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Metabolites
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Nicotiana spp. as Production Platforms for Bioproducts
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Nicotiana spp. as Production Platforms for Bioproducts

A special issue of Metabolites (ISSN 2218-1989). This special issue belongs to the section "Nutrition and Metabolism".

Deadline for manuscript submissions: closed (15 February 2023) | Viewed by 10720

Special Issue Editors

Dr. Olivia Costantina Demurtas

E-Mail Website
Guest Editor
Biotechnologies and Agroindustry Division, Department for Sustainability, ENEA, Italian National Agency for New Technologies, Energy and Sustainable Development, Casaccia Research Center, 00123 Rome, Italy
Interests: metabolic engineering; metabolomics; plant biotechnology; nutraceuticals; plant molecular farming; pathway compartimentalization
Dr. Sarah Frusciante

E-Mail Website
Guest Editor
Biotechnologies and Agroindustry Division, Department for Sustainability - ENEA (Italian National Agency for New Technologies, Energy and Sustainable Development), Rome, Italy
Interests: metabolic engineering; metabolomics; plant biotechnology; nutraceuticals; bioprospecting; pathway discovery

Special Issue Information

Dear Colleagues,

Metabolic engineering based on classical transgenesis has been widely reported in the genus Nicotiana for molecular farming purposes; more recently, the possibility to fine modulate the production of molecules of interest, thanks to New Plant Breeding Techniques (NPBT), has been successfully demonstrated. In this context, the use of non-food crops as biofactories to produce new pharmaceuticals, nutraceuticals, cosmetics, or biofuels, represent an innovative and efficient solution in a modern and sustainable bioeconomy.

This Special Issue of Metabolites will gather these frontier approaches by publishing reviews and original articles covering the latest developments in Nicotiana species as platforms for producing bioproducts, modulating the expression of genes involved in their pathway/regulation or by synthetic biology approaches.

Specific areas will include, but not be limited to, the use of NPBT to improve the content of desired compounds, to create novel biosynthetic pathways, or to generate improved Nicotiana chassis.

Dr. Olivia Costantina Demurtas
Dr. Sarah Frusciante
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Metabolites is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • metabolic engineering
  • nicotiana
  • plant molecular farming
  • synthetic biology
  • new plant breeding techniques (NPBT)
  • genome editing

Published Papers (6 papers)

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Research

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12 pages, 1807 KiB  
Article
Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor
by Olivia Costantina Demurtas, Maria Sulli, Paola Ferrante, Paola Mini, Maricarmen Martí, Verónica Aragonés, José-Antonio Daròs and Giovanni Giuliano
Metabolites 2023, 13(6), 729; https://doi.org/10.3390/metabo13060729 - 06 Jun 2023
Cited by 3 | Viewed by 1273
Abstract
Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro [...] Read more.
Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates. Full article
(This article belongs to the Special Issue Nicotiana spp. as Production Platforms for Bioproducts)
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13 pages, 4530 KiB  
Article
A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts
by Julia Legen, Sara Dühnen, Anton Gauert, Michael Götz and Christian Schmitz-Linneweber
Metabolites 2023, 13(3), 315; https://doi.org/10.3390/metabo13030315 - 21 Feb 2023
Cited by 3 | Viewed by 1549
Abstract
Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective [...] Read more.
Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. The objective is to demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts. Full article
(This article belongs to the Special Issue Nicotiana spp. as Production Platforms for Bioproducts)
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15 pages, 2174 KiB  
Article
Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage
by Luca Morelli, Laura García Romañach, Gaetan Glauser, Venkatasalam Shanmugabalaji, Felix Kessler and Manuel Rodriguez-Concepcion
Metabolites 2023, 13(2), 193; https://doi.org/10.3390/metabo13020193 - 28 Jan 2023
Cited by 3 | Viewed by 1308
Abstract
The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually [...] Read more.
The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in α-tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1). Full article
(This article belongs to the Special Issue Nicotiana spp. as Production Platforms for Bioproducts)
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22 pages, 4490 KiB  
Article
Metabolic Engineering of Nicotiana benthamiana to Produce Cannabinoid Precursors and Their Analogues
by Vaishnavi Amarr Reddy, Sing Hui Leong, In-Cheol Jang and Sarojam Rajani
Metabolites 2022, 12(12), 1181; https://doi.org/10.3390/metabo12121181 - 25 Nov 2022
Cited by 2 | Viewed by 1844
Abstract
In recent years, the perspective towards the use of cannabis has slowly shifted from being an illicit drug to a medicinal plant. The pathway and enzymes involved in the production of cannabinoids are known; however, studies evaluating the production of cannabinoids in heterologous [...] Read more.
In recent years, the perspective towards the use of cannabis has slowly shifted from being an illicit drug to a medicinal plant. The pathway and enzymes involved in the production of cannabinoids are known; however, studies evaluating the production of cannabinoids in heterologous plants and cell cultures are still limited. In this study, we assessed the potential use of N. benthamiana (Nicotiana benthamiana) plants as a heterologous host for producing natural and novel cannabinoids. Transgenic N. benthamiana plants expressing genes encoding cannabis acyl-activating enzyme and olivetol synthase were generated, which were then used for transiently expressing other downstream pathway genes. Production of olivetolic acid and divarinic acid, the universal precursors for major and minor cannabinoids, respectively, was observed in transgenic N. benthamiana plants. To produce novel cannabinoid precursors with different side chains, various fatty acids were infiltrated into the transgenic N. benthamiana plants and the production of novel derivatives was observed. Although we were not able to derive the core intermediate, cannabigerolic acid, from our transgenic plants, possibly due to the low production levels of the precursors, our transgenics plants still serve as a high-potential platform for further development and exploring the N. benthamiana chemical space for generating novel cannabinoids. Full article
(This article belongs to the Special Issue Nicotiana spp. as Production Platforms for Bioproducts)
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12 pages, 1805 KiB  
Article
The Biosynthesis of Non-Endogenous Apocarotenoids in Transgenic Nicotiana glauca
by Xin Huang, Lucía Morote, Changfu Zhu, Oussama Ahrazem, Teresa Capell, Paul Christou and Lourdes Gómez-Gómez
Metabolites 2022, 12(7), 575; https://doi.org/10.3390/metabo12070575 - 22 Jun 2022
Cited by 6 | Viewed by 1507
Abstract
Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified [...] Read more.
Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified in saffron, Buddleja, and gardenia. In this study, the enzyme from Buddleja, BdCCD4.1, was constitutively expressed in Nicotiana glauca, a tobacco species with carotenoid-pigmented petals. The transgenic lines produced significant levels of crocins in their leaves and petals. However, the accumulation of crocins was, in general, higher in the leaves than in the petals, reaching almost 302 µg/g DW. The production of crocins was associated with decreased levels of endogenous carotenoids, mainly β-carotene. The stability of crocins in leaf and petal tissues was evaluated after three years of storage, showing an average reduction of 58.06 ± 2.20% in the petals, and 78.37 ± 5.08% in the leaves. This study illustrates the use of BdCCD4.1 as an effective tool for crocin production in N. glauca and how the tissue has an important impact on the stability of produced high-value metabolites during storage. Full article
(This article belongs to the Special Issue Nicotiana spp. as Production Platforms for Bioproducts)
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Review

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16 pages, 2000 KiB  
Review
Current Status and De Novo Synthesis of Anti-Tumor Alkaloids in Nicotiana
by Md. Ahsan Habib, Md. Mobinul Islam, Md. Mukul Islam, Md. Mohidul Hasan and Kwang-Hyun Baek
Metabolites 2023, 13(5), 623; https://doi.org/10.3390/metabo13050623 - 30 Apr 2023
Cited by 1 | Viewed by 2222
Abstract
Alkaloids are the most diversified nitrogen-containing secondary metabolites, having antioxidant and antimicrobial properties, and are extensively used in pharmaceuticals to treat different types of cancer. Nicotiana serves as a reservoir of anti-cancer alkaloids and is also used as a model plant for the [...] Read more.
Alkaloids are the most diversified nitrogen-containing secondary metabolites, having antioxidant and antimicrobial properties, and are extensively used in pharmaceuticals to treat different types of cancer. Nicotiana serves as a reservoir of anti-cancer alkaloids and is also used as a model plant for the de novo synthesis of various anti-cancer molecules through genetic engineering. Up to 4% of the total dry weight of Nicotiana was found to be composed of alkaloids, where nicotine, nornicotine, anatabine, and anabasine are reported as the dominant alkaloids. Additionally, among the alkaloids present in Nicotiana, β-carboline (Harmane and Norharmane) and Kynurenines are found to show anti-tumor effects, especially in the cases of colon and breast cancers. Creating new or shunting of existing biosynthesis pathways in different species of Nicotiana resulted in de novo or increased synthesis of different anti-tumor molecules or their derivatives or precursors including Taxadiane (~22.5 µg/g), Artemisinin (~120 μg/g), Parthenolide (~2.05 ng/g), Costunolide (~60 ng/g), Etoposide (~1 mg/g), Crocin (~400 µg/g), Catharanthine (~60 ng/g), Tabersonine (~10 ng/g), Strictosidine (~0.23 mg/g), etc. Enriching the precursor pool, especially Dimethylallyl Diphosphate (DMAPP), down-regulating other bi-product pathways, compartmentalization or metabolic shunting, or organelle-specific reconstitution of the precursor pool, might trigger the enhanced accumulation of the targeted anti-cancer alkaloid in Nicotiana. Full article
(This article belongs to the Special Issue Nicotiana spp. as Production Platforms for Bioproducts)
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