Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Harnessing bioengineered microbes as a versatile platform for space nutrition.

Human enterprises through the solar system will entail long-duration voyages and habitation creating challenges in maintaining healthy diets. We discuss consolidating multiple sensory and nutritional attributes into microorganisms to develop customizable food production systems with minimal inputs, physical footprint, and waste. We envisage that a yeast collection bioengineered for one-carbon metabolism, optimal nutrition, and diverse textures, tastes, aromas, and colors could serve as a flexible food-production platform. Beyond its potential for supporting humans in space, bioengineered microbial-based food could lead to a new paradigm for Earth's food manufacturing that provides greater self-sufficiency and removes pressure from natural ecosystems.

Correction: Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pgen.1005824.].

Corrigendum: Evolutionary Recycling of Light Signaling Components in Fleshy Fruits: New Insights on the Role of Pigments to Monitor Ripening.

[This corrects the article DOI: 10.3389/fpls.2016.00263.].

An engineered extraplastidial pathway for carotenoid biofortification of leaves.

Carotenoids are lipophilic plastidial isoprenoids highly valued as nutrients and natural pigments. A correct balance of chlorophylls and carotenoids is required for photosynthesis and therefore highly regulated, making carotenoid enrichment of green tissues challenging. Here we show that leaf carotenoid levels can be boosted through engineering their biosynthesis outside the chloroplast. Transient expression experiments in Nicotiana benthamiana leaves indicated that high extraplastidial production of carotenoids requires an enhanced supply of their isoprenoid precursors in the cytosol, which was achieved using a deregulated form of the main rate-determining enzyme of the mevalonic acid (MVA) pathway. Constructs encoding bacterial enzymes were used to convert these MVA-derived precursors into carotenoid biosynthetic intermediates that do not normally accumulate in leaves, such as phytoene and lycopene. Cytosolic versions of these enzymes produced extraplastidial carotenoids at levels similar to those of total endogenous (i.e. chloroplast) carotenoids. Strategies to enhance the development of endomembrane structures and lipid bodies as potential extraplastidial carotenoid storage systems were not successful to further increase carotenoid contents. Phytoene was found to be more bioaccessible when accumulated outside plastids, whereas lycopene formed cytosolic crystalloids very similar to those found in the chromoplasts of ripe tomatoes. This extraplastidial production of phytoene and lycopene led to an increased antioxidant capacity of leaves. Finally, we demonstrate that our system can be adapted for the biofortification of leafy vegetables such as lettuce.

Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development.

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.

PHYTOCHROME-INTERACTING FACTOR 3 mediates light-dependent induction of tocopherol biosynthesis during tomato fruit ripening.

Tocopherols are important antioxidants exclusively produced in plastids that protect the photosynthetic apparatus from oxidative stress. These compounds with vitamin E activity are also essential dietary nutrients for humans. Although the tocopherol biosynthetic pathway has been elucidated, the mechanisms that regulate tocopherol production and accumulation remain elusive. Here, we investigated the regulatory mechanism underlying tocopherol biosynthesis during ripening in tomato fruits, which are an important source of vitamin E. Our results show that ripening under light conditions increases tocopherol fruit content in a phytochrome-dependent manner by the transcriptional regulation of biosynthetic genes. Moreover, we show that light-controlled expression of the GERANYLGERANYL DIPHOSPHATE REDUCTASE (SlGGDR) gene, responsible for the synthesis of the central tocopherol precursor phytyl diphosphate, is mediated by PHYTOCHROME-INTERACTING FACTOR 3 (SlPIF3). In the absence of light, SlPIF3 physically interacts with the promoter of SlGGDR, down-regulating its expression. By contrast, light activation of phytochromes prevents the interaction between SlPIF3 and the SlGGDR promoter, leading to transcriptional derepression and higher availability of the PDP precursor for tocopherol biosynthesis. The unraveled mechanism provides a new strategy to manipulate fruit metabolism towards improving tomato nutritional quality.

The Multiplanetary Future of Plant Synthetic Biology.

The interest in human space journeys to distant planets and moons has been re-ignited in recent times and there are ongoing plans for sending the first manned missions to Mars in the near future. In addition to generating oxygen, fixing carbon, and recycling waste and water, plants could play a critical role in producing food and biomass feedstock for the microbial manufacture of materials, chemicals, and medicines in long-term interplanetary outposts. However, because life on Earth evolved under the conditions of the terrestrial biosphere, plants will not perform optimally in different planetary habitats. The construction or transportation of plant growth facilities and the availability of resources, such as sunlight and liquid water, may also be limiting factors, and would thus impose additional challenges to efficient farming in an extraterrestrial destination. Using the framework of the forthcoming human missions to Mars, here we discuss a series of bioengineering endeavors that will enable us to take full advantage of plants in the context of a Martian greenhouse. We also propose a roadmap for research on adapting life to Mars and outline our opinion that synthetic biology efforts towards this goal will contribute to solving some of the main agricultural and industrial challenges here on Earth.

Interference with Clp protease impairs carotenoid accumulation during tomato fruit ripening.

Profound metabolic and structural changes are required for fleshy green fruits to ripen and become colorful and tasty. In tomato (Solanum lycopersicum), fruit ripening involves the differentiation of chromoplasts, specialized plastids that accumulate carotenoid pigments such as ß-carotene (pro-vitamin A) and lycopene. Here, we explored the role of the plastidial Clp protease in chromoplast development and carotenoid accumulation. Ripening-specific silencing of one of the subunits of the Clp proteolytic complex resulted in ß-carotene-enriched fruits that appeared orange instead of red when ripe. Clp-defective fruit displayed aberrant chromoplasts and up-regulated expression of nuclear genes encoding the tomato homologs of Orange (OR) and ClpB3 chaperones, most probably to deal with misfolded and aggregated proteins that could not be degraded by the Clp protease. ClpB3 and OR chaperones protect the carotenoid biosynthetic enzymes deoxyxylulose 5-phosphate synthase and phytoene synthase, respectively, from degradation, whereas OR chaperones additionally promote chromoplast differentiation by preventing the degradation of carotenoids such as ß-carotene. We conclude that the Clp protease contributes to the differentiation of chloroplasts into chromoplasts during tomato fruit ripening, acting in co-ordination with specific chaperones that alleviate protein folding stress, promote enzyme stability and accumulation, and prevent carotenoid degradation.

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light.

Light stimulates the biosynthesis of carotenoids and regulates the development of plastid structures to accommodate these photoprotective pigments. Work with Arabidopsis revealed molecular factors coordinating carotenoid biosynthesis and storage with photosynthetic development during deetiolation, when underground seedlings emerge to the light. Some of these factors also adjust carotenoid biosynthesis in response to plant proximity (i.e., shade), a mechanism that was readapted in tomato to monitor fruit ripening progression. While light positively impacts carotenoid production and accumulation in most cases, total carotenoid levels decrease in roots of colored carrot cultivars when illuminated. The recent discovery that such cultivars might be photomorphogenic mutants provides an explanation for this striking phenotype.

Rewiring carotenoid biosynthesis in plants using a viral vector.

Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers.

A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms.

A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments.

Regulation of Carotenoid Biosynthesis in Photosynthetic Organs.

A substantial proportion of the dazzling diversity of colors displayed by living organisms throughout the tree of life is determined by the presence of carotenoids, which most often provide distinctive yellow, orange and red hues. These metabolites play fundamental roles in nature that extend far beyond their importance as pigments. In photosynthetic lineages, carotenoids are essential to sustain life, since they have been exploited to maximize light harvesting and protect the photosynthetic machinery from photooxidative stress. Consequently, photosynthetic organisms have evolved several mechanisms that adjust the carotenoid metabolism to efficiently cope with constantly fluctuating light environments. This chapter will focus on the current knowledge concerning the regulation of the carotenoid biosynthetic pathway in leaves, which are the primary photosynthetic organs of most land plants.

Evolutionary Recycling of Light Signaling Components in Fleshy Fruits: New Insights on the Role of Pigments to Monitor Ripening.

Besides an essential source of energy, light provides environmental information to plants. Photosensory pathways are thought to have occurred early in plant evolution, probably at the time of the Archaeplastida ancestor, or perhaps even earlier. Manipulation of individual components of light perception and signaling networks in tomato (Solanum lycopersicum) affects the metabolism of ripening fruit at several levels. Most strikingly, recent experiments have shown that some of the molecular mechanisms originally devoted to sense and respond to environmental light cues have been re-adapted during evolution to provide plants with useful information on fruit ripening progression. In particular, the presence of chlorophylls in green fruit can strongly influence the spectral composition of the light filtered through the fruit pericarp. The concomitant changes in light quality can be perceived and transduced by phytochromes (PHYs) and PHY-interacting factors, respectively, to regulate gene expression and in turn modulate the production of carotenoids, a family of metabolites that are relevant for the final pigmentation of ripe fruits. We raise the hypothesis that the evolutionary recycling of light-signaling components to finely adjust pigmentation to the actual ripening stage of the fruit may have represented a selective advantage for primeval fleshy-fruited plants even before the extinction of dinosaurs.

Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid.

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution.

The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution.