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

Wallaart, TE; Bouwmeester, Harro J.; Hille, Jacques; Poppinga, Lena; Maijers, Nca; Maijers, Niels C.A.

doi:10.1007/s004250000428

Cited by 215 publications

(154 citation statements)

References 29 publications

(28 reference statements)

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“…We have now extended these observations to the expression plant terpene synthase genes in S. avermitilis. A synthetic gene encoding amorpha-4,11-diene synthase (ads) of Artemisia annua (27,28), in which the codon usage was optimized for expression in S. avermitilis, was expressed in S. avermitilis under control of the rpsJ promoter. The requisite farnesyl diphosphate precursor was provided by introducing a copy of the native of S. avermitilis ptlB gene (sav2997) encoding farnesyl diphosphate synthase downstream of the ads gene.…”

Section: Heterologeous Expression Of Exogenous Gene Clusters For Secomentioning

confidence: 99%

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Komatsu

Uchiyama

Ōmura

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

444

366

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To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.genome engineering | host development | natural products A prominent property of members of the genus Streptomyces is the ability to produce numerous secondary metabolites, including antibiotics and other biologically active compounds of proven value in human and veterinary medicine and agriculture; they are also useful as biochemical tools. These structurally diverse metabolites collectively express not only antibacterial, antifungal, antiviral, and antitumor activities but also antihypertensive and immunosuppressant properties. Streptomyces have been a rich source of pharmaceutical compounds in which common cellular intermediates, including amino acids, sugars, fatty acids, and terpenes, are combined to give more complex structures by defined biochemical pathways. Genomic analysis of three species of Streptomyces, S. avermitilis (1, 2), S. coelicolor A3(2) (3), and S. griseus (4), has revealed that these microorganisms each have large linear chromosomes that harbor over 20 secondary metabolic gene clusters encoding the biosynthesis of polyketides by polyketide synthases (PKSs), peptides by nonribosomal peptide synthetases (NRPSs), bacteriocins, terpenoids, shikimate-derived metabolites, aminoglycosides, and other natural products (5).T...

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Section: Heterologeous Expression Of Exogenous Gene Clusters For Secomentioning

confidence: 99%

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Komatsu

Uchiyama

Ōmura

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

444

366

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“…Interestingly the K m for farnesyl diphosphate as determined with heterologous protein (0.9 μM) is virtually the same as the K m reported for the purified protein (Bouwmeester et al 1999b;Mercke et al 2000). In the light of a possible use of this gene for alternative production systems for artemisinin, it is interesting to note that Wallaart et al transformed tobacco with the amorphadiene synthase (Wallaart et al 2001). This resulted indeed in the production of amorpha-4,11-diene in transgenic plants, but only at trace levels.…”

Section: Isolation and Characterization Of Genes From The Biosynthetisupporting

confidence: 69%

“…Following the biochemical identification of amorpha-4,11-diene synthase, which catalyses the first dedicated biosynthetic step in artemisinin biosynthesis (Bouwmeester et al 1999b), the corresponding gene was cloned by several groups almost in parallel (Chang et al 2000;Mercke et al 2000;Wallaart et al 2001) and a patent application was filed (Wallaart and Bouwmeester 1998). Interestingly, the three independently isolated cDNAs are exactly the same suggesting that there is only one copy of this gene present in the plant cell.…”

Section: Isolation and Characterization Of Genes From The Biosynthetimentioning

confidence: 99%

Research to Improve Artemisinin Production for use in the Preparation of Anti-Malarial Drugs

Bouwmeester

Bertea²,

Kraker³

et al.

Medicinal and Aromatic Plants

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Abstract. An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its semi-synthetically prepared derivatives. Because of the development of resistance against other malarial drugs, the demand for artemisinin has rapidly increased during the past decade. However, the supply of artemisinin is troublesome as neither total nor semi-synthesis are economically feasible and the only plant species known to produce artemisinin, Artemisia annua L., contains only low amounts of this compound. For a directed strategy to improve artemisinin production, one of the first requirements is to have insight into the regulation of its biosynthesis. Surprisingly, until some years ago hardly anything was known about the biosynthesis of artemisinin, particularly about the early enzymatic steps leading to (dihydro)artemisinic acid. To elucidate this important missing part in the pathway, we have analysed the terpenoids in A. annua leaves and the presence of sesquiterpene synthases, the enzyme class that should theoretically catalyse the first step in the formation of artemisinin. This led to the discovery of amorpha-4,11-diene synthase, which indeed catalyses the first step in artemisinin biosynthesis and for which the corresponding gene was cloned shortly afterwards. A subsequent search for oxidized derivatives of amorpha-4,11-diene -which could be postulated to be intermediates en route to artemisinin -revealed the presence of artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of the biosynthetic enzymes amorpha-4,11-diene hydroxylase, artemisinic alcohol dehydrogenase, artemisinic aldehyde reductase and dihydroartemisinic aldehyde dehydrogenase in A. annua. From these results, we conclude that the early steps in artemisinin biosynthesis involve: cyclization of farnesyl diphosphate by amorpha-4,11-diene synthase to amorpha-4,11-diene, hydroxylation of amorpha-4,11-diene to artemisinic alcohol, oxidation of artemisinic alcohol to artemisinic aldehyde, reduction of the C11-C13 double bond yielding dihydroartemisinic aldehyde, and oxidation of dihydroartemisinic aldehyde to dihydroartemisinic acid. During this biochemical research we have also managed to develop a trichome isolation protocol which has not only accelerated our biochemical work but also enables us to clone -in a very directed way -the genes that encode the enzymes responsible for artemisinin production. This should in the next few years result in several approaches to boost artemisinin production. 276H. BOUWMEESTER ET AL.

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“…benthamiana) have been also engineered to produce artemisinin precursors and even artemisinin per se. In a pioneering experiment, N. tabacum was transformed by amorphadiene synthase gene (ADS) cloned from A. annua, leading to the accumulation of amorpha-1,4-diene, a committed precursor of artemisinin, in trace amounts (Wallaart et al 2001). After years, an innovative strategy has been used to increase the substantial accumulation of amorpha-1,4-diene in N. tabacum, in which ADS gene from A. annua and farnesyl pyrophosphate synthase gene (FPS) from chicken were designed to be expressed in nucleus and targeted to plastid, resulting in 5,000-fold increases of amorpha-4,11-diene (Wu et al 2006).…”

Section: Introductionmentioning

confidence: 99%

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

Jiang

Gao

Liao

et al. 2015

Preprint

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Artemisinin is accumulated in wormwood (Artemisia annua) with uncertain ecological implications. Here, we suggest that artemisinin is generated in response to biotic/abiotic stress, during which dihydroartemisinic acid, a direct artemisinin precursor, quenches singlet oxygen ( 1 O2), one kind of reactive oxygen species. Evidence supporting artemisinin as a sink of 1 O2 emerges from that volatile isoprenoids protect plants from biotic/abiotic stress; biotic/abiotic stress induces artemisinin biosynthesis; and stress signaling pathways are involved in the biosynthesis of volatile isoprenoids among plants as well as the biosynthesis of artemisinin in A. annua. In this review, we address the ecological implication of glandular trichome-sequestered artemisinin as a sink sink of biotic/abiotic stress-triggered 1 O2, and also summarize the cumulating data on the transcriptomic and metabolic profiling of stress-enhanced artemisinin production upon eliciting 1 O2 omission from chloroplasts and initiating retrograde 1 O2 signaling from chloroplasts to nuclei.

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

Cited by 215 publications

References 29 publications

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Research to Improve Artemisinin Production for use in the Preparation of Anti-Malarial Drugs

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

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