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DOE OSTI2016

Consequences of Modification of Photosystem Stoichiometry and Amount in Cyanobacteria

The proposed research seeks to address two interconnected, important questions that impact photosynthetic processes and that reflect key differences between the photosynthetic systems of cyanobacteria and plants or algae. The first question is what are the reasons and consequences of the high photosystem I / photosystem II (PS I/PS II) ratio in many cyanobacteria, vs. a ratio that is close to unity in many plants and algae. The corresponding hypothesis is that most of PS I functions in cyclic electron transport, and that reduction in PS I will result primarily in a shortage of ATP rather than reducing power. This hypothesis will be tested by reducing the amount of PS I by changing the promoter region of the psaAB operon in the cyanobacterium Synechocystis sp. PCC 6803 and generating a range of mutants with different PS I content and thereby different PS I/PS II ratios, with some of the mutants having a PS II/PS I ratio closer to that in plants. The resulting mutants will be probed in terms of their growth rates, electron transfer rates, and P700 redox kinetics. A second question relates to a Mehler-type reaction catalyzed by two flavoproteins, Flv1 and Flv3, that accept electrons from PS I and that potentially function as an electron safety valve leading to no useful purpose of the photosynthesis-generated electrons. The hypothesis to be tested is that Flv1 and Flv3 use the electrons for useful purposes such as cyclic electron flow around PS I. This hypothesis will be tested by analysis of a mutant strain lacking flv3, the gene for one of the flavoproteins. This research is important for a more detailed understanding of the consequences of photosystem stoichiometry and amounts in a living system. Such an understanding is critical for not only insights in the regulatory systems of the organism but also to guide the development of biological or bio-hybrid systems for solar energy conversion into fuels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

From genomes to metabolomes: Understanding mechanisms of symbiosis and cell-cell signaling using the archaeal system Ignicoccus-Nanoarchaeum

The main objective of this project was to use symbiotic Nanoarchaeaota, a group of thermophilic Archaea that are obligate symbionts/parasites on other Archaea, to develop an integrated multi-omic approach to study inter-species interactions as well as to understand fundamental mechanism that enable such relationships. As part of this grant we have achieved a number of important milestone on both technical and scientific levels. On the technical side, we developed immunofluorescence labeling and tracking methods to follow Nanoarchaeota in cultures and in environmental samples, we applied such methods in conjunction with flow cytometry to quantify and isolate uncultured representatives from the environment and characterized them by single cell genomics. On the proteomics side, we developed a more efficient and sensitive method to recover and semi-quantitatively measure membrane proteins, while achieving high total cellular proteome coverage (70-80% of the predicted proteome). Metabolomic analyses used complementary NMR and LC/GC mass spectrometry and led to the identification of novel lipids in these organisms as well as quantification of some of the major metabolites. Importantly, using several informatics approaches we were also able to integrate the transcriptomic, proteomic and metabolomic datasets, revealing aspects of the interspecies interaction that were not evident in the single omic analyses (manuscript in review). On the science side we determined that N. equitans and I. hospitalis are metabolically coupled and that N. equitans is strictly dependent on its host both for metabolic precursors and energetic needs. The actual mechanism by which small molecules move across the cell membrane remains unknown. The Ignicoccus host responds to the metabolic and energetic burned by upregulating of key primary metabolism steps and ATP synthesis. The two species have co-evolved, aspect that we determined by comparative genomics with other species of Ignicoccus (manuscript in preparation) and by characterizing other similar Nanoarchaeota systems. Using a single cell genomics approach we characterized the first terrestrial geothermal Nanoarchaeota system, from Yellowstone National Park. That nanoarchaeon uses a different host, a species of Sulfolobales, and comparative genomics with N. equitans-Ignicoccus allowed us to come up with an evolutionary model for the evolution of this group of organisms across marine and terrestrial ecosystems. Based on metabolic inferences we were also able to isolate in culture the first such terrestrial nanoarchaeal system, also from Yellowstone, which involves a species of Acidilobus. The novel nanoarchaeal system was characterized using proteomics and it helped us better understand the metabolic capabilities of these organisms as well as how co-evolution shapes the genomes of interacting species. It was also one of the very few cases in which prior genomic data was used to successfully design an approach to culture an organism, which remains the gold standard in microbiology research. As a better understanding of interspecies interaction requires multiple model systems, we have pursued identification and genomic characterization or isolation of additional nanoarchaeal systems from geographically and geochemically distinct environments. Two additional nanoarchaeal systems are presently being characterized from hot springs in Yellowstone and Iceland and will be the subject to future publications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Final Report for research on The Glucose 6-Phosphate Shunt Around the Calvin-Benson Cycle

In this research, photosynthetic carbon metabolism was studied to identify mechanisms by which plants store energy from sunlight as carbon compounds, especially sugars. Conditions were identified in which carbon appeared to flow backwards from outside the photosynthetic compartment (chloroplast) back into it. A specific gene product was manipulated to make the flow bigger or smaller. Preventing the flow (by eliminating the gene) had little effect on plant growth but increasing the flow, by overexpressing the gene, caused the plants to become extremely sensitive to changes in light. Plants with the gene overexpressed had high rates of cyclic electron flow, the photosynthetic electron transport pathway that occurs when plants need more of the energy molecule ATP. These and other observations led us to conclude that a metabolic pathway that is normally turned off because it is counter-productive during photosynthesis, in fact occurs at about 10% of the rate of normal photosynthesis. This creates an inefficiency but may stabilize photosynthesis allowing it to cope with the very large and rapid changes that leaves experience such as the hundred-fold changes in light intensity that can occur in seconds on a partly cloudy day. We also concluded that the back flow of carbon into chloroplasts could be important at high rates of photosynthesis allowing increased rates of starch synthesis. Starch synthesis allows plants to store sugars during the day for use at night. At high rates of photosynthesis starch synthesis becomes very important to protect against end-product inhibition of photosynthesis. This research identified two metabolic pathways that extend the primary carbon fixation pathway called the Calvin-Benson cycle. These pathway extensions are now called the cytosolic bypass and the glucose 6-phosphate shunt. This improvement in our understanding of carbon metabolism of photosynthesis will guide efforts to increase photosynthesis to increase production of food, fuel, and fiber.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Molecular Dissection of the Arabidopsis 26S Proteasome

The 26S proteasome is a 2.5-MDa, ATP-dependent protease complex responsible for degrading many important cell regulators in plants and animals, especially those targeted by ubiquitylation. Its 64 or more subunits generate two subparticles: a 20S core protease (CP) that houses the proteolytic active sites and a 19S regulatory particle (RP) that binds to both ends of the CP and recruits appropriate substrates for breakdown. Despite its importance, we had only a rudimentary appreciation of the structure and functions of the core 26S proteasome from plants, and hints that the previously defined complex actually represents the nucleus of an even more elaborate and dynamic particle harboring multiple routes for substrate recognition and sophisticated mechanisms to control its assembly/location. Genomic analyses also suggested that plants in particular exploit this heterogeneity to generate a wide array of proteasome types, each with distinct compositions, locations, and/or functions/specificities. The goal of this DOE BES-funded research was to better define the 26S proteasome and its diversity in plants by a thorough analysis of the particle from Arabidopsis thaliana. During this completed work, we developed stringent affinity methods to rapidly isolate 26S proteasomes intact from Arabidopsis seedlings, and identified by mass spectrometry all the main CP and RP subunits along with a suite of interacting proteins. We also identified a number of post-translational modifications, and detected an alternative proteasome containing the CP capped by the PA200 protein. We then amassed a large collection of mutants eliminating most CP and RP subunits, and notable accessory proteins such as PA200. Phenotypic analyses of a few RP mutants support the notion that the Arabidopsis 26S proteasome is functionally heterogeneous. Through both genetic and biochemical analyses, we characterized a collection of shuttle proteins that help deliver ubiquitylated substrates, and discovered a subset of accessory proteins that likely act as chaperones that facilitate CP and RP assembly. These chaperone activities were supported by examining 26S proteasome assembly in plants missing these factors, which showed that the mutants accumulated high levels of partially assembled subparticles. In an effort to understand how the numerous genes encoding the various proteasome subunits are coordinately regulated to assemble the 64-subunit core complex and ultimately maintain adequate proteolytic capacity, we discovered by RNA-seq that most Arabidopsis genes required to synthesize the 26S proteasome are coordinately regulated transcriptionally. Through analysis of this ‘proteasome-stress’ regulon by protein-DNA binding, and reverse genetic methods, we identified the cis and trans-acting elements involved, that center around a pair of NAC transcription factors (NAC53 and NAC78) binding to a consensus proteasome-related cis element (PRCE) upstream of most proteasome-associated genes. And finally, we discovered that proteasomes are rapidly degraded by autophagy in response to nutrient stress or inactivation via a process we called proteaphagy. Whereas the former turnover is triggered by the ATG1-kinase complex working downstream of stress sensors such as the TOR kinase, the latter turnover is induced by substantial ubiquitylation of the complex followed by recognition of the appended ubiquitin moieties by the ubiquitin-binding autophagy receptor RPN10. Surprisingly, studies with RPN10 revealed that it represents the founding member of a new class of autophagy receptors that exploit a novel way to tether cargo to autophagic membranes. Instead of using the canonical AIM motif to bind ATG8 lining the autophagic membranes, it and relatives exploit a UIM motif to bind ATG8 at an alternative site. Studying the same inhibitor-induced proteaphagy in yeast revealed that an unrelated autophagy receptor Cue5 is required to recognize the bound ubiquitins. Strikingly, we found that carbon starvation also induces the accumulation novel cytoplasmic particles called proteasome storage granules that appear to protect proteasomes from autophagy. Their accumulation in yeast requires the CP-binding protein PA200 along with the RP-binding proteins Spg5. Taken together, this research revealed the composition of plant 26S proteasome, how it is assembled, and how is abundance is regulated both transcriptionally and by autophagy. Collectively, this research enhanced our appreciation of the plant 26S proteasome and its roles in ubiquitin-mediated protein turnover, and identified points of opportunity where its abundance or activity can be manipulated to improve food and biofuel crops.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Regulation of Thylakoid Lipid Biosynthesis in Plants (Final Technical Report)

The conversion of sunlight into chemical energy by photosynthesis drives life on earth as it provides food and feed, as well as industrial feed stocks for specialized chemicals and biofuels. Photosynthesis is a function of the photosynthetic membrane that forms the thylakoids inside plant chloroplasts. Thylakoid membranes harbor the protein complexes necessary for photosynthetic electron transport and ATP synthesis. These complexes are embedded into a specialized glycerolipid matrix. Two decades of research on the biochemistry of the glycerolipids of the photosynthetic membrane, their transport and remodeling in response to environmental cues and the underlying regulatory processes has provided novel fundamental insights into how photosynthetic membranes are assembled and protected in plant chloroplasts. An overview of the key findings is provided.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Ultra Efficient Light Duty Powertrain with Gasoline Low Temperature Combustion (Final Report)

This document details the work completed during Project DE-EE0006839 ‘Ultra Efficient Light Duty Powertrain with Gasoline Low Temperature Combustion’. All tasks from the project’s Statement of Project Objectives are discussed. Final demonstration data is also presented and discussed. Further project details can be found in the Quarterly Technicial Reports that were submitted during the course of the project and are stored on the EERE Project Management Center Website. The goal of this project was to develop, implement and demonstrate a low temperature combustion approach called Gasoline Direct Injection Compression Ignition (GDCI) that provides high thermal efficiency with low NOx and PM emissions. The project target was to demonstrate a 35% fuel economy improvement over the baseline vehicle while meeting Tier 3 emissions levels. At the start of this project, GDCI was a moderately mature combustion technology, due in large part to advances made during a Delphi Technologies program funded through an earlier Department of Energy Advanced Technology Powertrains (ATP) opportunity; under that earlier contract, DE-EE0003258, Delphi Technologies assembled a team with complementary industrial and academic experience, and led this team to advance the technology from the original fundamental work to the state of art embodied by a first-generation (Gen 1) multi-cylinder GDCI engine. Key activities during that project centered on engine and combustion chamber design, and development of fuel injection equipment, valvetrain and advanced controls. Performance results with the Gen 1 engine in a development vehicle demonstrated that GDCI had the capability to operate using Low Temperature Combustion (LTC) over a broad speed-load range. Based on benchmarking work, efficiency for the Gen 1 GDCI engine compared favorably overall to production light-duty diesel engines. The second DOE funded project, DE-EE0006839, focused on a number of technical risks and issues that were left to be resolved for GDCI to become a production-viable technology. These risks were:GDCI combustion system refinement to achieve near-ideal air/fuel mixture preparation for high efficiency and low HC and CO emissions; Transient control development for low temperature combustion with high EGR levels during real-world transient driving maneuvers and over a broader range of ambient conditions; Aftertreatment system design that is effective in dealing with the low-temperature challenges of a highly efficient engine. The project was executed in two phases. Phase 1 leveraged the development vehicle from the Advanced Technologies Powertrain 1 (ATP1) project, DE-EE0003258. That vehicle was used as an initial test bed in the development of a high performance low temperature exhaust aftertreatment system. The vehicle was also used to develop calibration refinements for improved exhaust emissions and fuel efficiency. Samples of a new GDCI development level engine, called Gen2 GDCI (and developed outside of the first DOE funded project), were also used during Phase 1 to develop refined controller hardware and control algorithms, as well as improved sensors and actuators. The Gen2 engine was then retrofitted into the development vehicle from the ATP1 project and used for controls refinement and calibration of GDCI to achieve improved cold start, drivability and robustness to real world environments and fuel variations. During Phase 2 of the project, Gen3 versions of the GDCI engine were designed and built specifically for this project based on experience from the earlier engines. These engines were designed to be production-like in construction and, when combined with refined control systems and project specific low temperature exhaust aftertreatment, were intended to support the goal of meeting Tier 3 emissions levels at a vehicle level. Though great progress had been made advancing the GDCI combustion process, and despite extensive presentations of our work on the technology at various conferences and in direct meetings with automotive companies, none of the US domestic automotive OEMs had shown an interest in adopting this combustion process as part of their technology planning as the project approached the Go/No-Go point at the end of Budget Period 3. As a result of this low-level of interest both the US Department of Energy and Delphi Technologies received in this technology from domestic auto manufactures, Delphi Technologies elected a ‘No-Go’ status for the gate between Budget Period 3 and Budget Period 4 which terminated the project without spending any Budget Period 4 funds. The project team concluded that the dynamometer mapping of the most recent level of hardware, combined with the vehicle level simulation by Argonne National Laboratory, was sufficient to document the capabilities and potential of the combustion process without spending the time and money to do the controls development and full calibration necessary for a demonstration vehicle.

33 ADVANCED PROPULSION SYSTEMS↗

DOE OSTI2019

Biological Electron Transfer and Catalysis Energy Frontier Research Center

The overarching objective of the BETCy EFRC project is to investigate the mechanisms and structural basis controlling electron bifurcation, electron-ion coupling, and redox catalysis in model enzymes. This detailed understanding of how biological systems control and exploit electron flow will be used to develop modular biochemical conversions for the production of hydrocarbon and hydrogen biofuels. This project will conduct an in-depth study of metalloenzyme structure-function relationships that enable retuning of catalytic sites, electron-transfer thermodynamics, and the evolution of tailor-designed chemical reactions involving two model enzymes: hydrogenase and nitrogenase. The center is organized into three major thrust areas: Thrust 1. Energy conservation and electron flow; Thrust 2. ATP-coupled electron transfer; and Thrust 3. Atomic level determinants of enzymatic redox properties and their relationship to catalytic bias. We will develop a collective knowledge in metalloenzymes by applying physical science and computational tools to characterize biochemical reactions catalyzed specifically by multi-subunit enzymes harboring iron-sulfur clusters and flavin cofactors, as models for redox reactions throughout biology. Understanding these mechanisms is central to overcoming the key thermodynamic barriers currently limiting production of reduced products and biofuels. The research plan of the BETCy EFRC has been designed to provide the basis to exploit unique biochemical mechanisms that have yet to be explored substantively in the context of bioenergy but have the potential for game-changing advancement. The successful outcome of this research will be a collection of fundamental principles that serve as a blueprint to enable the tailored re-design of biological systems and enzymes to control matter and energy at the level of electrons and molecules to provide the foundations to create new energy technologies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2019

October 2019 SAPHIRE 8 Software Quality Assurance Oversight Status Report

The software quality assurance oversight consists of updating and maintaining revision control of the System Analysis Program for Hands-on Integrated Reliability Evaluations (SAPHIRE) Version 8 quality assurance program documentation and of monitoring revision control of the SAPHIRE Version 8 source code. This report summarizes the oversight efforts through description of the revision control system (RCS) setup, operation and contents. Documents maintained under revision control include the Acceptance Test Plan (ATP), Configuration Management Plan, Quality Assurance Plan, Software Project Plan, Requirements Traceability Matrix (RTM), System Test Plan, Significance Determination Process (SDP) Interface Training Manual, and the SAPHIRE 8 “New Features and Capabilities Overview.” These documents are referred to and can be transmitted upon request of the Nuclear Regulatory Commission (NRC) Contracting Officer’s Representative (COR).

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2020

Productivity and bioproduct formation in phototropic knock/out mutants in micro algae

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m⁻²s⁻¹). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), Sidoheptulose 1,7 biophosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Where Have All of the Electrons Gone? Understanding the Role of Alternative Electron Flow and O 2 Reduction in Balancing Cellular Redox (Final Technical Report)

An informed understanding of the fundamentals of light-driven electron transfer reactions in photoautotrophic organisms is necessary to enable improved photosynthetic efficiencies for the production of renewable fuels and novel biomaterials. To obtain high photosynthetic yields, light energy must be efficiently coupled to the fixation of CO 2 . Sub-optimal environmental conditions and metabolic routing (caused by blocks in biosynthetic routes) can severely impact the conversion of light energy to biomass and lead to reactive oxygen production, which in turn can cause cellular damage and productivity losses. Hence, plants, algae, and photosynthetic bacteria have evolved a network of alternative outlets to sustain the flow of photosynthetically derived-electrons. Our research was focused on the nature and integration of these outlets. The data obtained in this project will inform efforts to rationally engineer crop plants and algae to improve photosynthetic yields by enhancing electron transfer reactions to CO 2 reduction and targeted biomass accumulation. A major project research thrust was focused on identifying and quantifying the flow of photosynthetic reductant through electron transfer circuits that result in the light-dependent reduction of O 2 . New energy management strategies were identified that are used when normal carbon assimilation pathways are compromised due to nutrient deprivation, and/or by a reduction in starch synthesis/carbon storage, all conditions resulting in highly reduced intracellular redox pools. The green alga Chlamydomonas reinhardtii, and likely many other algae, have multiple O 2 -reducing pathways that play critical roles in maintaining cellular metabolic balance and scavenging electrons that could potentially cause cellular damage, which in some instances leads to reduced photosynthetic yields but increased fitness. We explored the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (1) reduction of O 2 to H 2 O through Flavodiiron proteins (FLVs) and (2) Plastid Terminal Oxidases (PTOX), and (3) the synthesis of starch. Real-time measurements of O 2 exchange demonstrated that FLVs immediately engage during dark to light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified, for the first time, maximal FLV activity and its overall capacity to direct photosynthetic electrons towards O 2 reduction. However, when starch synthesis is compromised, a greater proportion of electrons is directed toward O 2 reduction through the FLVs, while PTOX, which is sensitive to the PQ pool redox state, is activated. This suggests, that starch synthesis has an important role in priming/regulating CBBC and electron transport. We also identified a biological ‘switch’ in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b 6 f complex when reductant and ATP generated by PET are in excess of the capacity of carbon metabolism to utilize these products; we specifically show a restriction at this switch when sta6 mutant cells, which cannot synthesize starch, are limited for nitrogen (growth inhibition) and subjected to a dark to light transition. This restriction causes diminished electron flow to PSI, which prevents PSI photodamage, and the plastid alternative oxidase (PTOX) becomes fully activated, serving as an electron valve that dissipates excitation energy absorbed by PSII, thereby lessening PSII photoinhibition. Furthermore, illumination of the cells following the dark acclimation gradually diminishes the restriction at the switch. Future engineering of these switches may allow more effective electron transfer to lipid (biofuel) pathways and diminish the number of electrons “wasted” in the reduction of O 2 to water. Lastly, we explored metabolic routing of electrons during algal fermentation and discovered multiple novel pathways that are activated when the preferred anoxic routes are blocked. These provide valuable products (e.g. lactate, glycerol) that can be used in broad portfolio of biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Energy Efficient Waste Heat Coupled Forward Osmosis for Effluent Water Management at Coal-Fired Power Plants

This project sought to evaluate the technical and economic viability of the Aquapod©, a transformational low energy (<200 kJ/kg water) waste heat coupled forward osmosis (FO) technology, to manage effluents, meet cooling water demands, and achieve water conservation in a coal-fired power plant environment. The Aquapod© process is innovative because it is heat-driven, avoids the evaporation of water, and uses no toxic chemicals such as ammonia or amines. The Aquapod© process accomplishes this using an aqueous two-phase system (ATPS) coupled FO process. The evaluation revealed that the Aquapod© process offers a pathway to exploit waste heat resources within a power plant to achieve flue-gas desulfurization (FGD) wastewater volume reduction and water recovery with minimal pretreatment. Water recovery of 80% from FGD wastewater was achieved with minimal pretreatment, exceeding the project target of 50%. The estimated electrical energy of 2.16 kWh/m 3 of water produced for the Aquapod© process met the project target of < 3.6 kWh/m 3 . The heat required for the process operation was approximately 186 kJ/kg of produced water, which was slightly lower than the project target of 200 kJ/kg. The estimated treatment cost for installing and operating the Aquapod© process in conjunction with a spray dryer evaporator to achieve zero-liquid discharge (ZLD) of the 100 gpm FGD wastewater was $13.24/m 3 over a 30-year lifetime. As a point of reference, this study’s host power plant currently incurs a cost of $3.70/m 3 – $8.87/m3 on a discharged volume basis to discharge its wastewater into the publicly owned treatment works (POTW) after physical-chemical treatment. Therefore, the Illinois power plant would incur an incremental cost increase of $4.37/m 3 – 9.54/m 3 to achieve ZLD using the Aquapod© and spray dryer combination. Ample opportunities exist to further lower the ZLD capital and operating costs in the next design iteration to attain pipe parity at the higher end of the site treatment costs.

01 COAL, LIGNITE, AND PEAT↗

DOE OSTI2021

Engineering Forisome Scaffolds: Elucidating Spatial Self-Assembly Patterning of Bio-inorganic Complexes

Organisms can synthesize biomaterials incorporating an array of naturally occurring elements while overcoming challenges and insults. Although, it is known that most cellular biomaterials are synthesized in specialized cellular compartments, there are knowledge gaps about how organic/inorganic biomaterial synthesis is orchestrated inside cells. In addition, there is great potential in understanding how individual monomers can self-assembly into organized patterns to form responsive biomaterials. Forisomes are a natural responsive biomaterial found in legume plants that serve as a plug sieve element in the plant phloem that undergo anisotropic conformational changes by rapid (<1 s) ATP-independent from condensed spindle to plug-like form, triggered by the influx of Ca 2+ . Addressing principles of forisome synthesis and assembly will determine how biomaterials containing inorganic elements self-assemble and conduct chemical modification to produce biomaterials or undergo biomineralization. We employ transcription and translation (TXTL) using cell-free expression systems for forisome monomer expression, self-assembly, and pattern probing. We conducted experiments to precisely control forisome proteins synthesis of various monomers SEO1, SEO2, SEO3, and SEO4 to explore self- assembly. We demonstrate forisome self-assembly of the SEO monomers is possible and indicate unique monomer fluorescent labeling patterns that require additional analysis. We investigated locations and linkers for adding tetracysteine tag fluorophore probes to determine impacts of self-assembly and anisotropic conformational changes.

42 ENGINEERING↗

DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product (Final Report)

The goal is to achieve a comprehensive understanding of the nature, mechanism of function and biogenesis of c-type cytochromes and their oligomeric complexes. These cytochrome are ubiquitous heme proteins that are required for cellular bioenergetics, including photosynthesis (Ps) and respiration (Res), which are central to chemical energy (ATP) production by oxidative- and photophosphorylation. We contribute to the molecular characterization of individual and membrane-integral cytochromes as parts of Ps and Res electron transport (ET) pathways, using the facultative phototrophic bacteria of as excellent models for multidisciplinary investigations. Supported by this grant we discovered novel membrane-bound cytochromes and cyts c complexes that are important for Ps- and Res-ET. We worked out their biogenesis including maturation (covalent heme addition) and assembly of mature subunits into complexes. These involve fundamental process found virtually in all organisms and are fundamental processes with major significance to the “Photosynthetic Systems” program of DOE-BES, because the cytochromes are integral components of natural energy production systems. Elucidation of basic principles, and the knowledge gained by their study greatly impact on our ability to design, engineer, and create new natural, bio-hybrid, bio-inspired and artificial systems to better capture light, transduce and store its energy with improved efficacy and are of broad significance.

14 SOLAR ENERGY↗

DOE OSTI2018

Development of Anabaena 33047, a Fast-Growing N 2 -Fixing Cyanobacterium, as a Carbon Neutral Bioproduction Platform

Cyanobacteria, oxygenic photosynthetic prokaryotes, are emerging as promising platforms for cost effective conversion of sunlight and CO 2 into high value end products. Anabaena sp. ATCC 33047, a heterocystous cyanobacterium, stands out in this category of fast-growing cyanobacteria for its ability to utilize atmospheric N 2 in addition to CO 2 and generate biomass at unprecedented rates. Developing such a strain as a chassis can eliminate the need for fixed nitrogen in bioproduction systems, a significant step towards replacement of petroleum derived products. Our work aimed to develop Anabaena 33047 into such a bioproduction platform. Despite its many appealing traits, Anabaena 33047 remained largely unexplored due to its recalcitrance to any genetic manipulation. During this project, we have developed a method that has enabled targeted genome modification of this strain. We also constructed a genome scale metabolic model i AnC892, which captures the diazotrophic life cycle of Anabaena 33047 by accounting for both vegetative and heterocyst cell types. The model provided insight into the importance of light dependent electron transport in the heterocyst and revealed pathway combinations that can supply reducing equivalents and ATP in the appropriate ratio for optimal N2 fixation. To validate the existing metabolic models and to have a better understanding of the metabolism of Anabaena 33047 under different growth conditions, we performed a series of stable-isotope tracing experiments and quantified metabolic fluxes using state-of-the-art tools for 13C-metabolic flux analysis. Using our novel engineering strategy and based on our model predictions, we performed targeted deletion in the strain and generated a mutant with modified light harvesting properties in heterocysts. The mutant exhibited better light utilization and increased rates of nitrogen fixation. Overall, this project has laid the foundation for developing the non-model cyanobacterium Anabaena 33047 into a highly efficient chassis for conversion of solar energy, atmospheric CO 2 and N 2 into high value bio products.

09 BIOMASS FUELS↗

DOE OSTI2023

Mechanistic Studies of a Primitive Homolog of Nitrogenase Involved in Coenzyme F430 Biosynthesis (Final Report)

03 NATURAL GAS↗

DOE OSTI2024

Mechanistic Studies of a Primitive Homolog of Nitrogenase Involved in Coenzyme F430 Biosynthesis

Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel or other high-value commodity chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction that converts Ni-sirohydrochlorin a , c -diamide to 15,17 3 -seco-F430-17 3 -acid. This remarkable transformation, which involves a 6-electron reduction of the isobacteriochlorin ring system, cyclization of the c -acetamide side chain to form a γ-lactam ring, and the formation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the ATP-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes that catalyze multi-electron redox reactions. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, the following Specific Aims will be pursued to determine the: 1) Identity of the CfbCD reaction product. The exact reaction catalyzed by CfbCD, including the number of electrons transferred and whether it involves enzymatic cyclization, is unclear. Several approaches, including reaction stoichiometry measurements, spectroelectrochemistry, and magnetic resonance spectroscopy will be applied to elucidate the structure of the reaction product and establish whether CfbCD is a reductase or reductive cyclase. 2) Structure, conformational dynamics, and oligomerization state changes of CfbCD. Significant insight into the mechanism and allosteric regulation of CfbCD can be obtained by assessing changes in the structure and dynamics of the complex during the catalytic cycle. To accomplish this, a combination of size-exclusion chromatography, hydrogen-deuterium exchange mass spectrometry, molecular dynamics simulations, and high-resolution structural methods will be employed. 3) Source, order, and stereochemistry of proton additions during CfbCD catalysis. Details regarding the order and stereochemistry of proton additions during the CfbCD reaction will be uncovered using a combined spectroscopic and computational approach. Complementary mechanistic studies employing site-directed mutagenesis and substrate analogs will establish the identity of active site acid residues and the possible involvement of substrate-assisted catalysis during the CfbCD reaction.

09 BIOMASS FUELS↗

DOE OSTI2021

Protein Motifs for Proton Transfers That Build the Transmembrane Proton Gradient

Biological membranes are barriers to polar molecules, so membrane embedded proteins control the transfers between cellular compartments. Protein controlled transport moves substrates and activates cellular signaling cascades. In addition, the electrochemical gradient across mitochondrial, bacterial and chloroplast membranes, is a key source of stored cellular energy. This is generated by electron, proton and ion transfers through proteins. The gradient is used to fuel ATP synthesis and to drive active transport. Here the mechanisms by which protons move into the buried active sites of Photosystem II (PSII), bacterial RCs (bRCs) and through the proton pumps, Bacteriorhodopsin (bR), Complex I and Cytochrome c oxidase (CcO), are reviewed. These proteins all use water filled proton transfer paths. The proton pumps, that move protons uphill from low to high concentration compartments, also utilize Proton Loading Sites (PLS), that transiently load and unload protons and gates, which block backflow of protons. PLS and gates should be synchronized so PLS proton affinity is high when the gate opens to the side with few protons and low when the path is open to the high concentration side. Proton transfer paths in the proteins we describe have different design features. Linear paths are seen with a unique entry and exit and a relatively straight path between them. Alternatively, paths can be complex with a tangle of possible routes. Likewise, PLS can be a single residue that changes protonation state or a cluster of residues with multiple charge and tautomer states.

cytochrome c oxidase↗

DOE OSTI2022

A Path-Based Analysis of Infected Cell Line and COVID-19 Patient Transcriptome Reveals Novel Potential Targets and Drugs Against SARS-CoV-2

Most transcriptomic studies of SARS-CoV-2 infection have focused on differentially expressed genes, which do not necessarily reveal the genes mediating the transcriptomic changes. In contrast, exploiting curated biological network, our PathExt tool identifies central genes from the differentially active paths mediating global transcriptomic response. Here we apply PathExt to multiple cell line infection models of SARS-CoV-2 and other viruses, as well as to COVID-19 patient-derived PBMCs. The central genes mediating SARS-CoV-2 response in cell lines were uniquely enriched for ATP metabolic process, G1/S transition, leukocyte activation and migration. In contrast, PBMC response reveals dysregulated cell-cycle processes. In PBMC, the most frequently central genes are associated with COVID-19 severity. Importantly, relative to differential genes, PathExt-identified genes show greater concordance with several benchmark anti-COVID-19 target gene sets. We propose six novel anti-SARS-CoV-2 targets ADCY2, ADSL, OCRL, TIAM1, PBK, and BUB1, and potential drugs targeting these genes, such as Bemcentinib, Phthalocyanine, and Conivaptan.

59 BASIC BIOLOGICAL SCIENCES↗