Genome Analyses And The Analysis Of Pinoresinol And Its Glucoside Derivatives Biosynthesis Pathway In Phomopsis Sp

Pinoresinol[?+?-pinoresinol]?Pin?anditsglycosidederivatives,?+?-pinoresinol-4-O-?-D glucopyranoside?POG?and Pinoresinol diglucoside[?+?-1-pinoresinol 4,4?-di-O-?-D-glucopyranoside]?PDG?are belong to furofurans lignans.They have multiple functions including antifungi,antitumor,and hypoglycemic action,but low in production in nature.Microbial biosynthesis of these compounds has great advantages over plants in aspects of short time,high efficiency and no season limitation.However,the key steps and enzymes for the biosynthesis of Pin and its glucoside derivatives,especially those involved in the glycosylation of Pin are rarely reported.In this paper,Phomopsis sp.XP-8,an endophytic fungus having capability to produce Pin and its glucoside derivatives was studied.In order to reveal the biosynthesis pathway for pin and its glucoside derivatives,especially the lignan biosynthesis pathway,genome and transcriptome analysis was used to carry out the study at gene levels;then,substrate-feeding and the addition of specific inhibitors was used to detect the mass flow in the pathway;Finally,isolation,purification,and characteristics of key enzymes and cloning of key genes were used to determine the key enzymes and the corresponding coding genes for the glycosylation of Pin.Finally,the Pin and its glucoside derivatives biosynthesis pathways were constructed and the key genes were confirmed.Main results are listed as the follows:?1?Genomic analysis revealed the diversity of metabolic pathway of Phomopsis sp.XP-8.The genome of Phomopsis sp.XP-8 was sequenced,assembled and annotated.As results,the genome size was obtained as 55.2 Mb,with the GC content of 53.5%and repeatitive sequence of 0.83%.The genome contained 17,094 protein-encoding genes and310 non-coding genes.Among them,16,025 genes were annotated as protein-encoding function in different databases,accounting for 93.75%of the total annotation protein genes.OrthoMCL homologous gene analysis showed the strain had 11,818 gene families,of which64 belong to unique family.A total of 1,140 unique genes,163 genes belong to the unique family.A total of 80 secondary metabolite gene clusters were detected.The genome exhibited tremendous Carbohydrate-Active Enzymes?CAZys?involved in carbohydrate metabolism,Pin and its glucoside derivatives synthesis pathways in the CAZymes Analysis Toolkit?CAT?.?2?Transcriptome analysis revealed the Pin and its glucoside derivatives biosynthesis pathway.The cDNA library was sequenced by the Illumina HiSeq2500 high throughput sequencing platform.Based on the de novo and reference genome assembly,the transcripts and the expression data of key genes were obtained.4.10 Gb clean data were obtained,and the Q30 base percentage was not less than 85.02%.Comparison to the reference genome,the annotation efficiency was 88.6 4%and 302 new genes were found.Of all predicted genes,13,557 and 16,111 genes were successfully annotated in function using the Nr,Swiss-Prot,GO,COGandKEGGdatabasesthroughthenoReference-Transcript and Reference-Transcript methods.Reference-Transcript resulted more annotation information than the noReference-Transcript method.A total of 2,921 were annotated in 117 pathways by searching the KEGG database.The transcriptomic mapped database showed the Pin biosynthesis may via the pathways of glycolysis,pentose phosphate pathway,phenylalanine biosynthesis,and phenylpropanoid biosynthesis.A total of 180 genes encoding 40 enzymes were annotated in the pinoresinol and its derivatives biosynthesis pathway.The glycosyltransferase genes that may be invovled in the biosynthesis of Pin glucoside derivatives were screened.?3?Enzyme inhibitor and precursor addition analysis indicated Pin and its derivaties biosynthesis pathway.The specific inhibitor of polyketide pathway-cerulenin and iodoacetamide had no effects,whereas,the addition of shikimate pathway inhibitor trimethylamine and EDTA strongly inhibited the PDG and Pin production.Precursors and intermediates of shikimate pathway and phenylpropanoid biosynthesis pathway could effectively improve the production of PDG and Pin.These results indicated the Pin and PDG biosynthesis pathway may involve the the shikimate and phenylpropanoid biosynthesis pathway,but not the polyketide pathway.?4?Isolation of the glycosyltransferase catalyzing the glycosylation of pinoresinol.The ammoniumsulfateprecipitation,DEAE-SepharoseFastFlowanion-exchange chromatography and Sephadex G-100-gel filtration were used in order to isolate and purify the enzyme.The molecular weight of the obtained enzyme was estimated to be about 49.5 kDa according to the results of SDS-PAGE gel electrophoresis.Product of the enzymes was identified as POG according to the mass spectrometry analysis.It was indicated that the purified enzyme?UGT?had the activity to convert glucose and Pin to POG.Three UGT genes were screened out according to the genome analysis and successfully cloned.The bioinformatics analysis showed these three UGT were all belonged to the GTB superfamily that had typical Rossmann foldswith lots of?-helix,about 46⁴9%in the N-terminal and C-terminal.The amino acid sequence of GT-4 shared high homology??75%?,but GT-1 and GT-2 shared low homology with other reported UGTs.The prokaryotic expression of GT-4was successfully constructed using pET28as and Escherichia coli BL21?DE3?.The expressed product was 50 kDa,consistent with the results obtained by protein purification.?5??-glucosidase with activities towards Pin glucoside derivatives.Purification and characterization of the intracellular?-D-glycosidase inside cells were carried out.Successive steps of 70%ammonium sulfate precipitation,Hitrap^Tm Butyl FF-Hydrophobic chromatography,and Superdex^Tm G200-gel filtration chromatography were applied to isolate and purify the?-glycosidases that have activities towards Pin glucoside derivates.Finally,a protein of 93.4 k Da was obtained according to the SDS-PAGE analysis and identified as?-D-glucosidase by MALDI-TOF-TOF.The gene was predicted as Gglean007197.1 in the genome of Phomopsis sp.XP-8.The enzyme was purified by 30.71 folds with an activity recovery rate of 17.04%,and the specific activity of 75.86 U/mg.With pNPG as the substrate,the?-glucosidase showed the optimal reaction temperature and pH value of 70?and 5.0,and high stability at 30-70?and pH 3.0-7.0.It was identified as a thermophilic?-glucosidase.Meanwhile,metal ions Na⁺and Mg²⁺showed promotion,while K⁺,Cu²⁺,and Li⁺showed inhibition on the enzyme activity.Mn²⁺,Zn²⁺,and Fe²⁺showed faint inhibition on the enzyme activity.The enzyme showed hydrolytic activity towards a wide range of?-1,4and?-1,2 linked glycosidic bonds,but not towards?-1,4 and?-1,1 bonds,with pNPG and cellobiose as the optimal substrates.The enzyme also had a high activity towards aromatic compounds with?-1,4 glycosidic bonds.More important,the enzyme had capability to catelyze the deoxygenation reaction of POG and PDG,indicating it was related to the reverse reaction of the biosynthesis of Pin glucoside derivatives.