Developing Multipurpose Nicotiana Crops for Molecular Farming using New Plant Breeding Techniques

Project Information

Newcotiana

Grant agreement ID: 760331

DOI

10.3030/760331

Project closed

EC signature date 4 November 2017

Start date 1 January 2018

End date 31 December 2022

Funded under

• INDUSTRIAL LEADERSHIP - Leadership in enabling and industrial technologies – Biotechnology

Total cost

€ 7 363 310,00

EU contribution

€ 7 199 560,00

Coordinated by

AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Spain

Periodic Reporting for period 4 - Newcotiana (Developing Multipurpose Nicotiana Crops for Molecular Farming using New Plant Breeding Techniques)

Reporting period: 2022-01-01 to 2022-12-31

Summary of the context and overall objectives of the project

NEWCOTIANA is a research project that uses New Breeding Techniques (NBTs) to develop new Nicotiana plant varieties producing end-value chemicals. Plants can be used as biofactories of biopharmaceuticals and other added-value chemicals at an agricultural scale, offering a sustainable solution for present and future manufacturing needs. Species of the genus Nicotiana, more specifically N. tabacum (cultivated tobacco) and N. benthamiana (an Australian relative), are among the most widely used plant biofactories due to their favourable attributes such as metabolic versatility, ease of cultivation, high yield, availability of genetic tools for trait manipulation, and non-food status, which minimizes the possibility of contamination of the food supply.
In contrast to its high appreciation as biofactory, traditional tobacco cultivation is in continuous decline in Europe due, among other reasons, to the bad reputation of a crop that is mainly used for manufacturing cigarettes and other smoking products. NEWCOTIANA aims to offer an alternative to tobacco farmers by breeding competitive Nicotiana biofactory varieties for high value non-smoking products. However, to become fully competitive and scalable biofactories, Nicotiana plants need to incorporate unconventional traits such as protein stability or metabolite fortification, which are not accessible to traditional breeding. Recently, NBTs have emerged that bring unprecedented opportunities for Plant Biotechnology. The driving hypothesis in NEWCOTIANA is that breeding tobacco biofactory varieties for high value non-smoking products is possible with the use of NPBTs. Furthermore, NEWCOTIANA goals include improving public awareness and understanding of NBTs by creating a two-way dialogue between science on the one hand and stakeholders, policymakers and the general public on the other.
NEWCOTIANA results demonstrate the great potential of NBT in developing new crops, specifically industrial crops, in response to current societal and agricultural challenges. New added-value plant varieties with improved composition were generated in the project. Field trials were conducted to assess the agronomic performance of new varieties. All possible areas of concern raised in the risk assessment analysis were addressed and no other safety issues arose from the trials. The objectives of the project were perceived very positively by stakeholders and well received by the public. Positive public opinion was the result of the perceived social benefits derived from the project and was facilitated by the non-food status of the crop. In light of these results, the NEWCOTIANA consortium considers that a lift of current technology-based restrictions in NBT, and its substitution by a new case-by-case regulation that focuses on products, is strongly needed to respond to producers’ needs while aligning with current public perception.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Newcotiana scientists were able to create plants that produce biologics more efficiently and with improved quality, such as human growth factors (hGFs) involved in cell regeneration or human secretory antibodies used to fight mucosal pathogens. In parallel, researchers were able to enrich the composition of tobacco plants in small molecules with high added value, namely anatabine and squalene, with applications in biomedicine as a potential anti-inflammatory drug and a vaccine adjuvant. One of the main outputs of the project was the conduction of three rounds of open field trials (2020-2022) to study the agronomic performance of the new tobacco varieties. The results of the trials were encouraging and the traits observed in the greenhouse were confirmed in the field. Field-grown enriched lines were subjected to an optimized biorefinery process where, on top of anatabine and squalene, other high-value chemicals were isolated from tobacco biomass leaving the remains for biogas production. Other important results include (i) generation of a multiomic resource for N. benthamiana including high-quality genome assemblies of two ecotypes, updated gene annotations, epigenomic and transposon landscapes; (ii) development of new tools for NBTs, including more efficient CRISPR enzymes, alternative methods for gRNA delivery, optimised methods for gene targeting, and new intragenic elements; (iii) generation of biofactory glycoengineered and non-flowering tobacco lines with better quality and safety profiles and increased biomass. Dozens of proprietary plant lines have been created, two of which are intended for registration, while a patent covering several others is being filed. Techno-economic models have been generated for protein products (hGFs, VLP vaccines and antibodies) and metabolites (anatabine and squalene) and combined production scenarios have been explored showing the potential financial viability of some of the products. A spin-off company has been founded aiming to exploit the intellectual property arising from the project.
There have been constant interactions with stakeholders, including policy makers, farmers' associations and industry, and the general public. More than 200 dissemination/communication activities have been undertaken, including conferences, workshops, 38 scientific publications (zenodo.org/communities/newcotiana) exhibitions, and videos (newcotiana.org/videos). Reports on stakeholder opinions and public perception on NBTs have been generated.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

NEWCOTIANA has advanced beyond the state-of-the-art in sequence-specific nuclease (SSN) technologies and platform optimization. We have developed alternative nucleases and more efficient gene editing/targeting methods. For platform optimization, the new N. benthamiana genome assembly has set a crucial milestone in plant biofactories that will facilitate future breeding strategies; the expanded collection of regulatory elements represents a crucial advance in the application of intragenesis in breeding; prototype Nicotiana lines incorporating new valuable biofactory traits have been obtained and validated at laboratory scale and in the open field. Briefly these are the main impacts arising from Newcotiana:
Scientific: the project has developed new resources for NBTs. It has deepened in the understanding of gene function in Nicotiana species and the interactions between the metabolome and agronomic behavior.
Technical: new prototype lines have been generated using NBTs and validated at pilot scale leading to the selection of elite plant varieties as biofactories. These elite lines show improved production of recombinant proteins (higher quality and/or yield) or added-value metabolites. Biorefinery cascades have been established for the full use of tobacco biofactories, meeting the requirements of the circular economy.
Social: In the EU circa 60.000 tobacco farmers and as many indirect jobs are threatened by the decline in the tobacco demand. NEWCOTIANA will bring the opportunity to maintain those jobs and generate new ones, revitalizing rural areas where tobacco is a traditional crop as well as new ones that will incorporate NEWCOTIANA. The scalability of the NEWCOTIANA biofactory will improve accessibility to biopharmaceuticals (vaccines, antibodies and others) whose availability is at risk due to high manufacturing costs and/or long production cycles. Furthermore, NEWCOTIANA aims to provide industry, policy-makers and consumers with experimental evidence that will facilitate the decision-making process for the adoption of NBTs.

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