Transcriptome-informed identification and characterization of Planococcus citri cis- and trans-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 Planococcus citri. We queried the available genomic and newly generated short- and long-read P. citri transcriptomic data and identified 18 putative IDS genes, whose phylogenetic analysis indicates several gene family expansion events. In vitro testing confirmed regular short-chain coupling activity with five gene products. With the candidate with highest IDS activity, we also detected low amounts of irregular coupling products, and determined amino acid residues important for chain-length preference and irregular coupling activity. This work therefore provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, including the sex pheromone biosynthesis in P. citri.

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. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

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. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.

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. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichia coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11-amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

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. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

Aromatic Decoration Determines the Formation of Anthocyanic Vacuolar Inclusions.

Anthocyanins are some of the most widely occurring secondary metabolites in plants, responsible for the orange, red, purple, and blue colors of flowers and fruits and red colors of autumn leaves. These pigments accumulate in vacuoles, and their color is influenced by chemical decorations, vacuolar pH, the presence of copigments, and metal ions. Anthocyanins are usually soluble in the vacuole, but in some plants, they accumulate as discrete sub-vacuolar structures. Studies have distinguished intensely colored intra-vacuolar bodies observed in the cells of highly colored tissues, termed anthocyanic vacuolar inclusions (AVIs), from more globular, membrane-bound anthocyanoplasts. We describe a system in tobacco that adds additional decorations to the basic anthocyanin, cyanidin 3-O-rutinoside, normally formed by this species. Using this system, we have been able to establish which decorations underpin the formation of AVIs, the conditions promoting AVI formation, and, consequently, the mechanism by which they form.

Ectopic expression of snapdragon transcription factors facilitates the identification of genes encoding enzymes of anthocyanin decoration in tomato.

Given the potential health benefits of polyphenolic compounds in the diet, there is a growing interest in the generation of food crops enriched with health-protective flavonoids. We undertook a series of metabolite analyses of tomatoes ectopically expressing the Delila and Rosea1 transcription factor genes from snapdragon (Antirrhinum majus), paying particular attention to changes in phenylpropanoids compared to controls. These analyses revealed multiple changes, including depletion of rutin and naringenin chalcone, and enhanced levels of anthocyanins and phenylacylated flavonol derivatives. We isolated and characterized the chemical structures of the two most abundant anthocyanins, which were shown by NMR spectroscopy to be delphinidin-3-(4'''-O-trans-p-coumaroyl)-rutinoside-5-O-glucoside and petunidin-3-(4'''-O-trans-p-coumaroyl)-rutinoside-5-O-glucoside. By performing RNA sequencing on both purple fruit and wild-type fruit, we obtained important information concerning the relative expression of both structural and transcription factor genes. Integrative analysis of the transcript and metabolite datasets provided compelling evidence of the nature of all anthocyanin biosynthetic genes, including those encoding species-specific anthocyanin decoration enzymes. One gene, SlFdAT1 (Solyc12g088170), predicted to encode a flavonoid-3-O-rutinoside-4'''-phenylacyltransferase, was characterized by assays of recombinant protein and over-expression assays in tobacco. The combined data are discussed in the context of both our current understanding of phenylpropanoid metabolism in Solanaceous species, and evolution of flavonoid decorating enzymes and their transcriptional networks in various plant species.