Catalytic Mechanism Of Hyoscyamine Dehydrogenase And Metabolic Engineering Of Hyoscyamine Production

Hyoscyamine is a representative natural medicine with anticholinergic activity,which is produced in medicinal plants of Solanaceae.The racemic mixture of hyoscyamine is atropine,a famous medicine.Both hyoscyamine and atropine belong to tropane alkaloids(TA).Clinically,hyoscyamine is used in the treatment of various disorders,such as gastrological disorders,analgesic spasmolysis,epilepsy and seasickness,etc.Medicinal plants of Solanaceae,such as Atropa belladonna,Datura stramonium,Anisodus acutangulus and Duboisia�hybrid,are the main natural resources for commercial production of hyoscyamine.Of these TA-producing medicinal plants,A.belladonna is the most widely cultivated plant to industrially produce hyoscyamine,which is listed in Pharmacopoeia of the People's Republic of China.The medicinal plants of Solanaceae are the sole source for industrial production of hyoscyamine,but the low content of hyoscyamine in these plants limits its supply and makes its price high.Therefore,it is of scientific importance to study hyoscyamine biosynthesis,identify hyoscyamine-synthesizing key enzymes and related genes,and elucidate the catalytic mechanism of the key enzymes.Such studies facilitate extending knowledge on TA biosynthesis.Furthermore,it is economically valuable to develop varieties of A.belladonna with high-yield hyoscyamine by using metabolic engineering technology,which would facilitate reducing the cost of hyoscyamine production and relieving the shortage of medicine supply.According to the reported studies,the scientists postulated that hyoscyamine aldehyde might be the direct precursor of hyoscyamine biosynthesis.Hyoscyamine aldehyde might be reduced to form hyoscyamine by a specific alcohol dehydrogenase,and this unknown enzyme might be a key enzyme to promote hyoscyamine production.Besides,it has been reported that hyoscyamine 6?-hydroxylase(H6H)catalyzes hyoscyamine to produce anisodamine and subsequently converts anisodamine to scopolamine through epoxidation.So,blocking H6H-catalyzed reactions might lead to higher accumulation of hyoscyamine.In this study,a novel alcohol dehydrogenase gene(named Hyoscyamine dehydrogenase,HDH)highly expressed in secondary roots of A.belladonna was functionally identified,and the catalytic activity and mechanism of HDH was studied in detail.Furthermore,the metabolic role of HDH was investigated in A.belladonna,and the Ab H6H gene was knocked out in A.belladonna to investigate its effect on TA biosynthesis.Based on the studies in this thesis,the results are listed below:1.Cloning of HDH gene and analysis of its expression patternThrough analyzing gene expression patterns involved in TA biosynthetic pathway,a candidate EST sequence of the putative HDH gene was screened from the transcriptomes of A.belladonna,and then its 1098 bp full-length c DNA was isolated using rapid amplification of c DNA ends(RACE)technology.Bioinformatic analysis showed that HDH belongs to the oxidoreductase family,encoding a 366-amino-acid polypeptide with a calculated molecular weight of 39 k Da.The heatmap of gene expression analysis showed that the tissue expression pattern of HDH is highly similar to other known TA biosynthetic genes,which is highly expressed in secondary roots of A.belladonna.Then,gene expression analysis by real-time quantitative PCR(q PCR)showed that HDH is specifically expressed in roots,with much higher expression level in secondary roots than in primary roots.To further investigate the expression pattern of HDH in secondary roots,a 1493 bp promoter region of HDH(p HDH)was isolated and used to drive the expression of the GUS reporter gene in hairy roots of A.belladonna.The GUS-stained results indicated that robust GUS signal was detected in pericycle and endodermis,and the cell expression patter of HDH is similar with the reported studies on pericycle-specific expression of PPAR and H6H,two TA biosynthesis genes.The above results suggested that the HDH gene might be a dehydrogenase gene in hyoscyamine biosynthesis.2.Study on the catalytic function of HDHTo study the catalytic activity of HDH,6�His-HDH recombinant protein was produced and purified from engineered E.coli,whose molecular weight is approximately42.9 k Da,consistent with its calculated one.Hyoscyamine aldehyde,the substrate of HDH,is extremely unstable,prone to Retro-Clainsen condensation.So,it is not possible to make hyoscyamine aldehyde in the lab or purchase commercial hyoscyamine aldehyde standard.A successive reaction method was used to validate the function of HDH in the reduction of hyoscyamine aldehyde.Firstly,littorine mutase(CYP80F1)was expressed in engineered yeast and microsomes with HDH enzyme was isolated;when littorine was added in microsomes with HDH,hyoscyamine aldehyde(m/z 288.1584)was produced and detected;then,after the purified HDH was added into the reaction system containing hyoscyamine aldehyde,hyoscyamine was detected.In control reaction,no hyoscyamine was detected.These enzymatic assays indicated that HDH is able to reduce hyoscyamine aldehyde to form hyoscyamine.In order to further study catalytic features of HDH as a reductase,phenylacetaldehyde,with a similar structure to hyoscyamine aldehyde,was used as the substrate to study the enzymatic kinetics of HDH.The maximum reduction activity of HDH was detected under p H 6.4 and 40�C.Under the optimal conditions,the HDH values of K_m,k_cat,k_cat/K_m for phenylacetaldehyde are 44.46�10.73?M,228.4�21.39 min^-1 and 5.14 min^-1??M^-1 respectively.Because HDH belongs to the oxidoreductase family,it might theoretically oxidize hyoscyamine to generate hyoscyamine aldehyde in certain condition.When hyoscyamine was added in the reaction systems containing purified HDH,hyoscyamine aldehyde was detected.The maximum activity of HDH as an oxidase was detected under p H 10.4 and at 45�C.Under optimal conditions,the HDH values of K_m,k_cat,k_cat/K_m for hyoscyamine are respectively 33.36�4.69?M,7.49�0.51min^-1 and 0.22 min^-1??M^-1.By comparing the k_cat/K_m values for HDH-mediated reduction and oxidation reactions,we found the reduction efficiency of HDH is 23.36 folds of its oxidation efficiency.The measurement of equilibrium constant for HDH oxidizing hyoscyamine indicated the oxidative activity of HDH is quite low under p H 7.2(physiological p H in plant cells).These results implied that HDH mainly functions as a reductase under physiological p H conditions in plant cells.3.Study on the catalytic mechanism of hyoscyamine dehydrogenaseIn order to study the catalytic mechanism of HDH reducing hyoscyamine aldehyde to hyoscyamine,the crystal structure of HDH with the resolution of 2.4?was obtained by structural biological method in this paper.The crystal structure analysis of HDH revealed that HDH is a Zn²⁺-dependent long chain dehydrogenase containing two domains,including one NADP(H)binding domain composed of Rossman folding and one substrate binding domain.The HDH catalytic activity site contains a tetrahedral structure composed of zinc ions,Cys52,His74,Glu75 and Cys168.Three non-polar amino acids,including Met124,Leu282 and Ala305,form the substrate binding pocket by shape-dependent van der Waals force control.Two polar amino acids,including Ser54 and Cys100,form hydrogen bonds with the carbonyl group of the substrate,which are the key residues for catalyzing the reduction of hyoscyamine aldehyde.The function of the key residues was further analyzed by site mutation.HDH lost its catalytic activity against hyoscyamine and phenylacetaldehyde after the site mutation of H74A.The activity of S54A mutant protein to reduce phenylacetaldehyde disappeared,and its oxidation activity was significantly decreased.The three mutations C100G/Y/F retained very low catalytic activity.The results of HDH site mutation showed that C100 may form the hydrogen bond with the carbonyl group of the substrate,thus stabilizing the orientation of the substrate.Ser54 may be involved in proton transfer during substrate reduction.The source of hydrogen for reducing aldehyde was studied by using[4-²H]NADPH labeled with deuterium.Only when[4R-²H]NADPH was used as the cofactor,the deuterium labeled reduction products could be detected.These experimental results showed that the pro-R hydrogen atoms in NADPH participated in the reduction reaction catalyzed by HDH.4.Study on the metabolic function of overexpressed scopolamine dehydrogenaseIn order to study the metabolic function of HDH in hyoscyamine biosynthesis,the hair roots of A.belladonna overexpressing HDH were obtained by genetic engineering technology.The hair roots were detected by PCR to distinguish between the control and HDH transformants.The 423-bp rol B gene fragment,795-bp kanamycin resistance gene(NPTII)fragment and 1184-bp 35S::HDH gene fragment were detected in the engineered strains C58C1(p Ri A4,p BI121-HDH),and these gene fragments were also detected in the transgenic hair roots of HDH.In the control Agrobacterium tumefaciens C58C1(p Ri A4,PBI121),the 423-bp rol B gene fragment and the 795-bp NPTII fragment were detected,but the 1184-bp 35S::HDH gene fragment was not detected.In the hair roots transformed with PBI121 as the control plasmid,the corresponding fragments of rol B and NPTII were detected,but the 35S::HDH fragment was not detected.The results of gene expression analysis showed that the expression of HDH in the HDH-transgenic hair roots was significantly increased compared with the control.The results of molecular analysis showed that the HDH gene was integrated into the genome of A.belladonna,with its expression level greatly increased,indicating that the HDH-mediated catalytic step was enhanced.In order to analyze the effect of HDH overexpression on TA synthesis in A.belladonna,LC-MS was used to detect TA content in hair root culture of A.belladonna.The results showed that the contents of hyoscyamine,anisodamine and scopolamine in control hair roots were 1.35 mg/g dry weight(DW),0.69 mg/g DW and 0.43 mg/g DW,respectively.The contents of hyoscyamine,anisodamine and scopolamine in different HDH-overexpression hair root lines were 1.83?4.03 mg/g DW,1.16?1.73 mg/g DW and 0.62?1.28 mg/g DW,respectively.These results showed that HDH overexpression was able to effectively promote the biosynthesis of hyoscyamine,anisodamine and scopolamine in A.belladonna hair roots.Although hair root culture is a powerful tool in metabolic engineering research,it is difficult to realize commercial application due to its immature large-scale culture technology.In this study,Agrobacterium EHA105 mediated genetic transformation method was further used to cultivate A.belladonna plants overexpressing HDH.The transgenic A.belladonna lines overexpressing HDH was identified by molecular detection.The level of hyoscyamine was significantly increased in HDH-overexpressing plants of A.belladonna.The results indicate that HDH is an important gene that can effectively increase hyoscyamine content in A.belladonna plants.5.Study on metabolic engineering for promoting hyoscyamine production by knocking out hyoscyamine 6?-hydroxylase geneH6H catalyzes 6?-hydroxylation of hyoscyamine to generate anisodamine and subsequently converts anisodamine to form scopolamine through an epoxidation reaction.Therefore,knocking out H6H gene might lead to the accumulation of hyoscyamine.In this study,H6H gene was knocked out using the CRISPR/Cas9 technology in A.belladonna,and the effect of H6H knock-out on the content of TA was analyzed.A guide RNA(g RNA)sequence,5'-TGGATTACCAGAAAAGCTGATGG-3',specifically targeted to the second exon of HDH,was designed and constructed in the plant expression vector,named p Cas9-H6H.In this vector,g RNA expression was driven by the U6 promoter of Arabidopsis thaliana;the expression of Cas9 enzyme from Streptococcus pyogenes was driven by the Ca MV 35S promoter.Agrobacterium EHA105 harboring p Cas9-H6H was used to genetically transform A.belladonna plants.Molecular detection indicated that there were 11 transgenic authentic plants,out of 15 generated plants.The assay of H6H gene mutation sites showed that 4 plants had not genome editing,and 7 had genome editing among the 11 transformants.So the mutation rate is 63.6%.H9,H14 and H15 had homozygous mutation in H6H;H1 had biallelic mutation in H6H;H4,H6 and H8 had heterozygous mutation in H6H.The contents of TA were analyzed in the plants of H9,H14 and H15 with homozygous mutation.Neither anisodamine nor scopolamine were detected in H9,H14 and H15,while the accumulation of hyoscyamine were detected in the roots and leaves of the three lines.The hyoscyamine content was increased by 3.68,4.21 and 4.28 folds respectively in H9,H14 and H15 roots,compared with that in roots of control plants.The hyoscyamine content was increased by 0.61,0.76 and 2.22 folds,respectively in H9,H14 and H15 leaves,compared with that in leaves of control plants.The results above showed that knocking out H6H gene in A.belladonna disrupted the conversion from hyoscyamine to anisodamine and scopolamine,thus elevating the accumulation of hyoscyamine in A.belladonna plants.To sum up,this paper includes two main research contents:1)molecular biological and biochemical research of hyoscyamine dehydrogenase(HDH);2)metabolic engineering of hyoscyamine.In the first part of the study,it is found that HDH was highly expressed in secondary roots of A.belladonna plants and mainly located in pericycle and endodermis.HDH has typical oxidoreductase catalytic characteristics,which can reduce hyoscyamine aldehyde to hyoscyamine and oxidize hyoscyamine to hyoscyamine aldehyde,but it mainly plays the function of reductase under the physiological p H condition in plant cells.The results of enzyme kinetics showed that HDH was the rate-limiting enzyme in the biosynthesis of hyoscyamine.HDH crystal structure,amino acid site mutation and deuterium-labeled NADPH feeding assay revealed the catalytic mechanism of this enzyme.The active center of the enzyme was a tetrahedral structure composed of Zn²⁺,His74,Glu75,Cys52 and Cys168.Three non-polar amino acids,Met124,Leu282 and Ala305,formed the substrate binding pocket.His74,Ser54 and Cys100 are important amino acids that maintain the catalytic activity of HDH.pro-R hydrogen atom at the C4 position of NADPH participates in the reduction reaction catalyzed by HDH.In the second part,we found that overexpression of HDH could increase the content of hyoscyamine in the hair roots and plants of A.belladonna.Knocking out H6H could block the conversion of hyoscyamine into anisodamine and scopolamine,and lead to the accumulation of hyoscyamine at higher level.The study of HDH expression and catalytic mechanism is of great significance to improve the theoretical knowledge of TA biosynthesis.The study on metabolic engineering of hyoscyamine and the acquisition of new varieties with increased content of hyoscyamine provided an important technical support for solving the problem of hyoscyamine resource supply.