Construction Of All Fermentable Sugars Assimilating Pathways And Inhibitor Tolerance Into Pediococcus Acidilactici For High Titer Chiral Lactic Acid Production From Lignocellulose

Cellulose is a homopolymer composed of glucose,hemicellulose is a heteropolymer of hexoses(glucose,mannose and galactose)and pentoses(xylose and arabinose).Hemicellulose-derived sugars account for about 40%(w/w)of the total carbohydrates in lignocellulose.The efficient utilization of lignocellulose-derived all fermentable sugars(glucose,xylose,arabinose,mannose and galactose)is one of the key scientific issues in lignocellulose biorefinery.However,in cellulosic chiral lactic acid researches,no practical solutions on all fermentable sugars fermentation has been given from the fermenting strains construction to the lignocellulose fermentation technology.Moreover,the pretreatment process in biorefinery inevitably produces many inhibitors such as furans,weak acids and phenolic aldehydes.The complete removal of these inhibitors is a prerequisite for the lignocellulose fermentation.This thesis studied the above two key scientific issues in lignocellulose biorefinery with the goal of high titer chiral lactic acid production from lignocellulose,based on the dry biorefinery process consisting of dry acid pretreatment,biodetoxification and high solids loading simultaneous saccharification and fermentation.The dry solid granular lignocellulose feedstock was obtained by dry acid pretreatment,the furans and weak acids in pretreated lignocellulose were quickly removed,and pentoses(xylose and arabinose)derived from hemicellulose were efficiently remained after biodetoxification.The large amount of pentoses existed in pretreated and biodetoficated lignocellulose provided an important prerequisite for the subsequent hexose and pentose co-fermentation strains construction as well as the simultaneous saccharification and hexose and pentose co-fermentation.On the other hand,some phenolic aldehydes derived from lignin still existed in the pretreated and biodetoxificated lignocellulose and had a significant negative impact on high titer and high productivity of chiral lactic acid fermentation.In our previous works,a high-temperature tolerant D,L-lactic acid producing strain Pediococcus acidilactici DQ2 with special adaptability to lignocellulose sysytem was isolated from cellulosic ethanol fementation broth.Two chiral lactic acid producing strains.acidilactici TY112(for L-lactic acid production)and P.acidilactici ZP26(for D-lactic acid production)were constructed by disrupting ldhD and ldh in P.acidilactici DQ2 genome and the following adaptive evolution,respectively.However,they have two disadvantages,one is that they can utilize hexose such as glucose and galactose,but cannot efficiently metabolize xylose or arabinose,another is that they have less tolerance on phenolic aldehydes.In this thesis,metabolic engineering was applied to construct the all fermentable sugars assimilating pathways and strengthen the tolerance of phenolic aldehydes into the above two strains.High solids loading simultaneous saccharification and all fermentable suagrs co-fermentation(SSCF)using dry acid pretreated and biodetoxificated lignocellulose feedstock were earied out and record high titer of D-lactic acid and L-lactic acid were achieved.In the first part,the xylose-assimilating pathways were constructed into the D-lactic acid producing strain R acidilactici ZP26 chromosome to obtain the glucose,xylose,arabinose,mannose and galactose co-fermentation strain,the high solids loading SSCF of lignocellulose was carried out and high titer D-lactic acid was achieved.The parental R acidilactici ZP26 can utilize arabinose and mannose with low yield of D-lactic acid,but cannot utilize xylose.The heterologous genes encoding xylose isomerase(xylA),xylulokinase(xylB),transketolase(tkt)and transaldolase(tal)were integrated into the.acidilactici ZP26 chromosome,and the endogenous phosphoketolase gene(pkf)in the phosphoketolase pathway(PK)was disrupted to reduce the byproduct acetic acid production.The xylose-assimilating ability of the newly constructed strain was significantly improved by adaptive evolution.Moreover,the xylose-assimilating pathways construction increased the yield of D-lactic acid from arabinose and mannose,and had no negative impact on the utilization of glucose and galactose.The resulting strain R acidilactici ZY15 can efficiently co-utilize all fermentable sugar for homo D-lactic acid production with the high optical purity of 99.2%.High solids loading SSCF was conducted using 30%(w/w)solids loading of the dry acid pretreated and biodetoxified wheat straw feedstock.Record high at 127.1 g/L ofD-lactic acid was obtained with 1.8 g/L/h of productivity.62.3%of yield and only 1.6 g/L of residue mixed sugars.In the second part,the xylose-assimilating pathways were constructed into the L-lactic acid producing strain.acidilactici TY112 to obtain the all fermentable sugars co-fermentation strain,the high solids loading SSCF of lignocellulose was carried out and high titer L-lactic acid was obtained.The parental.acidilactici TY112 can utilize arabinose and mannose with low yield of L-lactic acid,but cannot utilize xylose.According to the metabolic engineering and adaptive evolution used in the first part,the efficient xylose-assimilating pathways were successfully constructed into the chromosome of.acidilactici TY112 by introducing four heterologous genes(xy/j,xylB,tkt and tal),and disrupting two endogenous genes(pkt and ackA2),followed by adaptive evolution.Furthermore,the xylose-assimilating pathways construction increased the yield of L-lactic acid from arabinose and mannose,and had no negative impact on the fermentation of glucose and galactose.The resulting strain can co-utilize all fermentable sugars for homo L-lactic acid production with 99.6%of optical purity,named as.acidilactici ZY271.The engineered strain was applied to the 30%(w/w)solids loading SSCF of the dry acid pretreated and biodetoxified wheat straw.The L-lactic acid titer reached record high at 126.7 g/L with 1.8 g/L/h of productivity,62.3%of yield and 1.1 g/L of residue mixed sugars.In the third part,it focused on decreasing the inhibition of phenolic aldehydes on SSCF by enhancement of the phenolic aldehydes tolerance in.acidilactici ZY15 through improving the phenolic aldehydes conversion pathway.The tolerance of.acidilactici ZY15 on 4-hydroxybenzaldehyde,vanillin and syringaldehyde were assayed,respectively.Vanillin showed the most inhibition on R acidilactici ZY15.Then,the heterologous gene CGS9114 RS09725 encoding short-chain dehydrogenase was integrated into the chromosome of.acidilactici ZY15.The resulting strain.acidilactici ZY15-AackA2::CGS9114 RS09725 performed better fermentation than.acidilactici ZY15 under the stress of vanillin.It indicated that CGS9114 RS09725 integration strengthened the vanillin tolerance of.acidilactici ZY15.High solids loading SSCF using the dry acid pretreated and biodetoxified com stover was conducted,D-lactic acid fermentation performance of.acidilactici ZY15-?ackA2\:CGS9114_RS09725 with higher vanillin tolerance was much better than the parental.acidilactici ZY15.Conclusively,to achieve the efficient utilization of all fermentable sugars derived from lignocellulose and decrease the inhibition of phenolic aldehydes on.acidilactici strains.First?the efficient xylose-assimilating pathways were constructed into the R acidilactici strains with the metabolic engineering and adaptive evolution strategy.The resulting strains R acidilactici ZY271(for L-lactic acid production)and.acidilactici ZY15(for D-lactic acid production)can co-utilize glucose,xylose,arabinose,mannose and galactose for homo chiral lactic acid production,and high titer of D-lactic acid and L-lactic acid were obtained from lignocellulose by high solids loading simultaneous saccharification and all fermentable suagrs co-fermentation.Second,the vanillin tolerance of.acidilactici ZY15 was successfully enhanced by genome integration of an exogenous short-chain dehydrogenase gene,and resulted in better D-lactic acid fermentation performance from lignocellulose with phenolic aldehydes stress.This thesis provided important strains for chiral lactic acid fermentation from lignocellulose with efficient utilization on all fermentable sugar and high tolerance on phenolic aldehydes.