CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.

The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO), an inexpensive and widely used agricultural input.

A hybrid pathway for self-sustained luminescence.

The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants.

Plant virus-derived nanoparticles decorated with genetically encoded SARS-CoV-2 nanobodies display enhanced neutralizing activity.

Viral nanoparticles (VNPs) are a new class of virus-based formulations that can be used as building blocks to implement a variety of functions of potential interest in biotechnology and nanomedicine. Viral coat proteins (CP) that exhibit self-assembly properties are particularly appropriate for displaying antigens and antibodies, by generating multivalent VNPs with therapeutic and diagnostic potential. Here, we developed genetically encoded multivalent VNPs derived from two filamentous plant viruses, potato virus X (PVX) and tobacco etch virus (TEV), which were efficiently and inexpensively produced in the biofactory Nicotiana benthamiana plant.

A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology.

Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low-cost, and high-throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence.

Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content.

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly.

Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum.

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g.

The design of synthetic gene circuits in plants: new components, old challenges.

The fascination produced by the possibility of engineering plants with augmented capabilities has accompanied plant biotechnology since its origins. This prospect has become even more relevant in present times under the pressure imposed by climate change and population growth. Today's plant biotechnologists approach this challenge with the tools of synthetic biology, which facilitate the assembly of synthetic gene circuits (SGCs) from their modular components.

Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.

New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming.

Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators.

Transcriptional regulators based on CRISPR architecture expand our ability to reprogramme endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

Background: CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana.

Production of Volatile Moth Sex Pheromones in Transgenic Plants.

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing -11-hexadecenol (Z11-16OH) and -11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into plants.

Strong and tunable anti-CRISPR/Cas activities in plants.

CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana.

The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants.

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation.

Pilot Production of SARS-CoV-2 Related Proteins in Plants: A Proof of Concept for Rapid Repurposing of Indoor Farms Into Biomanufacturing Facilities.

The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world's capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions.

Engineering Metabolism in Nicotiana Species: A Promising Future.

Molecular farming intends to use crop plants as biofactories for high value-added compounds following application of a wide range of biotechnological tools. In particular, the conversion of nonfood crops into efficient biofactories is expected to be a strong asset in the development of a sustainable bioeconomy. The 'nonfood' status combined with the high metabolic versatility and the capacity of high-yield cultivation highlight the plant genus Nicotiana as one of the most appropriate 'chassis' for molecular farming.

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond.

Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate-based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host-specific features into its basic scheme.

A memory switch for plant synthetic biology based on the phage ϕC31 integration system.

Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been the tool of choice to build long-term and stable synthetic memory in other organisms, because they enable a shift between two alternative states registering the information at the DNA level.

Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions.

Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A.