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Pakrasi, Himadri B.

Publications and source records attributed to Pakrasi, Himadri B..

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). Here, to understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Photobiological production of high-value pigments via compartmentalized co-cultures using Ca-alginate hydrogels

Abstract Engineered cyanobacterium Synechococcus elongatus can use light and CO 2 to produce sucrose, making it a promising candidate for use in co-cultures with heterotrophic workhorses. However, this process is challenged by the mutual stresses generated from the multispecies microbial culture. Here we demonstrate an ecosystem where S. elongatus is freely grown in a photo-bioreactor (PBR) containing an engineered heterotrophic workhorse (either β-carotene-producing Yarrowia lipolytica or indigoidine-producing Pseudomonas putida ) encapsulated in calcium-alginate hydrogel beads. The encapsulation prevents growth interference, allowing the cyanobacterial culture to produce high sucrose concentrations enabling the production of indigoidine and β-carotene in the heterotroph. Our experimental PBRs yielded an indigoidine titer of 7.5 g/L hydrogel and a β-carotene titer of 1.3 g/L hydrogel, amounts 15–22-fold higher than in a comparable co-culture without encapsulation. Moreover, 13 C-metabolite analysis and protein overexpression tests indicated that the hydrogel beads provided a favorable microenvironment where the cell metabolism inside the hydrogel was comparable to that in a free culture. Finally, the heterotroph-containing hydrogels were easily harvested and dissolved by EDTA for product recovery, while the cyanobacterial culture itself could be reused for the next batch of immobilized heterotrophs. This co-cultivation and hydrogel encapsulation system is a successful demonstration of bioprocess optimization under photobioreactor conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advances in the Understanding of the Lifecycle of Photosystem II

Photosystem II is a light-driven water-plastoquinone oxidoreductase present in cyanobacteria, algae and plants. It produces molecular oxygen and protons to drive ATP synthesis, fueling life on Earth. As a multi-subunit membrane-protein-pigment complex, Photosystem II undergoes a dynamic cycle of synthesis, damage, and repair known as the Photosystem II lifecycle, to maintain a high level of photosynthetic activity at the cellular level. Cyanobacteria, oxygenic photosynthetic bacteria, are frequently used as model organisms to study oxygenic photosynthetic processes due to their ease of growth and genetic manipulation. The cyanobacterial PSII structure and function have been well-characterized, but its lifecycle is under active investigation. In this review, advances in studying the lifecycle of Photosystem II in cyanobacteria will be discussed, with a particular emphasis on new structural findings enabled by cryo-electron microscopy. These structural findings complement a rich and growing body of biochemical and molecular biology research into Photosystem II assembly and repair.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Psb27, a photosystem II assembly protein, enables quenching of excess light energy during its participation in the PSII lifecycle

Photosystem II (PSII), the enzyme responsible for oxidizing water into molecular oxygen, undergoes a complex lifecycle during which multiple assembly proteins transiently bind to and depart from PSII assembly intermediate complexes. Psb27 is one such protein. It associates with the CP43 chlorophyll-binding subunit of PSII to form a Psb27-PSII sub-complex that constitutes 7–10% of the total PSII pool. Psb27 remains bound to PSII assembly intermediates and dissociates prior to the formation of fully functional PSII. In this study, we compared a series of Psb27 mutant strains in the cyanobacterium Synechocystis sp. PCC 6803 with varied expression levels of Psb27: wild type (WT); psb27 genetic deletion (Del27), genetically complemented psb27 (Com27); and over-expressed Psb27 (OE27). The Del27 strain demonstrated decreased non-photochemical fluorescence quenching, while the OE27 strain showed increased non-photochemical quenching and tolerance to fluctuating light conditions. Multiple flashes and fluorescence decay analysis indicated that OE27 has the least affected maximum PSII quantum yield of the mutants. OE27 also displayed a minimal impact on the half-life of the fast component of QA – reoxidation over multiple flashes, indicating robust PSII function. Further, we propose that the close association between Psb27 and CP43, and the absence of a fully functional manganese cluster in the Psb27-PSII complex create a PSII sub-population that dissipates excitation energy prior to its recruitment into the functional PSII pool. Efficient energy dissipation prevents damage to this pre-PSII pool and allows for efficient PSII repair and maturation. Participation of Psb27 in the PSII life cycle ensures high-quality PSII assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A Novel Mode of Photoprotection Mediated by a Cysteine Residue in the Chlorophyll Protein IsiA

Oxygenic photosynthetic organisms have evolved a multitude of mechanisms for protection against high-light stress. IsiA, a chlorophyll a-binding cyanobacterial protein, serves as an accessory antenna complex for photosystem I. Intriguingly, IsiA can also function as an independent pigment protein complex in the thylakoid membrane and facilitate the dissipation of excess energy, providing photoprotection. The molecular basis of the IsiA-mediated excitation quenching mechanism remains poorly understood. In this study, we demonstrate that IsiA uses a novel cysteine-mediated process to quench excitation energy. The single cysteine in IsiA in the cyanobacterium Synechocystis sp. strain PCC 6803 was converted to a valine. Ultrafast fluorescence spectroscopic analysis showed that this single change abolishes the excitation energy quenching ability of IsiA, thus providing direct evidence of the crucial role of this cysteine residue in energy dissipation from excited chlorophylls. Under stress conditions, the mutant cells exhibited enhanced light sensitivity, indicating that the cysteine-mediated quenching process is critically important for photoprotection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Systems Level Study of a Novel Fast-Growing Cyanobacterial Strain for Next Generation Biofuel Production

The overall objective of this project is to use an integrated systems biology approach to develop a fast growing cyanobacterial strain as a platform organism for photobiological production of advanced biofuels and other useful chemicals. Cyanobacteria are oxygenic photosynthetic microbes that use light and CO 2 as feedstocks, and hence offer enormous promises for sustainable bioenergy production. One of the key issues in cyanobacterial biofuel production is the growth rates of these microbes. Compared to other oxygenic photosynthetic organisms such as plants and eukaryotic algae, many cyanobacterial strains have superior growth properties. However, they grow significantly slower than heterotrophic microbes such as E. coli and yeast that are commonly used in biofuel research. We have recently identified Synechococcus elongatus UTEX 2973, a unicellular cyanobacterium that has the ability of rapid photoautotrophic growth comparable to that of the yeast Saccharomyces cerevisiae. In this project, we will pursue a systems approach to develop Synechococcus 2973 as a versatile and useful model cyanobacterial host to be used by the bioenergy research community during the coming era. The knowledge base generated during this project will also be helpful for sustainable biofuel production by other cyanobacterial as well as eukaryotic photosynthetic hosts that use sunlight and CO 2 as renewable feedstocks.

09 BIOMASS FUELS↗
DOE OSTI2020

Engineering biology approaches for food and nutrient production by cyanobacteria

Here, as photoautotrophic organisms, cyanobacteria capture and store solar energy in the form of biomass. Cyanobacterial biomass has been an important component of diet and nutrition in several regions for centuries. Synthetic biology strategies are currently being applied to increase the yield and productivity of cyanobacterial biomass by optimizing solar energy utilization and CO 2 fixation rates for carbon storage. Likewise, engineering cyanobacteria as cellular factories to synthesize carbohydrates, amino acids, proteins, lipids and fatty acids is providing an attractive way to sustainably produce food and nutrients for human consumption. In this review, we have summarized recent progress in both aspects and prospective trends under development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Photosynthetic Co-production of Succinate and Ethylene in a Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801

Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of P cpcB , the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13 C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.

13C isotopic labelling↗
DOE OSTI2019

Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973

Cyanobacteria are photoautotrophic prokaryotes that serve as key model organisms to study basic photosynthetic processes and are potential carbon-negative production chassis for commodity and high-value chemicals. The development of new synthetic biology tools and improvement of current ones is a requisite for furthering these organisms as models and production vehicles. CRISPR interference (CRISPRi) allows for targeted gene repression using a DNase-dead Cas nuclease (“dCas”). Here, we describe a titratable dCas12a (dCpf1) CRISPRi system and apply it to repress key photosynthetic processes in the fast-growing cyanobacterium Synechococcus sp. UTEX 2973 (S2973). The system relies on a lac repressor system that retains tight regulation in the absence of inducer (0–10% repression) while maintaining the capability for >90% repression of high-abundance gene targets. We determined that dCas12a is less toxic than dCas9. We tested the efficacy of the system toward eYFP and three native targets in S2973: the phycobilisome antenna, glycogen synthesis, and photosystem I (PSI), an essential part of the photosynthetic electron transport chain in oxygenic photoautotrophs. PSI was knocked down indirectly by repressing the protein factor BtpA involved in stabilizing core PSI proteins. We could reduce cellular PSI titer by 87% under photoautotrophic conditions, and we characterized these cells to gain insights into the response of the strain to the low PSI content. The ability to tightly regulate and time the (de)repression of essential genes in trans will allow for the study of photosynthetic processes that are not accessible using knockout mutants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

A novel chlorophyll protein complex in the repair cycle of photosystem II

In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. It must undergo a repair and reassembly process following photodamage, many facets of which remain unknown. We have discovered a PSII subcomplex that lacks 5 key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment–protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low molecular mass subunits, and the assembly factor Psb27. Immunoblotting, mass spectrometry, and ultrafast spectroscopic results support the absence of a functional reaction center in this complex, which we call the “no reaction center” complex (NRC). Analytical ultracentrifugation and clear native PAGE analysis show that NRC is a stable pigment–protein complex and not a mixture of free CP47 and CP43 proteins. NRC appears in higher abundance in cells exposed to high light and impaired protein synthesis, and genetic deletion of PsbO on the PSII luminal side results in an increased NRC population, indicative that NRC forms in response to photodamage as part of the PSII repair process. Our finding challenges the current model of the PSII repair cycle and implies an alternative PSII repair strategy. Formation of this complex may maximize PSII repair economy by preserving intact PSII core antennas in a single complex available for PSII reassembly, minimizing the risk of randomly diluting multiple recycling components in the thylakoid membrane following a photodamage event.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Metabolic model guided strain design of cyanobacteria

Cyanobacteria are oxygenic photoautotrophs that serve as potential platforms for the production of biochemicals from cheap and renewable raw materials – sunlight, water, and carbon dioxide. Systems level analysis of the metabolic network of these organisms could enable the successful engineering of these organisms for the enhanced production of target chemicals. Metabolic modeling techniques including both stoichiometric and kinetic modeling with a genome-wide coverage enable a global assessment of metabolic capabilities. Recent studies guided by such modeling techniques have engineered strains for the enhanced production of valuable chemicals such as ethanol, n-butanol, 1,3-propanediol, glycerol, limonene, and isoprene from CO 2 .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Influence of Chemically Disrupted Photosynthesis on Cyanobacterial Thylakoid Dynamics in Synechocystis sp. PCC 6803

The photosynthetic machinery of the cyanobacterium Synechocystis sp. PCC 6803 resides in flattened membrane sheets called thylakoids, situated in the peripheral part of the cellular cytoplasm. Under photosynthetic conditions these thylakoid membranes undergo various dynamical processes that could be coupled to their energetic functions. Using Neutron Spin Echo Spectroscopy (NSE), we have investigated the undulation dynamics of Synechocystis sp. PCC 6803 thylakoids under normal photosynthetic conditions and under chemical treatment with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), an herbicide that disrupts photosynthetic electron transfer. Our measurements show that DCMU treatment has a similar effect as dark conditions, with differences in the undulation modes of the untreated cells compared to the chemically inhibited cells. We found that the disrupted membranes are 1.5-fold more rigid than the native membranes during the dark cycle, while in light they relax approximately 1.7-fold faster than native and they are 1.87-fold more flexible. The strength of the herbicide disruption effect is characterized further by the damping frequency of the relaxation mode and the decay rate of the local shape fluctuations. In the dark, local thicknesses and shape fluctuations relax twice as fast in native membranes, at 17% smaller mode amplitude, while in light the decay rate of local fluctuations is 1.2-fold faster in inhibited membranes than in native membranes, at 56% higher amplitude. The disrupted electron transfer chain and the decreased proton motive force within the lumenal space partially explain the variations observed in the mechanical properties of the Synechocystis membranes, and further support the hypothesis that the photosynthetic process is tied to thylakoid rigidity in this type of cyanobacterial cell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Diverse hydrocarbon biosynthetic enzymes can substitute for olefin synthase in the cyanobacterium Synechococcus sp. PCC 7002

Cyanobacteria are among only a few organisms that naturally synthesize long-chain alkane and alkene hydrocarbons. Cyanobacteria use one of two pathways to synthesize alka/enes, either acyl-ACP reductase (Aar) and aldehyde deformylating oxygenase (Ado) or olefin synthase (Ols). The genomes of cyanobacteria encode one of these pathways but never both, suggesting a mutual exclusivity. We studied hydrocarbon pathway compatibility using the model cyanobacterium Synechococcus sp. PCC 7002 (S7002) by co-expressing Ado/Aar and Ols and by entirely replacing Ols with three other types of hydrocarbon biosynthetic pathways. We find that Ado/Aar and Ols can co-exist and that slower growth occurs only when Ado/Aar are overexpressed at 38 °C. Furthermore, Ado/Aar and the non-cyanobacterial enzymes UndA and fatty acid photodecarboxylase are able to substitute for Ols in a knockout strain and conditionally rescue slow growth. Production of hydrocarbons by UndA in S7002 required a rational mutation to increase substrate range. Expression of the non-native enzymes in S7002 afforded unique hydrocarbon profiles and alka/enes not naturally produced by cyanobacteria. This suggests that the biosynthetic enzyme and the resulting types of hydrocarbons are not critical to supporting growth. Exchanging or mixing hydrocarbon pathways could enable production of novel types of CO 2 -derived hydrocarbons in cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗