-dG with significantly improved tolerance to hydrolysate was obtained.Additionally,the process of treating corncob hydrolysate with lime neutralization method was comprehensively optimized to make the hydrolysate more suitable for engineered strain to produce xylitol.The IS5-dG strain produced 136.7 g/L of xylitol after 78-h fermentation from lime-treated corncob hydrolysate in a 15-L bioreactor and the volumetric productivity was 1.75 g/L/h.These are the highest concentration and volumetric productivity of xylitol reported to date,produced by E.coli fermentation from hemicellulose hydrolysate without ion-exchange detoxification.In summary,a recombinant E.coli that can produce high purity xylitol from corncob hydrolysates without ion-exchange detoxification has been achieved.This laid a good foundation for the industrial production of xylitol.