Construction Of Recombinant Yarrowia Lipolytica And The Application In Degradation Of Stover

Lignocellulose is a kind of polysaccharide which is the most abundant biomass resources in the world.It widely exists in grains,beans,wheat and their by-products such as poultry feed.In China,over ten million tons of agricultural and processing products are produced every year.However the effective utilization efficiency of these cellulose resources is very low.For example,several million tons of plant straw is unused or directly burned as fuel every year.It is obvious that if we can open up a new protein resource by fermentation of plant straw and turn them into protein feed,it will have a profound impact for our country's feed industry and animal husbandry.Yarrowia liopolytica is a typical unconventional yeast,and generally regard as safe,it can be used in the food and drug production.The available substrates and carbon source which can be used by Yarrowia lipolytica is very wide range.Yarrowia lipolytica also has a strong secretory expression ability and without pathogenic,these characteristics makes it an ideal exogenous gene yeast expression system.The purpose of this study is to construct recombinant Yarrowia lipolytica strains that can express cellulose enzymes,used in the fermentation of plant straw.By degradation of cellulose in the straw,the content of single cell protein have increased Through this way we can increase the utilization efficiency of straw,and expand the application scope of plant straw in feed industry.Experimental process and conclusions of this study are as follows:1.Insert cellulose enzyme gene into pINA1297 plasmid vector which can express secretory proteins in Yarrowia lipolytica.There are three kinds of cellulose enzyme,including?-glucosidase(bg,Genbank:AF163097)comes form Dalbergia cochinchinensis,endoglucanase(eg,Genbank:EU169241)form synthetic and cellobiohydrolase(cbh,Genbank:AY861348)comes form Chaetomium thermophilum.PCR and restriction enzyme digestion was used to confirm the correctness of reconstructed plasmids.Finally,we get three reconstructed plasmids named pINA1297-BG,pjINA1297-EG and pINA1297-CBH,respectively?2.The control(empty piNA1297 vector)and three newly constructed plasmids were linearized by Not I restriction enzyme,and transformed into the genetically modified Yarrowia lipolytica strain,Polh.Transformation of reconstructed plasmids to Yarrowia lipolytica was performed using the lithium acetate chemical transformation method.The selective medium and colony PCR was first used to preliminary screen transformants.Than culture transformants,measure the enzyme activity change,the best transformants for the subsequent experiment was choosen by enzyme activity evaluate.In our experiments,the choosen recombinant strains are:Polh-1297-BG,Polh-1297-CBH and Polh-1297-EG.After fermentation for 13 days,1 mL crude enzyme culture were taken to measure the enzymatic activity of cellulose enzyme expressed by Yarrowia lipolytica with the DNS method at 50?,pH5.0.The enzymatic activity of three recombinant strains are 14.181U/mL,16.307U/mL,17.391U/mL respectively.3.Three recombinant strains were cultured in the same environment,equivalent volumes of three recombinant strains bacteria culture solution were mixed.In a sterile environment,mix the bacteria with substrates and ferment at 28 ?;sampling at the tenth and fifteenth day,measure the protein content by kjeldahl determination.After fermentation,the crude protein content in the cell culture was increased,after 15 days fermentation,the crude protein content in corn straw and rice straw are 16.23%and 14.75%,increased by 168.26%and 161.52%compared with their control group,respectively.Research conclusion:Three cellulose enzymes were successfully expressed in Yarrowia lipolytica.The enzymatic activity of three recombinant strains respectively are 14.181U/mL,16.307U/mL and 17.391U/mL.The mixed reconstructed Yarrowia lipolytica can directly use crop strover(corn straw and wheat straw)for effective bioconversion.The crude protein content increased significantly in straws,After 15 days' fermentation the protein content reached 16.23%and 14.75%,increased by 168.26%and 161.52%respectively.The further research should be done are as follow:1.The enzymatic activity of three recombinant strains is not high.Follow-up study should select highly activity recombinant strains from the angle of high copy expression and function screening;2.This study use the shallow tray solid-state fermentation method for bioconversion.This method is adverse to the cellulose enzyme's work for straw substrate,and may affect the efficiency of biological transformation.Follow-ups should attempt to use half solid or liquid fermentation method,and optimize the fermentation conditions,expect to further improve the protein content after straw translate.