Engineering Nicotiana benthamiana as a platform for natural product biosynthesis.

Nicotiana benthamiana is a model plant, widely used for research. The susceptibility of young plants to Agrobacterium tumefaciens has been utilised for transient gene expression, enabling the production of recombinant proteins at laboratory and commercial scales. More recently, this technique has been used for the rapid prototyping of synthetic genetic circuits and for the elucidation and reconstruction of metabolic pathways.

Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation.

Transcriptome-informed identification and characterization of - and -isoprenyl diphosphate synthase genes.

Insect physiology and reproduction depend on several terpenoid compounds, whose biosynthesis is mainly unknown. One enigmatic group of insect monoterpenoids are mealybug sex pheromones, presumably resulting from the irregular coupling activity of unidentified isoprenyl diphosphate synthases (IDSs). Here, we performed a comprehensive search for IDS coding sequences of the pest mealybug .

Tuning Plant Promoters Using a Simple Split Luciferase Method to Assess Transcription Factor-DNA Interactions.

Sequence features, including the affinity of binding motifs for their cognate transcription factors, are important contributors to promoter behavior. The ability to predictably recode affinity enables the development of synthetic promoters with varying levels of response to known cellular signals. Here we describe a luminescence-based microplate assay for comparing the interactions of transcription factors with short DNA probes.

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes.

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests.

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors.

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana.

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules.

Cas9-Mediated Targeted Mutagenesis in Plants.

Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target.

Biofoundry-assisted expression and characterization of plant proteins.

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols.

Insect pest management in the age of synthetic biology.

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown.

Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR.

Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations.

A biofoundry workflow for the identification of genetic determinants of microbial growth inhibition.

Biofoundries integrate high-throughput software and hardware platforms with synthetic biology approaches to enable the design, execution and analyses of large-scale experiments. The unique and powerful combination of laboratory infrastructure and expertise in molecular biology and automation programming, provide flexible resources for a wide range of workflows and research areas. Here, we demonstrate the applicability of biofoundries to molecular microbiology, describing the development and application of automated workflows to identify the genetic basis of growth inhibition of the plant pathogen by a strain isolated from a potato field Combining transposon mutagenesis with automated high-throughput antagonistic assays, the workflow accelerated the screening of 2880 mutants to correlate growth inhibition with a biosynthetic gene cluster within 2 weeks.

Evaluation of existing guidelines for their adequacy for the molecular characterisation and environmental risk assessment of genetically modified plants obtained through synthetic biology.

Synthetic Biology (SynBio) is an interdisciplinary field at the interface of engineering and biology aiming to develop new biological systems and impart new functions to living cells. EFSA has been asked by the European Commission to evaluate SynBio developments in agri-food with the aim of identifying the adequacy of existing guidelines for risk assessment and determine if updated guidance is needed. The scope of this opinion covers the molecular characterisation and environmental risk assessment of such genetically modified plants obtained through SynBio, meant to be for cultivation or food and feed purposes.

80 questions for UK biological security.

Multiple national and international trends and drivers are radically changing what biological security means for the United Kingdom (UK). New technologies present novel opportunities and challenges, and globalisation has created new pathways and increased the speed, volume and routes by which organisms can spread. The UK Biological Security Strategy (2018) acknowledges the importance of research on biological security in the UK.

Rational design of minimal synthetic promoters for plants.

Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression.

Phytobricks: Manual and Automated Assembly of Constructs for Engineering Plants.

Phytobricks are standardized DNA parts for plants that can be assembled hierarchically into transcriptional units and, subsequently, into multigene constructs. Phytobricks each contain the sequences of one or more functional elements that comprise eukaryotic transcription units, with sequence features that enable them to be used interchangeably in one-step cloning reactions to facilitate combinatorial assembly. The simplicity and efficiency of this one-step reaction has enabled Phytobrick assembly to be miniaturized and automated on liquid handing platforms.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe.

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study.

Bioengineering horizon scan 2020.

Horizon scanning is intended to identify the opportunities and threats associated with technological, regulatory and social change. In 2017 some of the present authors conducted a horizon scan for bioengineering (Wintle et al., 2017).

Beyond natural: synthetic expansions of botanical form and function.

Powered by developments that enabled genome-scale investigations, systems biology emerged as a field aiming to understand how phenotypes emerge from network functions. These advances fuelled a new engineering discipline focussed on synthetic reconstructions of complex biological systems with the goal of predictable rational design and control. Initially, progress in the nascent field of synthetic biology was slow due to the ad hoc nature of molecular biology methods such as cloning.

Systematic Tools for Reprogramming Plant Gene Expression in a Simple Model, .

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities.