The genome of the Arctic snow-alga Limnomonas spitsbergensis (Chlamydomonadales).

Snow-algae are a diverse group of extremophilic microeukaryotes found on melting polar and alpine snowfields. They play an important role in the microbial ecology of the cryosphere, and their propagation on snow and ice surfaces may in part accelerate climate-induced melting of these systems. High quality snow-algae genomes are needed for studies on their unique physiology, adaptive mechanisms and genome evolution under multiple forms of stress, including cold temperatures and intense sunlight. Here we assembled and annotated the genome of Limnomonas spitsbergensis, a cryophilic biciliate green alga originally isolated from melting snow on Svalbard, in the Arctic. The L. spitsbergensis genome assembly is based primarily on the use of PacBio long reads and secondly Illumina short reads, with an assembly size of 260.248 Mb in 124 contigs. A combination of three alternative annotation strategies were used including protein homology, RNA-seq evidence and PacBio full length transcript isoforms. The best merged set of annotations identified 18,277 protein-coding genes, which were 95.2% complete based on BUSCO analysis. We also provide the annotated mitogenome, which is a relatively large 77.942 kb circular mapping sequence containing extensive repeats. The L. spitsbergensis genome will provide a new resource for research on snow-algae adaptation, behavior and natural selection in unique, low-temperature terrestrial environments that are under threat from climate change.

Advances in light system engineering across the phototrophic spectrum.

Current work in photosynthetic engineering is progressing along the lines of cyanobacterial, microalgal, and plant research. These are interconnected through the fundamental mechanisms of photosynthesis and advances in one field can often be leveraged to improve another. It is worthwhile for researchers specializing in one or more of these systems to be aware of the work being done across the entire research space as parallel advances of techniques and experimental approaches can often be applied across the field of photosynthesis research. This review focuses on research published in recent years related to the light reactions of photosynthesis in cyanobacteria, eukaryotic algae, and plants. Highlighted are attempts to improve photosynthetic efficiency, and subsequent biomass production. Also discussed are studies on cross-field heterologous expression, and related work on augmented and novel light capture systems. This is reviewed in the context of translatability in research across diverse photosynthetic organisms.

Proximate biomass characterization of the high productivity marine microalga Picochlorum celeri TG2.

Microalgae are compelling renewable resources with applications including biofuels, bioplastics, nutrient supplements, and cosmetic products. Picochlorum celeri is an alga with high industrial interest due to exemplary outdoor areal biomass productivities in seawater. Detailed proximate analysis is needed in multiple environmental conditions to understand the dynamic biomass compositions of P. celeri, and how these compositions might be leveraged in biotechnological applications. In this study, biomass characterization of P. celeri was performed under nutrient-replete, nitrogen-restricted, and hyper-saline conditions. Nutrient-replete cultivation of P. celeri resulted in protein-rich biomass (∼50% ash-free dry weight) with smaller carbohydrate (∼12% ash-free dry weight) and lipid (∼11% ash-free dry weight) partitions. Gradual nitrogen depletion elicited a shift from proteins to carbohydrates (∼50% ash-free dry weight, day 3) as cells transitioned into the production of storage metabolites. Importantly, dilutions in nitrogen-restricted 40 parts per million (1.43 mM nitrogen) media generated high-carbohydrate (∼50% ash-free dry weight) biomass without substantially compromising biomass productivity (36 g ash-free dry weight m-2 day-1) despite decreased chlorophyll (∼2% ash-free dry weight) content. This strategy for increasing carbohydrate content allowed for the targeted production of polysaccharides, which could potentially be utilized to produce fuels, oligosaccharides, and bioplastics. Cultivation at 2X sea salts resulted in a shift towards carbohydrates from protein, with significantly increased levels of the amino acid proline, which putatively acts as an osmolyte. A detailed understanding of the biomass composition of P. celeri in nutrient-replete, nitrogen-restricted, and hyper saline conditions informs how this strain can be useful in the production of biotechnological products.

Simultaneous CAS9 editing of cpSRP43, LHCA6, and LHCA7 in Picochlorum celeri lowers chlorophyll levels and improves biomass productivity.

High cellular pigment levels in dense microalgal cultures contribute to excess light absorption. To improve photosynthetic yields in the marine microalga Picochlorum celeri, CAS9 gene editing was used to target the molecular chaperone cpSRP43. Depigmented strains (>50% lower chlorophyll) were generated, with proteomics showing attenuated levels of most light harvesting complex (LHC) proteins. Gene editing generated two types of cpSRP43 transformants with distinct lower pigment phenotypes: (i) a transformant (Δsrp43) with both cpSRP43 diploid alleles modified to encode non-functional polypeptides and (ii) a transformant (STR30309) with a 3 nt in-frame insertion in one allele at the CAS9 cut site (non-functional second allele), leading to expression of a modified cpSRP43. STR30309 has more chlorophyll than Δsrp43 but substantially less than wild type. To further decrease light absorption by photosystem I in STR30309, CAS9 editing was used to stack in disruptions of both LHCA6 and LHCA7 to generate STR30843, which has higher (5-24%) productivities relative to wild type in solar-simulating bioreactors. Maximal productivities required frequent partial harvests throughout the day. For STR30843, exemplary diel bioreactor yields of ~50 g m-2 day-1 were attained. Our results demonstrate diel productivity gains in P. celeri by lowering pigment levels.

Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Restricting electron flow at cytochrome b6f when downstream electron acceptors are severely limited.

Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of PSI and PSII, resulting in a decline in primary productivity. This work describes a biological "switch" in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production (potentially sustaining PSII repair and nonphotochemical quenching [NPQ]). The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how PET responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved.

Adaptive laboratory evolution for increased temperature tolerance of the diatom Nitzschia inconspicua.

Outdoor microalgal cultivation for the production of valuable biofuels and bioproducts typically requires high insolation and strains with high thermal (>37°C) tolerance. While some strains are naturally thermotolerant, other strains of interest require improved performance at elevated temperatures to enhance industrial viability. In this study, adaptive laboratory evolution (ALE) was performed for over 300 days using consecutive 0.5°C temperature increases in a constant temperature incubator to attain greater thermal tolerance in the industrially relevant diatom Nitzschia inconspicua str. Hildebrandi. The adapted strain was able to grow at a constant temperature of 37.5°C; whereas this constant temperature was lethal to the parental control, which had an upper-temperature boundary of 35.5°C before adaptive evolution. Several high-temperature clonal isolates were obtained from the evolved population following ALE, and increased temperature tolerance was observed in the clonal, parent, and non-clonal adapted cultures. This ALE method demonstrates the development of enhanced industrial algal strains without the production of genetically modified organisms (GMOs).

Cas9 deletion of lutein biosynthesis in the marine alga Picochlorum celeri reduces photosynthetic pigments while sustaining high biomass productivity.

Domestication of algae for food and renewable biofuels remains limited by the low photosynthetic efficiencies of processes that have evolved to be competitive for optimal light capture, incentivizing the development of large antennas in light-limiting conditions, thus decreasing efficient light utilization in cultivated ponds or photobioreactors. Reducing the pigment content to improve biomass productivity has been a strategy discussed for several decades and the ability to reduce pigment significantly is now fully at hand thanks to the widespread use of genome editing tools. Picochlorum celeri is one of the fastest growing marine algae identified and holds particular promise for outdoor cultivation, especially in saline water and warm climates. We show that while chlorophyll b is essential to sustain high biomass productivities under dense cultivation, removing Picochlorum celeri's main carotenoid, lutein, leads to a decreased total chlorophyll content, higher a/b ratio, reduced functional LHCII cross section and higher maximum quantum efficiencies at lower light intensities, resulting in an incremental increase in biomass productivity and increased PAR-to-biomass conversion efficiency. These findings further strengthen the existing strategies to improve photosynthetic efficiency and biomass production in algae.

The Genome of the Haptophyte Diacronema lutheri (Pavlova lutheri, Pavlovales): A Model for Lipid Biosynthesis in Eukaryotic Algae.

Haptophytes are biogeochemically and industrially important protists with underexplored genomic diversity. We present a nuclear genome assembly for the class Pavlovales, which was assembled with PacBio long-read data into highly contiguous sequences. We sequenced strain Diacronema lutheri NIVA-4/92, formerly known as Pavlova lutheri, because it has established roles in aquaculture and has been a key organism for studying microalgal lipid biosynthesis. Our data show that D. lutheri has the smallest and most streamlined haptophycean genome assembled to date, with an assembly size of 43.503 Mb and 14,446 protein-coding genes. Together with its high nuclear GC content, Diacronema is an important genus for investigating selective pressures on haptophyte genome evolution, contrasting with the much larger and more repetitive genome of the coccolithophore Emiliania huxleyi. The D. lutheri genome will be a valuable resource for resolving the genetic basis of algal lipid biosynthesis and metabolic remodeling that takes place during adaptation and stress response in natural and engineered environments.

Picochlorum celeri as a model system for robust outdoor algal growth in seawater.

With fast growth rates, broad halotolerance and the ability to thrive at high temperatures, algae in the genus Picochlorum are emerging as promising biomass producers. Recently, we isolated a remarkably productive strain, Picochlorum celeri, that attains > 40 g m-2 day-1 productivities using simulated outdoor light. To test outdoor productivities, Picochlorum celeri was cultivated in 820 L raceway ponds at the Arizona Center for Algae Technology and Innovation. Picochlorum celeri demonstrated the highest outdoor biomass productivities reported to date at this testbed averaging ~ 31 g m-2 day-1 over four months with a monthly (August) high of ~ 36 g m-2 day-1. Several single day productivities were > 40 g m-2 day-1. Importantly for sustainability, Picochlorum celeri achieved these productivities in saline water ranging from seawater to 50 parts per thousand sea salts, without any biocides or pond crashes, for over 143 days. Lastly, we report robust genetic engineering tools for future strain improvements.

Phased Diploid Genome Sequence for the Fast-Growing Microalga Picochlorum celeri.

Picochlorum celeri is a fast-growing marine microalga with high biomass productivity. Here, we report the use of PacBio sequencing to assemble the phased diploid genome of P. celeri.

The complete mitogenome and plastome of the haptophyte Pavlova lutheri NIVA-4/92.

The complete mitochondrial and plastid genomes of the microalga Pavlova lutheri strain NIVA-4/92 are reported. The circular-mapping mitogenome is 36,202 bp in length, contains 22 protein-coding genes, 24 tRNAs, and has a GC content of 37.5%. Like other haptophytes the mitogenome contains a single large, complex repeat region of approximately 5.4 kbp. The plastome is 95,281 bp in length and has a GC content of 35.6%. It contains 111 protein-coding genes and 27 tRNAs.

Development of a high-productivity, halophilic, thermotolerant microalga Picochlorum renovo.

Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, Picochlorum renovo. This microalga displays a rapid growth rate and high diel biomass productivity (34 g m-2 day-1), with a composition well-suited for downstream processing. P. renovo exhibits broad salinity tolerance (growth at 107.5 g L-1 salinity) and thermotolerance (growth up to 40 °C), beneficial traits for outdoor cultivation. We report complete genome sequencing and analysis, and genetic tool development suitable for expression of transgenes inserted into the nuclear or chloroplast genomes. We further evaluate mechanisms of halotolerance via comparative transcriptomics, identifying novel genes differentially regulated in response to high salinity cultivation. These findings will enable basic science inquiries into control mechanisms governing Picochlorum biology and lay the foundation for development of a microalga with industrially relevant traits as a model photobiology platform.

Alternative outlets for sustaining photosynthetic electron transport during dark-to-light transitions.

Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have 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 maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.

Down-Selection and Outdoor Evaluation of Novel, Halotolerant Algal Strains for Winter Cultivation.

Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona. Three strains, from the genera Micractinium, Chlorella, and Scenedesmus, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m2/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m2/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.

Unlocking the Constraints of Cyanobacterial Productivity: Acclimations Enabling Ultrafast Growth.

UNLABELLED: Harnessing the metabolic potential of photosynthetic microbes for next-generation biotechnology objectives requires detailed scientific understanding of the physiological constraints and regulatory controls affecting carbon partitioning between biomass, metabolite storage pools, and bioproduct synthesis. We dissected the cellular mechanisms underlying the remarkable physiological robustness of the euryhaline unicellular cyanobacterium Synechococcus sp. strain PCC 7002 (Synechococcus 7002) and identify key mechanisms that allow cyanobacteria to achieve unprecedented photoautotrophic productivities (~2.5-h doubling time). Ultrafast growth of Synechococcus 7002 was supported by high rates of photosynthetic electron transfer and linked to significantly elevated transcription of precursor biosynthesis and protein translation machinery. Notably, no growth or photosynthesis inhibition signatures were observed under any of the tested experimental conditions. Finally, the ultrafast growth in Synechococcus 7002 was also linked to a 300% expansion of average cell volume. We hypothesize that this cellular adaptation is required at high irradiances to support higher cell division rates and reduce deleterious effects, corresponding to high light, through increased carbon and reductant sequestration. IMPORTANCE: Efficient coupling between photosynthesis and productivity is central to the development of biotechnology based on solar energy. Therefore, understanding the factors constraining maximum rates of carbon processing is necessary to identify regulatory mechanisms and devise strategies to overcome productivity constraints. Here, we interrogate the molecular mechanisms that operate at a systems level to allow cyanobacteria to achieve ultrafast growth. This was done by considering growth and photosynthetic kinetics with global transcription patterns. We have delineated putative biological principles that allow unicellular cyanobacteria to achieve ultrahigh growth rates through photophysiological acclimation and effective management of cellular resource under different growth regimes.

Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase.

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Biochemical and Structural Properties of a Thermostable Mercuric Ion Reductase from Metallosphaera sedula.

Mercuric ion reductase (MerA), a mercury detoxification enzyme, has been tuned by evolution to have high specificity for mercuric ions (Hg(2+)) and to catalyze their reduction to a more volatile, less toxic elemental form. Here, we present a biochemical and structural characterization of MerA from the thermophilic crenarchaeon Metallosphaera sedula. MerA from M. sedula is a thermostable enzyme, and remains active after extended incubation at 97°C. At 37°C, the NADPH oxidation-linked Hg(2+) reduction specific activity was found to be 1.9 µmol/min⋅mg, increasing to 3.1 µmol/min⋅mg at 70°C. M. sedula MerA crystals were obtained and the structure was solved to 1.6 Å, representing the first solved crystal structure of a thermophilic MerA. Comparison of both the crystal structure and amino acid sequence of MerA from M. sedula to mesophillic counterparts provides new insights into the structural determinants that underpin the thermal stability of the enzyme.