CRISPR/Cas9-mediated disruption of the PYRROLIDINE KETIDE SYNTHASE gene reduces the accumulation of tropane alkaloids in Atropa belladonna hairy roots

2021 ◽  
Author(s):  
Fumihito Hasebe ◽  
Honoka Yuba ◽  
Takashi Hashimoto ◽  
Kazuki Saito ◽  
Nobutaka Funa ◽  
...  
Keyword(s):  
Hairy Roots ◽  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Tropane Alkaloids ◽  
Tropane Alkaloid ◽  
Atropa Belladonna ◽  
Type Iii Polyketide Synthase ◽  
Canonical Type ◽  
Solanaceae Family

Abstract Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A non-canonical type III polyketide synthase (PKS), pyrrolidine ketide synthase (PYKS), catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

A Type I/Type III Polyketide Synthase Hybrid Biosynthetic Pathway for the Structurally UniqueansaCompound Kendomycin

ChemBioChem ◽  
2008 ◽  
Vol 9 (16) ◽  
pp. 2711-2721 ◽  
Author(s):  
Silke C. Wenzel ◽  
Helge B. Bode ◽  
Irene Kochems ◽  
Rolf Müller
Keyword(s):  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Type I ◽  
Type Iii Polyketide Synthase ◽  
Type Iii

Differential Production of Tropane Alkaloids in Hairy Roots and in vitro Cultured Two Accessions of Atropa belladonna L. under Nitrate Treatments

2010 ◽  
Vol 65 (5-6) ◽  
pp. 373-379 ◽  
Author(s):  
Najmeh Ahmadian Chashmi ◽  
Mozafar Sharifi ◽  
Farah Karimi ◽  
Hasan Rahnama
Keyword(s):  
Secondary Metabolites ◽  
Hairy Roots ◽  
Nitrate Concentration ◽  
Tropane Alkaloids ◽  
Atropa Belladonna ◽  
Remarkable Effect ◽  
Aerial Parts ◽  
Potential Source ◽  
Stable Production

Plants are a potential source of a large number of valuable secondary metabolites. In vitro cultures are being considered as an alternative to agricultural processes for studying valuable secondary metabolites. In this way, nutritive factors are important parameters influencing the production of these compounds in plants. Effects of nitrate concentrations (KNO3) on the production of two tropane alkaloids, hyoscyamine and scopolamine, and the growth of aerial parts and roots of two in vitro propagated accessions of Atropa belladonna and hairy roots were investigated. As hairy roots cultures are able to keep a stable production of alkaloids over long periods of subculturing, they are considered as an interesting option for the study of alkaloid biosynthesis. A hairy roots culture of Atropa belladonna was established by transformation with Agrobacterium rhizogenes strain AR15834. The results of our study showed that a rise in KNO3 concentration caused a decline in hairy roots growth, and had a remarkable effect on the alkaloid content. The alkaloid concentrations obtained in the hairy roots were 3 - 20 times higher than that in the plants at 35 mM of KNO3. Increasing the nitrate concentration in the medium of hairy roots also improved the hyoscyamine/scopolamine ratio, while it increased the scopolamine/hyoscyamine ratio in the studied plants.

scholarly journals Tropane alkaloids biosynthesis involves an unusual type III polyketide synthase and non-enzymatic condensation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jian-Ping Huang ◽  
Chengli Fang ◽  
Xiaoyan Ma ◽  
Li Wang ◽  
Jing Yang ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Tropane Alkaloids ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Unusual Type

scholarly journals Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3161-3169 ◽  
Author(s):  
Stephen F. Haydock ◽  
Anthony N. Appleyard ◽  
Tatiana Mironenko ◽  
John Lester ◽  
Natasha Scott ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Building Blocks ◽  
Biosynthetic Gene Cluster ◽  
Sequence Motif ◽  
Biosynthetic Gene ◽  
Concanamycin A ◽  
Modular Polyketide Synthase

The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 4′-O-carbamoyl-2′-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA- from methoxymalonyl-ACP-specific AT domains in natural PKSs.

scholarly journals Enhancement of Phytosterol and Triterpenoid Production in Plant Hairy Root Cultures—Simultaneous Stimulation or Competition?

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2028
Author(s):  
Agata Rogowska ◽  
Anna Szakiel
Keyword(s):  
Hairy Roots ◽  
Biosynthetic Pathway ◽  
Experimental Models ◽  
Regulatory Mechanisms ◽  
Root Cultures ◽  
Efficient Tool ◽  
Common Precursor ◽  
The Common

Plant in vitro cultures, including hairy roots, can be applied for controlled production of valuable natural products, such as triterpenoids and sterols. These compounds originate from the common precursor squalene. Sterols and triterpenoids distinctly differ in their functions, and the 2,3-oxidosqualene cyclization step is often regarded as a branch point between primary and secondary (more aptly: general and specialized) metabolism. Considering the crucial role of phytosterols as membrane constituents, it has been postulated that unconstrained biosynthesis of triterpenoids can occur when sterol formation is already satisfied, and these compounds are no longer needed for cell growth and division. This hypothesis seems to follow directly the growth-defense trade-off plant dilemma. In this review, we present some examples illustrating the specific interplay between the two divergent pathways for sterol and triterpenoid biosynthesis appearing in root cultures. These studies were significant for revealing the steps of the biosynthetic pathway, understanding the role of particular enzymes, and discovering the possibility of gene regulation. Currently, hairy roots of many plant species can be considered not only as an efficient tool for production of phytochemicals, but also as suitable experimental models for investigations on regulatory mechanisms of plant metabolism.

Role of the Active Site Cysteine of DpgA, a Bacterial Type III Polyketide Synthase†

Biochemistry ◽  
2004 ◽  
Vol 43 (4) ◽  
pp. 970-980 ◽  
Author(s):  
Claire C. Tseng ◽  
Shaun M. McLoughlin ◽  
Neil L. Kelleher ◽  
Christopher T. Walsh
Keyword(s):  
Active Site ◽  
Polyketide Synthase ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Active Site Cysteine ◽  
Bacterial Type

Alkaloid production and capacity for methyljasmonate induction by hairy roots of two species in Tribe Anthocercideae, family Solanaceae

2015 ◽  
Vol 42 (8) ◽  
pp. 792 ◽  
Author(s):  
Suzanne M. Ryan ◽  
Kathleen D. DeBoer ◽  
John D. Hamill
Keyword(s):  
Hairy Roots ◽  
Tropane Alkaloids ◽  
Tropane Alkaloid ◽  
Root Cultures ◽  
Stress Hormone ◽  
Alkaloid Synthesis ◽  
Meja Treatment ◽  
Extant Species ◽  
Wound Stress ◽  
Key Genes

In addition to producing medicinally important tropane alkaloids, some species in the mainly Australian Solanaceous tribe Anthocercideae, sister to genus Nicotiana, are known to also contain substantial levels of the pyridine alkaloids nicotine and nornicotine. Here, we demonstrate that axenic hairy root cultures of two tribe Anthocercideae species, Cyphanthera tasmanica Miers and Anthocercis ilicifolia ssp. ilicifolia Hook, contain considerable amounts of both nicotine and nornicotine (~0.5–1% DW), together with lower levels of the tropane alkaloid hyoscyamine (<0.2% DW). Treatment of growing hairy roots of both species with micromolar levels of the wound stress hormone methyl-jasmonate (MeJa) led to significant increases (P < 0.05) in pyridine alkaloid concentrations but not of hyoscyamine. Consistent with previous studies involving Nicotiana species, we also observed that transcript levels of key genes required for pyridine alkaloid synthesis increased in hairy roots of both Anthocercideae species following MeJa treatment. We hypothesise that wound-associated induction of pyridine alkaloid synthesis in extant species of tribe Anthocercideae and genus Nicotiana was a feature of common ancestral stock that existed before the separation of both lineages ~15 million years ago.

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