Discovery And Functional Identification Of Three Novel Genes Involved In The Biosynthesis Of Tropane Alkaloids

Natural products are one of the most important sources for pharmaceuticals that play vital roles in curing diseases,maintaining health and improving life quality.Tropane alkaloids?TAs?are a particular group of alkaloids containing a unique tropane skeleton.Of them,hyoscyamine and scopolamine are the representedative anticholinesterase agents,which are clinically used for anesthesia before surgical operation,easing pain,relieving cough and asthma,curing motion sickness,treating pesticide poisoning and etc.Some medicinal plants of the Solanaceae,such as Atropa belladonna,Datura species and Duboisia×hybrid,are the main sources for commercially producing the pharmaceutical TAs.Atropa belladonna,listed in Chinese Pharmacopoeia and many other countries'Pharmacopoeia,is the most important plant widely cultivated for commercially producing TAs.Unfortunately,the levels of hyoscyamine and scopolamine are very low in the leaves of A.belladonna,approximately 0.2%dry weight?DW?for hyoscyamine and 0.02%DW for scopolamine.The market demand for hyoscyamine and scopolamine is huge,while their production is limited,resulting the shortage of pharmaceutical resources.Metabolic engineering and synthetic biology are considered to be the efficient approaches to solve the difficulties on pharmaceutical resource shortage.But,either metabolic engineering or synthetic biology is absolutely dependent on identification of biosynthetic genes or their encoded enzymes.To date,nine TAs biosynthesis genes have been functionally identified,but a few TAs biosynthesis genes remain to be discovered,which are related with the following enzymatic reactions,including the reduction of phenylpyruvate,the activation of phenyllactate,the formation of littorine and the reduction of hyoscyamine aldehyde.In the present study,three novel genes involved in TAs biosynthesis,including phenylpyruvate reductase?PPR?,phenyllactate UDP-glycosyltransferase?PLA-UGT?and littorine synthase?LS?,are discovered and functionally identified from A.belladonna,using the integrated methods of molecular biology,bioinformatics,biochemistry and plant biotechnology.The main findings of this study are as follows.1.Cloning and functional identification of phenylpyruvate reductase?PPR??1?Cloning and bioinformatics analysis of PPR.In the transcriptomes of A.belladonna,16 unigenes,belonging to glycerate dehydrogenase?GDH?family,were identified using hmmsearch program.Tissue profiles of the 16 GDH unigenes and all the characterized TAs biosynthesis genes were analyzed,resulting in a sole candidate highly expressed in secondary roots,which was designated PPR.PPR,microbial PPRs and plant hydroxyphenylpyruvate reductases?HPPR?had a common ancestor belonging to NADP?H?-dependent dehydrogenase.However,the sequence of PPR was highly different from those of microbial PPRs and plant HPPRs at the amino acid levels.?2?Enzymatic assays of PPR and its kinetics.6×His-tagged recombinant PPR was purified engineered E.coli and used for enzymatic assays.Biochemical results indicated that PPR was able to reduce phenylpyruvate?PPA?to form phenyllactate?PLA?,and also reduce p-hydroxyphenylpyruvate?p-HPPA?to produce p-hydroxyphenyllactate?p-HPLA?.The PPR Km values for PPA and p-HPPA were 2.77±0.39 mM and 2.25±0.34 mM,respectively.The similar Km values suggested that PPR has very similar affinity with the two substrates,PPA and p-HPPA.The PPR Kcat/Km values for PPA and p-HPPA were respectively 108.30±4.33S^-1·M^-1 and 12.88±0.44 S^-1·M^-1,implying that the efficiency of PPR-mediated reduction of PPA was 8.41 fold of its efficiency in reduction of p-HPPA.These data indicated that the main function of PPR was to reduce PPA to form PLA.?3?Expression analysis of PPR in different organs/tissues.The expression levels of PPR were analyzed in secondary roots,primary roots,stems and leaves of A.belladonna,and the results showed that PPR was specifically expressed in roots,with much a much higher level in secondary roots than in primary roots.Tissue profile of PPR was similar to those of the previously characterized TAs biosynthesis genes.To investigate the expression of PPR in secondary roots of A.belladonna,its 1195-bp promoter?pPPR?was cloned and used to drive the expression GUS gene in transgenic hairy roots of A.belladonna.The GUS-stained cross-section of hairy roots,with pPPR::GUS transformation,indicated that pPPR was specifically expressed in pericycle and endodermis.Cell-type specific expression of pPPR was highly similar with that of the putrescine N-methyltransferase gene?PMT?and hyoscyamine 6?-hydroxylase?H6H?gene of A.belladonna.?4?Metabolite analysis in A.belladonna hairy roots with PPR suppressed by RNAi.To further study the role of PPR in TAs biosynthesis,transgenic hairy root cultures of A.belladonna were developed,in which PPR was suppressed using RNAi technology.Molecular detection showed that the expression levels of PPR in PPR-RNAi hairy root lines were 18.74%to 41.92%of that in control hairy roots.The PLA contents in all the PPR-RNAi hairy root lines were greatly reduced,showing 0.48%to 6.41%of the PLA content in control hairy roots.When PPR was suppressed,the contents of hyoscyamine,anisodamine and scopolamine were also significantly reduced,compared with those in control hairy roots.The hyoscyamine contents in PPR-RNAi hairy root lines were 0.49%to 9.14%of control.The anisodamine contents in PPR-RNAi hairy root lines were 2.29%to 21.56%of control.The scopolamine contents in PPR-RNAi hairy root lines were 4.51%to 40.60%of control.These data indicated that suppression of PPR substantially disrupted the biosynthesis of PLA and the TAs,suggesting the involvement of PPR in the TAs biosynthesis.2.Cloning and functional identification of phenyllactate UDP-glycosyltransferase?PLA-UGT??1?Cloning and bioinformatics analysis of PLA-UGT.102 UGT unigenes were identified from A.belladonna transcriptomes.Tissue profiles of the 102 UGT unigenes and all the characterized TAs biosynthesis genes were analyzed,resulting in 5 candidates highly expressed in secondary roots.Of the five UGT unigenes,aba_locus₁9485 was clustered with AT4G15480.1 and AT3G21560.1,of which were identified to catalyze the esterification between phenylpropanoids and UDP-glucose.Phylogenetic analysis suggested that aba_locus₁9485 might have similar biochemical functions to the two Arabidopsis UGTs.Therefore,it was postulated that aba_locus₁9485 might be PLA-UGT.?2?Expression analysis of PLA-UGT in different organs/tissues.Tissue profile of the PLA-UGT gene was highly similar to those of PPR and all the previously characterized TAs biosynthesis gene from A.belladonna,with specific expression in roots and with a much higher level in secondary roots than in primary roots.To study the PLA-UGT gene expression in secondary roots of A.belladonna,its 1329-bp promoter?pPLA-UGT?was cloned and used to drive the expression GUS gene in transgenic hairy roots of A.belladonna.The GUS-stained cross-section of hairy roots,genetically transformed with pPLA-UGT::GUS,indicated that pPLA-UGT expressed in pericycle and endodermis.?3?Enzymatic assays of PLA-UGT.6×His-tagged recombinant PLA-UGT proteins were purified from engineered E.coli and used for enzymatic assays.Because authentic phenyllactylglucose is not available,high-resolution mass spectrum was used to identify metabolites.When PLA and UDP-glycose were fed to purified PLA-UGT,a new product was detected at the retention time of 3.81 min,and its m/z value was 327.1084,the same as the theoretical m/z value of phenyllactylglucose ion in negative ion form.The enzymatic assay results indicated that PLA-UGT was able to catalyze the formation of phenyllactylglucose using PLA and UDP-glucose.?4?Effects of PLA-UGT suppression on the production and accumulation of related metabolites.To study the role of PLA-UGT in the TAs biosynthesis,the PLA-UGT gene was silenced using the virus induced gene silencing?VIGS?method in seedlings of A.belladonna.The expression level of PLA-UGT was 9.89%of control in the VIGS secondary roots of A.belladonna.The hyoscyamine content in the PLA-UGT-silencing plants was 30.88%of control.Gene silencing suggested that PLA-UGT might be involved in the TAs biosynthesis.To further confirm the involvement of PLA-UGT in the TAs biosynthesis,transgenic hairy root lines of A.belladonna,with PLA-UGT suppression using RNAi,were developed.Molecular detection showed that the transcript levels of PLA-UGT in RNAi hairy root lines were 7.55%to 11.99%of control.The contents of the TAs,including littorine,hyoscyamine,anisodamine and scopolamine,were detected using mass spectrum.Suppression of PLA-UGT significantly reduced the contents of the four types of TAs in hairy roots.The littorine contents in PLA-UGT-RNAi hairy root lines were 7.12%to 21.50%of control,the hyoscyamine contents in PLA-UGT-RNAi hairy root lines were 3.65%to 14.29%of control,the anisodamine contents in PLA-UGT-RNAi hairy root lines were 7.20%to 35.20%of control and the scopolamine contents in PLA-UGT-RNAi hairy root lines were 6.02%to25.30%of control.The accumulation of tropine reached higher levels in PLA-UGT-RNAi hairy root lines than that in control,tropine contents were 2.33-3.03 times of control.These results indicated that suppression of PLA-UGT significantly reduced the production of TAs,suggested that PLA-UGT participated in the TAs biosynthesis.3.Cloning and functional identification of littorine synthase?LS??1?Cloning and bioinformatic analysis of LS.33 serine carboxypeptidase unigenes were identified from A.belladonna transcriptomes.Tissue profiles of the 33 serine carboxypeptidase unigenes and all the characterized TAs biosynthesis genes were analyzed,resulting in aba_locus₁7884 as the sole candidates for further study.Phylogenetic analysis showed that aba_locus₁7884 was clustered with AtSAT,AtSCT,AtSMT,AtSST,BnSCT1,BnSCT2 and CtAT1.All of the these genes,belonging to serine peptidase-like acyltransferase?SCPL-AT?family,were identified to catalyze the condensation between glycosylated phenylpropanoids and acyl acceptors through esterification.It was predicted that aba_locus₁7884 might have the catalytic function of esterification and designated littorine synthase?LS?.?2?Expression analysis of LS in different organs/tissues.The LS was also specifically expressed in roots of A.belladonna,with a much higher level in secondary roots than in primary roots.To study the LS gene expression in secondary roots of A.belladonna,its1629-bp promoter?pLS?was cloned and used to drive the expression GUS gene in transgenic hairy roots of A.belladonna.The GUS-stained cross-section of hairy roots,genetically transformed with pLS::GUS,indicated that pLS was highly expressed in endodermis and pericycle.?3?Effects of LS suppression on the production and accumulation of related metabolites.To unveil the LS role in the TAs biosynthesis,the LS gene was silenced using VIGS in A.belladonna seedlings.The LS expression level was 19.95%of control in the VIGS secondary roots.The hyoscyamine content in the LS-silencing plants was 30.04%of control.Gene silencing suggested that LS might participated in the TAs biosynthesis.To further study the involvement of LS in the TAs biosynthesis,transgenic hairy root lines of A.belladonna,with LS suppression using RNAi,were developed.Molecular detection showed that the transcript levels of LS in RNAi hairy root lines were 3.79%to 20.35%of control.Suppression of LS markedly reduced the production of littorine,hyoscyamine,anisodamine and scopolamine in hairy root lines of A.belladonna.Especially,the production of the four types of TAs were detected with trace amounts in two LS-RNAi root lines,including ILS-1and ILS-5.Metabolite detection also showed that suppression of LS significantly led to the accumulation of the TAs precursors,including tropine and phenyllactate,at significantly higher levels,compared with control.These date showed that suppression of LS substantially disrupted the TAs biosynthesis and suggested that LS was involved in the TAs biosynthesis.?4?Functional identification of LS using synthetic biology methods.LS belonged to SCPL-AT family.Previous studies indicated that SCPL-ATs with post translational processes had catalytic activities.When LS fused an HA tag at its C-terminal was ectopically expressed in tobacco leaves,western blot detection indicated that LS was processed into different peptides.The western blot detection result was one of the major reasons to functionally identify LS using synthetic biology methods in tobacco,and the other reason was that authentic phenyllactylglucose was not available.Littorine biosynthesis was performed in tobacco by transiently co-expressing PLA-UGT and LS,with injection of tropine and phenyllactate.When LS was solely expressed in tobacco leaves,littorine was detected in trace amounts;when LS and PLA-UGT in combination was co-expressed in tobacco leaves,the littorine production was detected in much higher levels,nearly 8.5 folds of solely expressing LS.When YFP?negative control?or PLA-UGT was solely expressed in tobacco leaves,no littorine was detected.These data indicated that the function of LS was to produce littorine.In summary,three novel genes involved in the TAs biosynthesis,including PPR,PLA-UGT and LS,were functionally identified from A.belladonna.The three TAs genes had similar tissue profiles,with specific or high expression in secondary roots.PPR,PLA-UGT and LS was highly expressed in pericycle and endodermis.PPR was shown to efficiently catalyze the reduction of PPA to form PLA,PLA-UGT was able to catalyze the formation of phenyllactylglucose using PLA and UDP-glucose,and the LS function was to produce littorine.Separate suppression of PPR,PLA-UGT and LS significantly reduced the TAs production.The discoveries and functional identification of PPR,PLA-UGT and LS not only led to an insight into the three enzymatic reactions of the TAs biosynthesis,but also provided novel genes for producing pharmaceutical TAs using metabolic engineering and synthetic biology approaches.