Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

Overexpression of PSR1 in Chlamydomonas reinhardtii induces luxury phosphorus uptake.

Remediation using micro-algae offers an attractive solution to environmental phosphate (PO4 3-) pollution. However, for maximum efficiency, pre-conditioning of algae to induce 'luxury phosphorus (P) uptake' is needed. To replicate this process, we targeted the global regulator PSR1 (Myb transcription factor: Phosphate Starvation Response 1) for over-expression in algae. Manipulating a single gene (PSR1) drove uptake of both PO4 3- and a Mg2+ counter-ion leading to increased PolyP granule size, raising P levels 4-fold to 8% dry cell weight, and accelerated removal of PO4 3- from the medium. Examination of the gene expression profile showed that the P-starvation response was mimicked under P-replete conditions, switching on luxury uptake. Hyper-accumulation of P depended on a feed-forward mechanism, where a small set of 'Class I' P-transporter genes were activated despite abundant external PO4 3- levels. The transporters drove a reduction in external PO4 3- levels, permitting more genes to be expressed (Class II), leading to more P-uptake. Our data pointed toward a PSR1-independent mechanism for detection of external PO4 3- which suppressed Class II genes. This model provided a plausible mechanism for P-overplus where prior P-starvation elevates PSR1 and on P-resupply causes luxury P-uptake. This is because the Class I genes, which include P-transporter genes, are not suppressed by the excess PO4 3-. Taken together, these discoveries facilitate a bio-circular approach of recycling nutrients from wastewater back to agriculture.

Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches.

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little information is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP-P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Exploring the Impact of Terminators on Transgene Expression in Chlamydomonas reinhardtii with a Synthetic Biology Approach.

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.

Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas.

Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5'UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.

Responses of a Newly Evolved Auxotroph of Chlamydomonas to B12 Deprivation.

The corrinoid B12 is synthesized only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions, and we previously demonstrated that experimental evolution of the non-B12-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species, which preceded rapid cell death. Survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to coping with B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B12 limitation better.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

Synthetic biology approaches for the production of plant metabolites in unicellular organisms.

Synthetic biology is the repurposing of biological systems for novel objectives and applications. Through the co-ordinated and balanced expression of genes, both native and those introduced from other organisms, resources within an industrial chassis can be siphoned for the commercial production of high-value commodities. This developing interdisciplinary field has the potential to revolutionize natural product discovery from higher plants, by providing a diverse array of tools, technologies, and strategies for exploring the large chemically complex space of plant natural products using unicellular organisms. In this review, we emphasize the key features that influence the generation of biorefineries and highlight technologies and strategic solutions that can be used to overcome engineering pitfalls with rational design. Also presented is a succinct guide to assist the selection of unicellular chassis most suited for the engineering and subsequent production of the desired natural product, in order to meet the global demand for plant natural products in a safe and sustainable manner.

ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed.

Elucidation of the carotenoid biosynthetic pathway has enabled altering the composition and content of carotenoids in various plants, but to achieve desired nutritional impacts, the genetic components regulating carotenoid homeostasis in seed, the plant organ consumed in greatest abundance, must be elucidated. We used a combination of linkage mapping, genome-wide association studies (GWAS), and pathway-level analysis to identify nine loci that impact the natural variation of seed carotenoids in Arabidopsis (Arabidopsis thaliana). ZEAXANTHIN EPOXIDASE (ZEP) was the major contributor to carotenoid composition, with mutants lacking ZEP activity showing a remarkable 6-fold increase in total seed carotenoids relative to the wild type. Natural variation in ZEP gene expression during seed development was identified as the underlying mechanism for fine-tuning carotenoid composition, stability, and ultimately content in Arabidopsis seed. We previously showed that two CAROTENOID CLEAVAGE DIOXYGENASE enzymes, CCD1 and CCD4, are the primary mediators of seed carotenoid degradation, and here we demonstrate that ZEP acts as an upstream control point of carotenoid homeostasis, with ZEP-mediated epoxidation targeting carotenoids for degradation by CCD enzymes. Finally, four of the nine loci/enzymatic activities identified as underlying natural variation in Arabidopsis seed carotenoids also were identified in a recent GWAS of maize (Zea mays) kernel carotenoid variation. This first comparison of the natural variation in seed carotenoids in monocots and dicots suggests a surprising overlap in the genetic architecture of these traits between the two lineages and provides a list of likely candidates to target for selecting seed carotenoid variation in other species.

Redefining the metabolic continuity of chloroplasts and ER.

As a hub for plant metabolism, plastids extensively exchange metabolites with the extraplastid environment. For polar metabolites, membrane transporters mediate this exchange, but for many plastid-synthesized nonpolar compounds, such transporters remain elusive. Here, we discuss recent data from transorganellar complementation studies that demonstrate that enzymes in one organelle can directly access nonpolar metabolites from a companion organelle. We propose that a mechanism, based on hemifused-membranes at plastid-endoplasmic reticulum (ER) contact sites, facilitates interorganellar interactions and allows enzymes direct, transporter-independent access to a range of nonpolar compounds in both organelle membranes. In a wider context, interorganellar metabolism at hemifusion interfaces would allow evolution of membrane-spanning pathways for the many thousands of nonpolar metabolites in the plant kingdom to be uncoupled from coevolution with nonpolar metabolite transporters.

Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts.

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.

Engineering starch accumulation by manipulation of phosphate metabolism of starch.

A new understanding of leaf starch degradation has emerged in the last 10 years. It has been shown that starch phosphorylation and dephosphorylation are critical components of this process. Glucan, water dikinase (GWD) (and phosphoglucan, water dikinase) adds phosphate to starch, and phosphoglucan phosphatase (SEX4) removes these phosphates. To explore the use of this metabolism to manipulate starch accumulation, Arabidopsis (Arabidopsis thaliana) plants were engineered by introducing RNAi constructs designed to reduce expression of AtGWD and AtSEX4. The timing of starch build-up was altered with ethanol-inducible and senescence-induced gene promoters. Ethanol induction of RNAi lines reduced transcript for AtGWD and AtSEX4 by 50%. The transgenic lines had seven times more starch than wild type at the end of the dark period but similar growth rates and total biomass. Elevated leaf starch content in maize leaves was engineered by making an RNAi construct against a gene in maize that appeared to be homologous to AtGWD. The RNAi construct was expressed using the constitutive ubiquitin promoter. Leaf starch content at the end of a night period in engineered maize plants was 20-fold higher than in untransformed plants with no impact on total plant biomass. We conclude that plants can be engineered to accumulate starch in the leaves with little impact on vegetative biomass.

A central role for gamma-glutamyl hydrolases in plant folate homeostasis.

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development.

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.

Folate biofortification in food plants.

Folate deficiency is a global health problem affecting many people in the developing and developed world. Current interventions (industrial food fortification and supplementation by folic acid pills) are effective if they can be used but might not be possible in less developed countries. Recent advances demonstrate that folate biofortification of food crops is now a feasible complementary strategy to fight folate deficiency worldwide. The genes and enzymes of folate synthesis are sufficiently understood to enable metabolic engineering of the pathway, and results from pilot engineering studies in plants (and bacteria) are encouraging. Here, we review the current status of investigations in the field of folate enhancement on the eve of a new era in food fortification.