Metagenomic Mining Novel Lignocellulosic Enzymes And Their Effects On Cellobionic Acid Production

As one major type of lignocellulose biomass,food and agricultural waste represents the most widespread abundant and renewable resource in nature,which is economically valuable and highly under-exploited.The valorization of them into bio-based platform fuel and chemicals has been one of the most important developments to meet high worldwide demand for energy and concerns over global climate change.However,major obstacles impeding the economic applicability of lignocellulosic biorefinery is the absence of efficient lignocellulosic enzymes and economically affordable conversion strategy.Lignocellulosic enzymes mainly include cellulase,hemicellulase,pectinase and ligninolytic enzymes.Especially pectinase,which has long been regarded as a class of unimportant auxiliary enzymes,has been seriously under-estimated.In addition,cellobionic acid(CBA)is a high-value but low-volume organic acid and has numerous promising applications.Therefore,in order to excavate efficient and novel lignocellulosic enzymes as well as reduce the cost of CBA production process,a compost habitat with high lignocellulose-degrading capacity was constructed and its metagenomics analysis was conducted,novel lignocellulosic enzymes were found and expressed,and their effect in CBA production were explored as well as the pretreatment unit in consolidated bioprocessing CBA were investigated.The main innovations and work of the thesis include the following six aspects:1.Establishment of efficient lignocellulosic compost habitat and metagenomics analysisA metagenomic sequence-guided strategy combined with enrichment culture technique was used to targetedly discover the lignocellulolytic microbes and enzymes from an apple pomace-adapted compost microbial community(APACMC).During the 30-day enrichment,the temperature of APACMC reached 68? and underwent four typical stages of compost: initial,mesophilic,thermophilic and maturation.Analyses of 16 S r DNA high-throughput sequencing showed that microbial communities changed dramatically,indicating that the microbes were successfully enriched.Metagenomic sequencing generated 16.2 Gb highquality reads and 272,516 open reding frames was obtained after quality filtering.Taxonomic assignment revealed that APACMC was dominated by Proteobacteria,Bacteroidetes,Actinobacteria and Firmicutes.Analyses of functional classification showed that the major functions were amino acid and carbohydrate metabolism.Additionally,APACMC exhibited highly diverse,abundant and widely distributed genes encoding carbohydrate-active enzymes.2.Metagenomic mining cellulase,hemicellulase and ligninolytic enzymesCongo red agar plate test and(hemi)cellulase activities showed APACMC had high lignocellulose-degrading capacity.Phylogenetic origins of these lignocellulosic enzymes showed that most of them affiliated with organisms from Proteobacteria,Actinobacteria and Bacteroidetes by db CAN.Functional and metabolic annotations revealed a wide diversity of genes related to carbohydrate metabolism and potential biofuel synthesis pathways.Annotations based on CAZy identified 3,882 genes encoding a broad array of enzymes involved in lignocellulose degradation.Furthermore,94 genes encoding laccase and multicopper oxidase were found according to the Lcc ED database.Besides,abundant genes encoding multifunctional enzymes,thermozymes and novel lytic polysaccharide monooxygenases from the bacterial source were also found by catalytic domain analysis.3.Discovery of pectinolytic microbes,enzymes and their isolation,purification and characterizationBoth ruthenium red agar plate test and pectin content test proved APACMC had high pectinolytic activities.Function analysis showed that APACMC harbored a broad spectrum of genes involved in the metabolism of pectin,which accounted for 25.8% of the carbohydrate metabolism.A total of 1,756 entries were identified as encoding pectinolytic enzymes and many of them were identified as novel with low identity to any enzyme.Taxonomic assignment of key pectinolytic enzymes showed that majority of them were originated from the most abundant bacteria of APACMC.In addition,36 strains of pectinolytic thermophiles were isolated from APACMC,among which Bacillus subtilis Z10 showed the highest pectinolytic activity.One 1,263 bp gene encoding pectinase was obtained after fusion primer and nested integrated-PCR.Based on its predications of theoretical p I and molecular weight,one pectate lyase Bs Pel-Z10 was successfully purified by ion exchange and gel filtration chromatography.Biochemical characterization showed that Bs Pel-Z10 was a thermophilic pectate lyase with high stability.4.Heterologous expression and analysis of novel lignocellulolytic enzymes from metagenomeFive full-length genes which share less than 75% identity and belong to AA10,CE15,GH43,laccase and PL11 family were chosen and synthesized after several process such as subtraction of signal sequence.The gene encoding AA10 was seamlessly cloned into p ET-22b(+)expression vector after the downstream of the pel B coding sequence;other genes were cloned into p ET-28a(+)vector by double enzymatic digestion.The successful constructs were transformed into E.coli BL21 and the resulted engineered strains were cultured for enzyme production.All the five recombinant enzymes were successfully expressed,purified and showed corresponding enzymatic activity.In addition,analyses of sequence,phylogenetic tree and tertiary structural showed that all of them exhibited high novelty.5.Synergy effect of lignin,CDH and laccase for cellobionic acid productionWhen using Avicel as the substrate,redox mediator like ABTS was needed for high yield CBA production whereas ABTS was unnecessary when the substrate was switched to wheat straw.The addition of genuine lignin prepared from wheat straw into Neurospora crassa HL10 fermentation system and CDH-laccase cell-free system showed that lignin can promote CBA production in the presence of laccase.Furthermore,lignin has no effect on CDH and laccase activity.Electron paramagnetic resonance spectroscopy revealed that laccase catalyzed lignin to lignin radicals and these radicals can be consumed by reduced CDH,which indicated that lignin can be act as redox mediator in CDH-laccase system and thus promote CBA production.Besides,stopped-flow spectroscopy showed that laccase can accept electron from reduced CDH.6.Consolidated bioprocessing CBA from pretreated wheat straw by engineered N.crassa HL10The consolidated bioprocess of CBA from three pretreated wheat straw,i.e.alkaline,dilute acid and hydrothermal,by engineered strain N.crassa HL10 was compared.Fermentation results showed that alkaline pretreated wheat straw yielded highest CBA concentration(45.722 m M).Furthermore,three lignin were isolated from the solid residue of three pretreated wheat straw and alkaline pretreated wheat straw lignin(AP-WS-L)exhibited the best redox capacity both in N.carassa HL10 fermentation system(Avicel as the substrate)and CDH-laccase cell-free system.Analyses of FTIR,XRD and GPC found that the substrate structure,cellulose crystallinity and molecular weight of these three pretreated wheat straw and their corresponding linin were quite different,which can be accounted for their difference in CBA yield.