Construction Of Cellulase-producing Recombinant Lactococcus Lactis And Its Effect And Mechanism On Straw Silage Quality

The annual output of crop straw in China is huge and the resources are abundant,but the utilization rate is very low.There is a serious shortage of forage in China.How to improve the nutritional quality of straw plays an important role in promoting the sustainable development of grain saving animal husbandry and agriculture.In this study,two Celgene were obtained from Bacillus subtilis and transferred into L.lactis NZ9000 gene engineering bacterium for heterologous expression.The enzymatic characteristics of recombinant enzyme,cellulose degradation mechanism,synergistic effects of two Celand saccharification effects on different straw were analyzed.The effects of COS on the growth and metabolism of L.lactis NZ9000 was clarified,the suitable culture conditions for the production of enzyme by recombinant bacteria was conducted,and the degradation of straw by liquid fermentation of recombinant bacteria under the optimized culture conditions was determinated.The effects of the recombinant bacteria on the nutritional quality,fermentation parameters,straw microstructure and microbial community structure of the straw feed were analyzed by adding recombinant bacteria to micro-storage fermentation of rape straw.Thereby,the optimal addition amount of recombinant bacteria was obtained for making straw silage alone or in combination,and the regulation mechanism of the effects of recombinant bacteria on the quality of straw silage was elucidated.The purpose of this study is to provide some new theoretical and technical basis for promoting the efficient utilization of straws.The experiment was divided into four parts.Experiment 1:This experiment aims to construct lactic acid engineering bacteria producing Cel.In this study,possible cellulase genes CelA1805 and CelA3258 from Bacillus subtilis B111 and B110 strains were selected for BLAST alignment of homologous sequences,domain prediction and homologous modeling.The two genes were cloned and the recombinant L.lactis harboring cellulase gene was constructed.The expression of the recombinant protein was verified by the CMC containing plate,which was expressed in large quantities and purified,laying a foundation for the further study of the enzyme properties and functions of Cel.The results showed that CelA1805 belongs to GH8 family with 76.8%identity and 52.4 KDa molecular weight,CelA3258 belongs to GH5 family and contains CBM3 domain,shared 99.8%homology and with 53.2 KDa molecular weight.Two Celgenes were successfully transferred into the genome of L.lactis NZ9000.The target protein was identified by SDS-PAGE and Western blot in the fermentation supernatant of the expressing strain,and the molecular weight was consistent with the predicted value.Meanwhile,cellulase was further verified by CMC plate.These results indicated that the cellulase-producing recombinant lactobacillus was successfully constructed and could be used for further experiments.Experiment 2:This experiment was conducted to study the enzymatic characteristics,cellulose hydrolysis mechanism and possible synergistic effect of recombinant Cel.The optimal p H and temperature were determined by measuring the enzyme activity under different p H and temperature.The stability of p H and temperature was analyzed by measuring the enzyme activity under different p H and temperature for different time within48 h.Adding different metal ions with 1 m M concentration and different concentrations of chemical reagents to the enzyme reaction system to determine the effects of the enzyme activity.The hydrolysis products of the two enzymes with different degree of cello-oligosaccharides and pure fiber substrates were determined by TLC plates.The synergistic effects of the two enzymes on pure fiber substrates and five kinds of crop straw cellulose were determined,and the saccharification effects by the reaction of the two enzymes with separately or jointly were further confirmed.The results showed that the two enzyme have wide reaction p H and temperature,with good p H stability and thermostability,and widely substrate specificity.CelA1805 is endo-typeβ-1,4-glucanase,CelA3258 is endo and exo difunctional glucanase.The smallest oligosaccharide that can be hydrolyzed by CelA1805and CelA3258 was G₄ and G₃respectively.The two enzymes had synergistic effects on the hydrolysis of pure fiber substrate and straws,compared with single enzyme treatments,the least improvement rate of reducing sugar contents of CMC,PASC,Avicel and filter paper were 24.3%、8.1%、47.3%and85.2%,respectively,the least improvement rate of reducing sugar contents of wheat straw,rape straw,rice straw,peanut straw and corn straw were 48.4%、55.2%、68.7%、39.4%and 5.2%respectively.In conclusion,both cellulases have excellent enzymatic characteristics,which can meet the requirements of most industrial production,especially for silage production.Experiment 3:This experiment was conducted to study the effects of COS on the growth and metabolism of L.lactis,the optimal culture conditions for enzyme-producing of the two recombinant bacterias and the enzymolysis effects of the recombinant bacteria on straws by liquid fermentation with straw as substrate.G,G₂,COS and CMC were selected as carbon sources to analyze the effects of different carbon sources on the OD value and lactic acid yield of L.Lactis NZ9000.The OD value,colony number and enzyme activity of the two recombinant bacteria were analyzed under different culture temperature,p H and time to determine the optimal culture conditions.Two recombinant bacterias were added separately or in combination,with corn stover and rape stover as substrates for liquid fermentation,p H value,DMD and yield of lactic acid and acetic acid were measured,and enzymatic hydrolysis effects were analyzed.The results showed that COS,the two recombinant enzymes hydrolysis product with CMC,could promote the growth of L.lactis NZ9000 and increase the yield of lactic acid,and the promoting effects of cellobiose was similar to that of glucose.The optimum temperature,p H and time for enzyme production were respectively.Under the optimum condition（30℃,7.0 and 24 h）,the enzyme activity of CelA1805 and CelA3258 was 0.146 U/m L and 0.132 U/m L respectively.Fermented with corn stover and rape stover as substrates,the p H values of the recombinant strains were lower than those of L.Lactis NZ9000,and the DMD and lactic acid yields were higher than L.Lactis NZ9000.The effects of compound inoculation were better than that of single inoculation group.When corn stover and rape stover were used as substrates,DMD of compound inoculation group were 37.6%（P<0.05）and 17.4%（P<0.01）higher than that of control group,and lactic acid yield were 16.2%and 23.8%（P<0.01）higher than that of control group respectively.The results showed that the recombinant bacterias produced celluloses,that promote the DMD of staws,on the other hand,the production of COS could promote the growth of L.Lactis NZ9000 and the yield of lactic acid.Experiment 4:In this study,the effects of different adding methods and doses of recombinant bacterias on the quality of rape straw silage were analyzed.The microstructure of rape straw was analyzed,and the dynamic change of microbial community were examined by 16S r DNA high-throughput sequencing technology,so as to clarify the mechanism of recombinant bacteria regulating the quality of rape straw silage.Rape straw was used as fermentation substrate,three treatments supplemented with LC1,LC3 and LC1+3 were conducted.Each treatment was setted with 1×10⁷,1×10⁸ and 1×10⁹ CFU/g of additive amount,natural fermentation group was setted as control.All the treatments were fermented in anaerobic bags for 60 d.According to nutritional quality and fermentation parameters,single agent group,compound agent group and control group with the greatest difference were selected for SEM,FT-IR,XRD and microbial community analysis.The results showed that supplemented with recombinant L.Lactis,the contents of CP,NDF,ADF and CEL were significantly increased,p H value and ammonia concentration were decreased,and lactic acid content was tended to be increased.The additive and fermentation days had extremely significant interaction effects on CP content,lactic acid content and ammonia nitrogen concentration（P<0.01）,and LC1+3×10⁷ group effects best on silage quality among all treatments.The surface of straw fiber of recombinant bacteria group had different degrees of corrosion,skin peeling and tearing,while the surface of straw fiber of control group was relatively smooth and intact.The recombinant bacteria promoted the break of hydroxyl,methyl and methylene in cellulose carbon chain,and significantly reduced the crystallinity of straw fiber,and the crystallinity of the compound bacteria treatment group was the lowest.The dominant taxon distributions enriched in the control group mainly included Proteobacteria,Weissella and Enterobacter,in LC1 group were Enterococcus and Lactobacillales,in LC3 group were Firmicutes、Lactococcus、Prevotela、Pediococcus、Bacteroides,and in LC1+3 group were Agrobacterium、Sphingomonas、Devosia、Stenotrophomonas、Brevibacillus.The abundance of Proteobacteria was positively correlated with NDF,ADF,CEL content and ammonia nitrogen concentration.Enterobacter abundance was negatively correlated with CP content,positively correlated with NDF,ADF and CEL content,ammonia nitrogen concentration and p H value.Enterrococcus abundance was negatively correlated with NDF content and ammonia nitrogen concentration.Lactococcus abundance was significantly positively correlated with CP content,negatively correlated with NDF,CEL content and p H value,and negatively correlated with ADF content and ammonia nitrogen concentration.Pseudomonadaceae＿Pseudomonas and Ralstonia abundance was significantly positively correlated with CP and LA content,negatively correlated with p H value.The results showed that the microsilage fermentation of the recombinant bacteria could change the microstructure of straw by cracking the CH₂,CH and hydrogen bonds in the cellulose carbon chain and reducing the crystallinity of cellulose,and improve the nutritional quality of straw feed by enriching the abundance of the dominant bacteria that improved the silage quality.The above results indicated that the silage fermentation of rape straw by recombinant L.lactis could change the microstructure of straw by cracking the CH₂,CH and hydrogen bonds in cellulose carbon chain and reducing the crystallinity of cellulose.The nutrient quality of straw feed was improved by enriching the abundance of the dominant bacteria which improved the microstorage quality.The microstorage quality of LC1+3×10⁷group was the best,and the fermentation time of 60 days was the best.In conclusion,CelA1805 has typical features of endo-type cellulase,while CelA3258has dual functions of endo-type and exo-type cellulase,both of which have obvious synergistic effects on cellulose degradation.When adding recombinant bacteria for straw silage,cellulase was used to break the molecular structure of cellulose and reduce the crystallinity of straw fiber,thus reducing the content of cellulose.Compound addition can improve the quality of silage by regulating the abundance of dominant microflora related to silage quality.The two recombinant strains could be used as microbial agents to improve straw cellulose degradation efficiency and feed nutritional quality.