Amorpha-4,11-diene synthase: mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate

Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-(2)H]FPP, (1S)-[1-(2)H]FPP, and [1,1-(2)H2]FPP. GC-MS analysis of amorpha-4,11-diene formed from the deuterated FPPs shows that the deuterium atoms are retained in the product. Furthermore, analysis of the MS-spectra obtained with the differently labeled substrate indicates that the H-1si-proton of FPP is transferred during the cyclization reaction to carbon 10 of amorphadiene while the H-1re-proton of FPP is retained on C-6 of the product. Proton NMR and COSY experiments proved that the original H-1si-proton of FPP is located at C-10 of amorpha-4,11-diene as a result of a 1,3-hydride shift following initial 1,6-ring closure. The results obtained support the previously suggested mechanism for the cyclization of farnesyl diphosphate by amorph-4,11-diene synthase involving isomerization of FPP to (R)-nerolidyl diphosphate (NPP), ionization of NPP, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure to generate the amorphane skeleton, and deprotonation at either C-12 or C-13 to afford the final product (1S,6R,7R,10R)-amorpha-4,11-diene.     

在大肠杆菌内引入甲羟戊酸途径高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯

以青蒿素为基础的联合药物疗法 (ACTs) 被认为是目前治疗恶性疟疾的最有效方法。然而青蒿素供应不足且价格昂贵,限制了ACTs的广泛使用。采用基因工程手段构建异源类异戊二烯生物合成途径,利用大肠杆菌发酵能高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯。首先在大肠杆菌Escherichia coli DHGT7中引入人工合成的紫穗槐-4,11-二烯合酶基因,利用大肠杆菌内源的法尼基焦磷酸,成功获得了紫穗槐-4,11-二烯。为提高前体供给,引入粪肠球菌的甲羟戊酸途径,紫穗槐-4,11-二烯的产量提高了13     

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight. Received: 8 June 2000 / Accepted: 21 August 2000     

High-Level Production of Amorpha-4,11-Diene,a Precursor of the Antimalarial Agent Artemisinin,in Escherichia coli

Background

Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.

Methodology/ Principal Findings

By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.

Conclusions/ Significance

Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.     

Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L

In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.     

Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis

The endoperoxide sesquiterpene lactone artemisinin and its derivatives are a promising new group of drugs against malaria. Artemisinin is a constituent of the annual herb Artemisia annua L. So far only the later steps in artemisinin biosynthesis--from artemisinic acid--have been elucidated and the expected olefinic sesquiterpene intermediate has never been demonstrated. In pentane extracts of A. annua leaves we detected a sesquiterpene with the mass spectrum of amorpha-4,11-diene. Synthesis of amorpha-4,11-diene from artemisinic acid confirmed the identity. In addition we identified several sesquiterpene synthases of which one of the major activities catalysed the formation of amorpha-4,11-diene from farnesyl diphosphate. This enzyme was partially purified and shows the typical characteristics of sesquiterpene synthases, such as a broad pH optimum around 6.5-7.0, a molecular mass of 56 kDa, and a K(m) of 0.6 microM. The structure and configuration of amorpha-4,11-diene, its low content in A. annua and the high activity of amorpha-4,11-diene synthase all support that amorpha-4,11-diene is the likely olefinic sesquiterpene intermediate in the biosynthesis of artemisinin.     

Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L

A cDNA clone encoding amorpha-4,11-diene synthase from Artemisia annua was subcloned into a bacterial expression vector in frame with a His6-tag. Recombinant amorpha-4,11-diene synthase was produced in Escherichia coli and purified to apparent homogeneity. The enzyme showed pH optimum at pH 6.5, and a minimum at pH 7.5. Substantial activity was observed in the presence of Mg2+, Mn2+ or Co2+ as cofactor. The enzyme exhibits a low activity in the presence of Ni2+ and essentially no activity with Cu2+ or Zn2+. The sesquiterpenoids produced from farnesyl diphosphate in the presence of Mg2+ were analyzed by GC-MS. In addition to amorpha-4,11-diene, 15 sesquiterpenoids were produced. Only small quantitative differences in product pattern were observed at pH 6.5, 7.5, or 9.5. Amorpha-4,11-diene synthase showed significant increased product selectivity in the presence of Mn2+ or Co2+. Km for farnesyl diphosphate was 3.3, 8.0, and 0.7 microM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 x 10(-3) s(-1), respectively. Km and kcat for geranyl diphosphate were 16.9 microM and 7.0 x 10(-4) s(-1), respectively, at pH 6.5, in the presence of Mn2+.     

Amorpha-4,11-diene synthase of Artemisia annua: cDNA isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis

Artemisia annua, an indigenous plant to Korea, contains an antimalarial sesquiterpene, artemisinin. The first committed step of artemisinin biosynthesis is the cyclization of farnesyl diphosphate by a sesquiterpene synthase to produce an amorphane-type ring system. The aims of this research were to molecularly clone and express amorpha-4,11-diene synthase for metabolic engineering. PCR amplification of genomic DNA with a pair of primers, designed from the conserved regions of sesquiterpene synthases of several plants, produced a 184-bp DNA fragment. This fragment was used in Northern blot analysis as a probe, showing approximately 2.2 kb of a single band. Its sequence information was used to produce 2106 bp of a full-length cDNA sequence including 1641 bp of open reading frame for 546 amino acids (kcs12) through a rapid amplification of cDNA ends (RACE). The deduced amino acid sequence displayed 36% identity with 5-epi-aristolochene synthase of Nicotiana tabacum. A soluble fraction of Escherichia coli harboring kcs12 catalyzed the cyclization of farnesyl diphosphate to produce a sesquiterpene, which was identified through GC-MS analysis as amorpha-4,11-diene.     

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Δ11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1α was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.     

The improvement of amorpha-4,11-diene production by a yeast-conform variant

Aims:  To investigate the effect of the yeast-conform variant of the Artemisia annua gene encoding for amorpha-4,11-diene synthase (ADS) on the production of amorpha-4,11-diene in a transformed yeast.
Methods and Results:  The ADS gene was mutated to the yeast-conform variant ADSm . The ADSm synthesis was performed based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial ADSm gene contained codons preferred by the yeast translation machinery. The sequence was then integrated into a yeast expression vector pYeDP60. The fusion construct was active and the transformed yeast cells produced higher level of amorpha-4,11-diene compared with the plant gene-transformed yeast cells.
Conclusions:  Strains transformed with the yeast-conform allele ( ADSm ) were more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene.
Significance and Impact of the Study:  We demonstrated that yeast-conform allele of foreign genes by serial PCR reactions can be a solution to low efficiency of heterologous gene expression in Saccharomyces cerevisiae cells.     

Farnesol production in Escherichia coli through the construction of a farnesol biosynthesis pathway – application of PgpB and YbjG phosphatases

Farnesol is a sesquiterpenoid alcohol that has important industrial and medical potential. It is usually synthesized from farnesyl diphosphate (FPP) by farnesol synthase in plants. FPP accumulation can cause up‐regulation of phosphatases capable of FPP hydrolysis, resulting in farnesol production in Escherichia coli. We found that PgpB and YbjG, two integral membrane phosphatases, can hydrolyze FPP into farnesol. Overexpression of FPP synthase (IspA) and PgpB, along with a heterologous mevalonate pathway, enabled recombinant E. coli to produce 526.1 mg/L of farnesol. This result indicates that the phosphatases PgpB and YbjG can be used to construct a novel farnesol synthesis pathway for mass production in E. coli.     

Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation. Revisions requested 14 November 2005; Revisions received 17 January 2006     

Nicotiana benthamiana as a production platform for artemisinin precursors

Background

Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment.

Methodology/Principal Findings

Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg⁻¹ fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana.

Conclusion/Significance

This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.     

Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4′-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.     

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 — a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 — an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.     

Artemisinin biosynthesis in growing plants of Artemisia annua. A 13CO2 study

Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of ¹³CO₂ for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by ¹³C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation.     

Targeted proteomics for metabolic pathway optimization: application to terpene production

Successful metabolic engineering relies on methodologies that aid assembly and optimization of novel pathways in microbes. Many different factors may contribute to pathway performance, and problems due to mRNA abundance, protein abundance, or enzymatic activity may not be evident by monitoring product titers. To this end, synthetic biologists and metabolic engineers utilize a variety of analytical methods to identify the parts of the pathway that limit production. In this study, targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in Escherichia coli strains engineered to produce the sesquiterpene, amorpha-4,11-diene. From this analysis, two mevalonate pathway proteins, mevalonate kinase (MK) and phosphomevalonate kinase (PMK) from Saccharomyces cerevisiae, were identified as potential bottlenecks. Codon-optimization of the genes encoding MK and PMK and expression from a stronger promoter led to significantly improved MK and PMK protein levels and over three-fold improved final amorpha-4,11-diene titer (>500 mg/L).     

Expression of a synthetic Artemesia annua amorphadiene synthase in Aspergillus nidulans yields altered product distribution

A gene encoding a plant terpene cyclase, Artemisia annua amorpha-4,11-diene synthase (ADS), was expressed in Aspergillus nidulans under control of a strong constitutive promoter, (p)gpdA. The transformants produced only small amounts of amorphadiene, but much larger amounts of similar sesquiterpenes normally produced as minor by-products in planta. In contrast, expression of ADS in Escherichia coli produced almost exclusively amorpha-4,11-diene. These results indicate that the host environment can greatly impact the terpenes produced from terpene synthases.     

紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用

为了构建高产的紫穗槐-4,11-二烯酵母工程菌,主要探究了含紫穗槐-4,11-二烯合酶基因的不同表达载体在酵母工程菌中是否存在协同效应。首先构建了含紫穗槐-4,11-二烯合酶基因的酵母表达载体pGADADS,分别将pGADADS和pYeDP60/G/ADS转入酿酒酵母W303-1B和WK1中,获得6种能产生紫穗槐-4,11-二烯的酵母工程菌：W303B[pGADADS]、W303B[pYGADS]、W303B[pYGADS+pGADADS]、WK1[pGADADS]、WK1[pYGADS]和WK1[pYG