Missing Genomic Resources for the Next Generation of Environmental Risk Assessment.

Environmental risk assessment traditionally relies on a wide range of in vivo testing to assess the potential hazards of chemicals in the environment. These tests are often time-consuming and costly and can cause test organisms' suffering. Recent developments of reliable low-cost alternatives, both in vivo- and in silico-based, opened the door to reconsider current toxicity assessment. However, many of these new approach methodologies (NAMs) rely on high-quality annotated genomes for surrogate species of regulatory risk assessment. Currently, a lack of genomic information slows the process of NAM development. Here, we present a phylogenetically resolved overview of missing genomic resources for surrogate species within a regulatory ecotoxicological risk assessment. We call for an organized and systematic effort within the (regulatory) ecotoxicological community to provide these missing genomic resources. Further, we discuss the potential of a standardized genomic surrogate species landscape to enable a robust and nonanimal-reliant ecotoxicological risk assessment in the systems ecotoxicology era.

A synthetic C4 shuttle via the ß-hydroxyaspartate cycle in C3 plants.

Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO2 fixation. However, especially in C3 plants, photosynthetic yield is reduced by formation of 2-phosphoglycolate, a toxic oxygenation product of Rubisco, which needs to be recycled in a high-flux-demanding metabolic process called photorespiration. Canonical photorespiration dissipates energy and causes carbon and nitrogen losses. Reducing photorespiration through carbon-concentrating mechanisms, such as C4 photosynthesis, or bypassing photorespiration through metabolic engineering is expected to improve plant growth and yield. The ß-hydroxyaspartate cycle (BHAC) is a recently described microbial pathway that converts glyoxylate, a metabolite of plant photorespiration, into oxaloacetate in a highly efficient carbon-, nitrogen-, and energy-conserving manner. Here, we engineered a functional BHAC in plant peroxisomes to create a photorespiratory bypass that is independent of 3-phosphoglycerate regeneration or decarboxylation of photorespiratory precursors. While efficient oxaloacetate conversion in Arabidopsis thaliana still masks the full potential of the BHAC, nitrogen conservation and accumulation of signature C4 metabolites demonstrate the proof of principle, opening the door to engineering a photorespiration-dependent synthetic carbon-concentrating mechanism in C3 plants.

Mechanistic understanding of photorespiration paves the way to a new green revolution.

Photorespiration is frequently considered a wasteful and inefficient process. However, mutant analysis demonstrated that photorespiration is essential for recycling of 2-phosphoglycolate in C3 and C4 land plants, in algae, and even in cyanobacteria operating carboxysome-based carbon (C) concentrating mechanisms. Photorespiration links photosynthetic C assimilation with other metabolic processes, such as nitrogen and sulfur assimilation, as well as C1 metabolism, and it may contribute to balancing the redox poise between chloroplasts, peroxisomes, mitochondria and cytoplasm. The high degree of metabolic interdependencies and the pleiotropic phenotypes of photorespiratory mutants impedes the distinction between core and accessory functions. Newly developed synthetic bypasses of photorespiration, beyond holding potential for significant yield increases in C3 crops, will enable us to differentiate between essential and accessory functions of photorespiration.

The impact of synthetic biology for future agriculture and nutrition.

Global food production needs to be increased by 70% to meet demands by 2050. Current agricultural practices cannot cope with this pace and furthermore are not ecologically sustainable. Innovative solutions are required to increase productivity and nutritional quality. The interdisciplinary field of synthetic biology implements engineering principles into biological systems and currently revolutionizes fundamental and applied research. We review the diverse spectrum of synthetic biology applications that started impacting plant growth and quality. We focus on latest advances for synthetic carbon-conserving pathways in vitro and in planta to improve crop yield. We highlight strategies improving plant nutrient usage and simultaneously reduce fertilizer demands, exemplified with the engineering of nitrogen fixation in crops or of synthetic plant-microbiota systems. Finally, we address engineering approaches to increase crop nutritional value as well as the use of photoautotrophic organisms as autonomous factories for the production of biopharmaceuticals and other compounds of commercial interest.

In Vitro Analysis of Metabolite Transport Proteins.

The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.