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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein’s catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. In conclusion, the reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family ( e . g ., CcoA) are required for Cu import and heme-Cu B center biogenesis of the cbb 3 -type cytochrome c oxidases ( cbb 3 -Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo 3 -type quinol oxidases ( bo 3 -Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo 3 -Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E . coli bo 3 -Qox. When respiratory growth is dependent on bo 3 -Qox, because of deletion of the bd -type Qox enzymes, three candidate genes, yhjE , ydiM , and yfcJ , were found to be critical for E . coli growth. Radioactive metal uptake assays showed that Δ ydiM has a slower 64 Cu uptake, whereas Δ yhjE accumulates reduced 55 Fe in the cell, while no similar uptake defect is associated with Δ ycfJ . Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Glucuronidase Function in Clinical Samples

The purpose of this CRADA is to expand upon the work previously performed through CRADA No. PNNL/381. As explained in the previous CRADA, Glucuronidase enzymes are important for contributing to enterohepatic recycling of numerous therapeutic drug compounds, resulting in impaired or prolonged drug activity. Further, other pathways within the gut, such as bile acid metabolism through the action of bile salt hydrolase enzymes (BSH), have shown to impact both the development of colon cancers and impact recovery. Adapting the activity-based protein profiling (ABPP) technologies developed at PNNL to a microtiter plate-based chemical probe assay was the first step in demonstrating the clinical usability of the technology. With assay proof-of-concept complete through CRADA No. PNNL/381, clinical cohort testing using the developed assay is the next step towards commercialization of this technology. Some chemotherapeutic agents are mediated by gut-produced microbiota through enterohepatic circulation. For example, irinotecan, the first line treatment for many colon and other cancers, can be recycled into a patient’s system through gut microbiota produced glucuronidase enzymes. A significant percentage of patients subjected to irinotecan treatment experience dose-limiting diarrhea and gastric distress that limits treatment options and delays effective intervention. Relatedly, bile salt hydrolases modify ratios of bile acids in the gut, which alter nutrient and pharmaceutical uptake by the intestines and impact patient response to treatment. Coupled together these gut-mediated disorders can significantly decrease patient outcomes. In this project, we propose to investigate a clinical cohort of samples from colon cancer patients to better understand gut microbiome activities surrounding microbiota produced glucuronidases to aid clinician decisions and enhance therapeutic intervention. Our project will measure glucuronidase activity of individual patients and compare this activity to a variety of factors, including patient medical history, treatment response, and overall outcomes. Data will be compared with metagenomic, metabolomic, and proteomic analyses to fully defined the entire gut microbiome space and correlate results with the glucuronidase activity. Resultant analysis is expected to provide actionable information to direct decision making towards therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Increased activity of core photorespiratory enzymes and CO 2 transfer conductances are associated with higher and more optimal photosynthetic rates under elevated temperatures in the extremophile Rhazya stricta

Increase photorespiration and optimising intrinsic water use efficiency are unique challenges to photosynthetic carbon fixation at elevated temperatures. To determine how plants can adapt to facilitate high rates of photorespiration at elevated temperatures while also maintaining water-use efficiency, we performed in-depth gas exchange and biochemical assays of the C 3 extremophile, Rhazya stricta. These results demonstrate that R. stricta supports higher rates of photorespiration under elevated temperatures and that these higher rates of photorespiration correlate with increased activity of key photorespiratory enzymes; phosphoglycolate phosphatase and catalase. The increased photorespiratory enzyme activities may increase the overall capacity of photorespiration by reducing enzymatic bottlenecks and allowing minimal inhibitor accumulation under high photorespiratory rates. Additionally, we found the CO 2 transfer conductances (stomatal and mesophyll) are re-allocated to increase the water-use efficiency in R. stricta but not necessarily the photosynthetic response to temperature. These results suggest important adaptive strategies in R. stricta that maintain photosynthetic rates under elevated temperatures with optimal water loss. The strategies found in R. stricta may inform breeding and engineering efforts in other C 3 species to improve photosynthetic efficiency at high temperatures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A review on bacteria-derived antioxidant metabolites: their production, purification, characterization, potential applications, and limitations

Abstract Antioxidants are organic molecules that scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), thereby maintaining cellular redox balance in living organisms. The human body synthesizes endogenous antioxidants, whereas humans obtain exogenous antioxidants from other organisms such as plants, animals, fungi, and bacteria. This review primarily focuses on the antioxidant potential of natural metabolites and extracts from five major bacterial phyla, including the well-studiedActinobacteriaandCyanobacteria, as well as less-studiedBacteroides,Firmicutes, andProteobacteria.The literature survey revealed that the metabolites and the extracts with antioxidant activity can be obtained from bacterial cells and their culture supernatants. The metabolites with antioxidant activity include pigments, phycobiliproteins, polysaccharides, mycosporins-like amino acids, peptides, phenolic compounds, and alkaloids. Both metabolites and extracts demonstrate in vitro antioxidant capacity through radical-scavenging, metal-reducing, and metal-chelating activity assays. In in vivo models, they can scavenge ROS and RNS directly and/or indirectly eliminate them by enhancing the activities of antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase. Due to their antioxidant activities, they may find applications in the cosmetic industry as anti-aging agents for the skin and in medicine as drugs or supplements for combating oxidative stress-related disorders, such as neurodegenerative diseases and diabetes. The literature survey also elucidated that some metabolites and extracts with antioxidant activity also exhibited strong antimicrobial properties. Therefore, we consider that they may have future applications in the treatment of infectious diseases, the preparation of pathogen-free healthy foods, and the extension of food shelf life.

Pharmacology & Pharmacy↗
DOE OSTI2022

The rate of microtubule breaking increases exponentially with curvature

Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.

42 ENGINEERING↗
DOE OSTI2021

No evidence of top–down effects by ants on litter decomposition in a temperate grassland

Ants play multiple roles in ecosystems, but their ability to affect decomposition processes in temperate grasslands is relatively unknown. We investigated whether the suppression of ant populations influenced litter decomposition in grasslands via predation of some decomposers (e.g., mites and springtails) and/or microbial activity and composition. We performed two successful ant suppression treatments (seven weeks, 37% suppression, year 1, 10 weeks, 70% suppression, year 2) over the course of a 59-week experiment. We then assayed the effects of ant suppression using coarse- and fine-mesh litterbags and evaluated litter chemistry, microbial and arthropod communities, and microbial enzyme activity. Ant suppression efforts reduced ant abundance and altered ant, arthropod decomposer, and non-ant predator community composition. However, ant suppression did not affect decomposer arthropod abundance, litter mass loss, microbial composition, or enzyme activity in litterbags. Litterbag mesh size did not alter microbial composition, perhaps due to a failure to exclude decomposers, as mites and springtails were more or equally abundant in fine-mesh bags. Nevertheless, mesh size did change litter chemistry, suggesting that mesh size-mediated microenvironments affect decomposition environment regardless of invertebrate exclusion. Coarse-mesh litterbags had higher concentrations of microbial sugars, lignin, and N and lower concentrations of litter C and crystalline cellulose than fine-mesh litterbags. Litterbag mesh size may alter decomposition processes irrespective of invertebrate abundance. We found no evidence that ant predation was an important driver of decomposer populations or decomposition in these systems, and we suspect redundancy at the top of the detrital food web dilutes the role of ants.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS1987

An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.

Farrington, Marianne A.↗
DOE OSTI2023

Aim18p and Aim46p are chalcone isomerase domain–containing mitochondrial hemoproteins in Saccharomyces cerevisiae

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid–binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Plant-specific features of respiratory supercomplex I + III2 from Vigna radiata

Abstract The last steps of cellular respiration—an essential metabolic process in plants—are carried out by mitochondrial oxidative phosphorylation. This process involves a chain of multi-subunit membrane protein complexes (complexes I–V) that form higher-order assemblies called supercomplexes. Although supercomplexes are the most physiologically relevant form of the oxidative phosphorylation complexes, their functions and structures remain mostly unknown. Here we present the cryogenic electron microscopy structure of the supercomplex I + III 2 from Vigna radiata (mung bean). The structure contains the full subunit complement of complex I, including a newly assigned, plant-specific subunit. It also shows differences in the mitochondrial processing peptidase domain of complex III 2 relative to a previously determined supercomplex with complex IV. The supercomplex interface, while reminiscent of that in other organisms, is plant specific, with a major interface involving complex III 2 ’s mitochondrial processing peptidase domain and no participation of complex I’s bridge domain. The complex I structure suggests that the bridge domain sets the angle between the enzyme’s two arms, limiting large-scale conformational changes. Moreover, complex I’s catalytic loops and its response in active-to-deactive assays suggest that, in V. radiata , the resting complex adopts a non-canonical state and can sample deactive- or open-like conformations even in the presence of substrate. This study widens our understanding of the possible conformations and behaviour of complex I and supercomplex I + III 2 . Further studies of complex I and its supercomplexes in diverse organisms are needed to determine the universal and clade-specific mechanisms of respiration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

Background: Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available. Results: We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein-ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. Conclusions: This method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Multiplexed profiling of transcriptional regulators in plant cells

Transcriptional regulators play key roles in plant growth, development and environmental responses; however, understanding how their regulatory activity is encoded at the protein level has been hindered by a lack of multiplexed large-scale methods to characterize protein libraries in planta. Here we present enrichment of nuclear trans-elements reporter assay in plants with sequencing (ENTRAP-seq), a high-throughput method that introduces protein-coding libraries into plant cells to drive a nuclear magnetic sorting-based reporter, enabling multiplexed measurement of regulatory activity from thousands of protein variants. Using ENTRAP-seq and machine learning, we screen 1,495 plant viruses and identify hundreds of putative transcriptional regulatory domains found in structural proteins and enzymes not associated with gene regulation. In addition, we combine ENTRAP-seq with machine-guided design to engineer the activity of a plant transcription factor in a semirational fashion. Our findings demonstrate how scalable protein function assays deployed in planta will enable the characterization of natural and synthetic coding diversity in plants.

Alamos, Simon↗
DOE OSTI2024

Aryl hydrocarbon receptor-dependent toxicity by retene requires metabolic competence

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds frequently detected in the environment with widely varying toxicities. Many PAHs activate the aryl hydrocarbon receptor (AHR), inducing the expression of a battery of genes, including xenobiotic metabolizing enzymes like cytochrome P450s (CYPs); however, not all PAHs act via this mechanism. We screened several parent and substituted PAHs in in vitro AHR activation assays to classify their unique activity. Retene (1-methyl-7-isopropylphenanthrene) displays Ahr2-dependent teratogenicity in zebrafish, but did not activate human AHR or zebrafish Ahr2, suggesting a retene metabolite activates Ahr2 in zebrafish to induce developmental toxicity. To investigate the role of metabolism in retene toxicity, studies were performed to determine the functional role of cyp1a, cyp1b1, and the microbiome in retene toxicity, identify the zebrafish window of susceptibility, and measure retene uptake, loss, and metabolite formation in vivo. Cyp1a-null fish were generated using CRISPR-Cas9. Cyp1a-null fish showed increased sensitivity to retene toxicity, whereas Cyp1b1-null fish were less susceptible, and microbiome elimination had no significant effect. Zebrafish required exposure to retene between 24 and 48 hours post fertilization (hpf) to exhibit toxicity. After static exposure, retene concentrations in zebrafish embryos increased until 24 hpf, peaked between 24 and 36 hpf, and decreased rapidly thereafter. We detected retene metabolites at 36 and 48 hpf, indicating metabolic onset preceding toxicity. This study highlights the value of combining molecular and systems biology approaches with mechanistic and predictive toxicology to interrogate the role of biotransformation in AHR-dependent toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

In Vivo Assay Reveals Microbial OleA Thiolases Initiating Hydrocarbon and β-Lactone Biosynthesis

Microbially produced β-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into β-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a β-lactone decarboxylase, OleB. Both β-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris . In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction

Abstract Background Plant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities. Results To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum ( Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea , Filomicrobium , and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes. Conclusions The two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization

Successes in biocatalytic polyester recycling have raised the possibility of deconstructing alternative polymers enzymatically, with polyamide (PA) being a logical target due to the array of amide-cleaving enzymes present in nature. Here, we screen 40 potential natural and engineered nylon-hydrolyzing enzymes (nylonases), using mass spectrometry to quantify eight compounds resulting from enzymatic nylon-6 (PA6) hydrolysis. Comparative time-course reactions incubated at 40-70 °C showcase enzyme-dependent variations in product distributions and extent of PA6 film depolymerization, with significant nylon deconstruction activity appearing rare. The most active nylonase, a NylC K variant we rationally thermostabilized (an N-terminal nucleophile (Ntn) hydrolase, NylC K -TS, T m = 87.4 °C, 16.4 °C higher than the wild-type), hydrolyzes 0.67 wt% of a PA6 film. Reactions fail to restart after fresh enzyme addition, indicating that substrate-based limitations, such as restricted enzyme access to hydrolysable bonds, prohibit more extensive deconstruction. Overall, this study expands our understanding of nylonase activity distribution, indicates that Ntn hydrolases may have the greatest potential for further development, and identifies key targets for progressing PA6 enzymatic depolymerization, including improving enzyme activity, product selectivity, and enhancing polymer accessibility.

09 BIOMASS FUELS↗
NASA NTRS2004

Enzyme-Cascade Analysis of the Rio Tinto Subsurface Environment: A Biosensor Experiment

The Portable Test System (PTS), designed & developed by Charles Rivers Laboratories, Inc. (Charleston, SC) is a portable instrument that was designed to perform analysis of enzymatic assays related to rapid assessment of microbial contamination (Wainwright, 2003). The enzymatic cascade of Limulus Amebocyte Lysate (LAL) is known to be one of the most sensitive techniques available for microbial detection, enabling the PTS to be evaluated as a potential life detection instrument for in situ Astrobiology missions. In the summer of 2003 the system was tested as a part of the Mars Astrobiology Research and Technology Experiment (MARTE) ground truth science campaign in the Rio Tinto Analogue environment near Nerva, Spain. The preliminary results show that the PTS analysis correlates well with the contamination control tests and the more traditional lab-based biological assays performed during the MARTE field mission. Further work will be conducted on this research during a second field campaign in 2004 and a technology demonstration of a prototype instrument that includes autonomous sample preparation will occur in 2005.

McKay, David S.↗
DOE OSTI2022

Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin

Fucoxanthin is a major light-harvesting pigment in ecologically important algae such as diatoms, haptophytes, and brown algae (Phaeophyceae). Therefore, it is a major driver of global primary productivity. Species of these algal groups are brown colored because the high amounts of fucoxanthin bound to the proteins of their photosynthetic machineries enable efficient absorption of green light. While the structure of these fucoxanthin-chlorophyll proteins has recently been resolved, the biosynthetic pathway of fucoxanthin is still unknown. Here, we identified two enzymes central to this pathway by generating corresponding knockout mutants of the diatom Phaeodactylum tricornutum that are green due to the lack of fucoxanthin. Complementation of the mutants with the native genes or orthologs from haptophytes restored fucoxanthin biosynthesis. We propose a complete biosynthetic path to fucoxanthin in diatoms and haptophytes based on the carotenoid intermediates identified in the mutants and in vitro biochemical assays. It is substantially more complex than anticipated and reveals diadinoxanthin metabolism as the central regulatory hub connecting the photoprotective xanthophyll cycle and the formation of fucoxanthin. Moreover, our data show that the pathway evolved by repeated duplication and neofunctionalization of genes for the xanthophyll cycle enzymes violaxanthin de-epoxidase and zeaxanthin epoxidase. Brown algae lack diadinoxanthin and the genes described here and instead use an alternative pathway predicted to involve fewer enzymes. Our work represents a major step forward in elucidating the biosynthesis of fucoxanthin and understanding the evolution, biogenesis, and regulation of the photosynthetic machinery in algae.

59 BASIC BIOLOGICAL SCIENCES↗