Screening Of Genes Involved In The Biosynthesis Of 2-(2-phenylethyl) Chromones And Research On The Function Of Polyketide Synthases In Aquilaria Sinensis (Lour.) Gilg

Agarwood is the fragrant resin-containing wood formed in Aquilaria spp.or Gyrinops spp plants in the Thymelaeaceae family.It has been used in medicines,perfumes,religious and cultural practices,or as handicrafts with a long history.Aquilaria sinensis(Lour.)Gilg is the only source of medicinal agarwood listed in the Chinese Pharmacopoeia.The agarwood formation is occurred only in plants after being injured and is a slow process of the synthesis and accumulation of sesquiterpenes and 2-(2-phenylethyl)chromones(PECs).PECs are the characteristic and main active components of agarwood,which possess a wide range of biological activities,such as neuroprotection,acetylcholinesterase inhibition,anti-inflammatory,and antibacterial activities.Besides the source species of agarwood,only six other plant species have been reported to produce PECs.A.sinensis has two types of germplasm,ordinary and Chi-Nan.The Chi-Nan germplasm has the characteristics of easy agarwood induction and the high PECs content in the produced agarwood,which are beneficial for the researches related to PECs.In recent years,although the biosynthesis pathway of sesquiterpenes in agarwood has been elucidated,the biosynthesis pathway of PECs remains totally unknown,which seriously restricts the comprehensive understanding of agarwood formation mechanisms.PECs,which are derivatives of chromones substituted by 2-phenylethyl,belong to the class of polyketides.The skeleton structure of PECs(C6-C5-C6)is similar to that of flavonoids(C6-C3-C6),suggesting that the biosynthesis pathway of PECs may be similar to that of flavonoids:type Ⅲ polyketide synthase(PKS)catalyze the condensation of intermediate metabolites of phenylpropanoid metabolism and malonyl-CoA to form the PEC skeleton structure.Hence,this study based on previous genome data of A.sinensis and performed a transcriptome-metabolome analysis of the two types of A.sinensis to screen for potential PKS genes that may catalyze the synthesis of PEC skeleton structure and phenylpropanoid metabolism pathway genes involved in PECs biosynthesis.In addition,systematic analysis of AsPKS gene family were conducted to further target the candidate AsPKS genes that were involved in the synthesis of PEC skeleton structure.Then functional identification for the candidate AsPKS genes by reactions with various substrate combinations were carried out not only to identify the key enzymes involved in PEC biosynthesis,but also to explore the functional promiscuity of these enzymes.The main research findings are as follows:(1)The compositions and accumulation patterns of PECs,as well as the gene expression patterns in ordinary A.sinensis(OA)and Chi-Nan A.sinensis(CN)under wound were significantly different.Metabolome analysis revealed that the metabolic processes of CN under wound were more active than those of OA.The compositions and accumulation patterns of PECs showed notable variations between OA and CN,and a considerably higher amount of 2-[2-(4-methoxy-phenyl)ethyl]chromone(PEC33)were found in CN.The full-length transcriptome analysis showed that both germplasms contained a large number of differentially expressed genes(DEGs)at 6 h after wound compared to the healthy samples.However,as time elapsed.the number of DEGs in OA decreased sharply.and the expression of most genes had returned to healthy level by 60 d.while there were still a large number of DEGs in CN at other time points.In particular.most of the injury-induced genes in phenylpropanoid metabolism pathway had higher expression levels in CN.(2)Four PKS genes involved in the synthesis of PEC skeleton structure and 47 phenylpropanoid metabolism pathway genes involved in the biosynthetic pathway of PECs were screened.Weighted gene co-expression network analysis identified four and five gene modules with high correlation with PECs from OA and CN,respectively.Gene annotation results showed that the four modules positively correlated with PECs in OA contained 32 genes involved in the phenylpropanoid metabolism pathway and one PKS gene(AsPKS8),while the five modules in CN contained 36 genes involved in the phenylpropanoid metabolism pathway and three PKS genes(AsPKS3,AsPKS4,and AsPKS5).After merging and removing duplicates,a total of 47 phenylpropanoid metabolism pathway genes related to PEC biosynthesis and four PKSs(AsPKS3,AsPKS4,AsPKS5,and AsPKS8)for PEC skeleton structure synthesis were obtained.(3)A comprehensive analysis of the type Ⅲ PKS gene family in A.sinensis was performed,and seven candidate genes that encode crucial enzymes involved in PEC biosynthesis were finally selected.The AsPKSs were analyzed based on bioinformatic analysis,expression analysis,and subcellular localization analysis.The phylogenetic analysis revealed that the 13 AsPKSs could be classified into two distinctive types.chalcone-producing synthases(CHSs)and nonchalcone-producing synthases(non-CHSs).Among them,AsPKS3.4,5,and 6 were found to cluster together within the non-CHS group,while AsPKS7,8,and 11 were observed to cluster together within the CHS group.The gene promoter regions of AsPKSs contained many defense-related cis-regulatory elements,such as light-responsive,abiotic stress-responsive,and hormone-responsive elements.AsPKSs exhibited different tissue expression patterns and induction expression patterns under Agar-Wit(a whole-tree agarwood induction technique)treatment,suggesting that their potential involvement in various biological processes of A.sinensis.Moreover,AsPKS3-5 were identified as the most auspicious genes that potentially encoded the enzymes for synthesizing the PEC skeleton structure.Additionally,AsPKS3,4,5,6,7,and 11 showed a stronger up-regulating effect compared to other genes under MeJA or SA treatment.Based on this finding and in combination with AsPKS3,4,5,and 8 selected by transcriptome-metabolome analysis,AsPKS3,4,5,6,7,8,and 11 were finally targeted as candidate key enzyme genes for PEC skeleton synthesis.Subcellular localization analysis showed that all proteins encoded by these seven genes alone were localized in the cytoplasm,and moreover,non-CHSs AsPKS3,4,5,and 6 were also localized in the nucleus.(4)The in vitro enzymatic activity investigation revealed that the non-CHS type AsPKS3-6 and CHS type AsPKS7,8,11 could all catalyze the synthesis of PEC precursor(diarylpentanoid)as well as a series of other compounds with diverse structures,including benzalacetones,quinolones,pyrones and diarylheptanoids,and AsPKS7,8,11 also exhibited CHS activity.The seven candidate AsPKSs were each cloned into Escherichia coli to express the fusion proteins.Then the fusion proteins were purified and used for enzymatic reaction in vitro.The products of enzymatic reactions were determined by LC-MS detection.The results showed that all the seven AsPKSs could condense benzoyl-CoA with β-keto acid to produce a diarylpentanoid that is a precursor of PECs.Moreover,all AsPKSs had multifunctions while those belonging to the same branch in the phylogenetic tree showed similar catalytic activities.Non-CHS type AsPKS3-6 could catalyze the generation of benzalacetones or pyrones from various aromatic acylCoAs with malonyl-CoA,quinolones from N-methylanthraniloyl-CoA and malonylCoA/β-keto acid,curcumin from the iterative condensations of feruloyl-CoA and malonylCoA,and diarylheptanoids from different aromatic acyl-CoAs and β-keto acid.Additionally,AsPKS6 could also catalyze the synthesis of CTAL-type pyrone and benzophenone from benzoyl-CoA and malonyl-CoA.CHS type AsPKS7,8,and 11 clustered in another clade could also catalyze the synthesis of structurally diverse benzalacetones,quinolones,benzophenone,and diarylheptanoids.Unlike AsPKS3-6,AsPKS7,8,11 were able to condense different aromatic acyl-CoAs with malonyl-CoA to produce a series of flavonoids and two other types of products(BNY-type pyrones and CTAL-type pyrones)commonly found in the in vitro enzymatic reactions of CHS,showing obvious CHS activity.AsPKS7,8,11 could also condense N-methylanthraniloyl-CoA with three malonyl-CoA units to produce CTALtype alkaloid.On the other hand,AsPKS7,8,11 could not catalyze the condensation of feruloyl-CoA and malonyl-CoA to produce curcumin as AsPKS3-6 did.Amino acid sequence alignment and protein three-dimensional structure analysis revealed that the active-site residues Thr132,Ser133,Thr194,Thr197,Phe265,and Ser338 in Medicago sativa chalcone synthase(MsCHS)were substituted in APKS3-6(non-CHS type)with Ser134,Ala135,Asn196,Asn199/His199,Pro267/Ala267 and Phe340,respectively.As a result,the active-site entrances and adjacent spaces of the AsPKS3-6 were larger than those of MsCHS.In contrast,the active-site residues and active-site architectures of AsPKS7,8,and 11(CHS type)were identical to MsCHS.Additionally,the pH and temperature optima for the p-hydroxybenzalacetone or 4-hydroxy-N-methyl2(1H)-quinolone production of APKS3,AsPKS4,and AsPKS5 were found to be different.Real-time quantitative PCR showed that the expression levels of the seven AsPKSs were significantly up-regulated by salt stress.Based on the findings mentioned above,this study successfully cloned and identified seven novel PKSs that were capable of synthesizing PEC precursor and also exhibited other multiple enzymatic activities.It was the first study which found that the CHS type PKSs(AsPKS7,8 and 11)could catalyze the synthesis of the PEC precursor,as well as the alkaloids in plants.This study not only contributes to the comprehensive elucidation of the biosynthetic pathways of PECs and the molecular mechanism of agarwood formation,but also provides the foundations for the studies in biosynthetic pathways of flavonoids,benzalacetones,and quinolones.It is also helpful for the investigation of catalytic mechanisms of plant type III PKSs.