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NASA NTRS2003

Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

Bernhardsdotter, Eva C. M. J.↗

NASA NTRS2010

Utilization of Alternate Chirality Enantiomers in Microbial Communities

Our previous study of chirality led to interesting findings for some anaerobic extremophiles: the ability to metabolize substrates with alternate chirality enantiomers of amino acids and sugars. We have subsequently found that not just separate microbial species or strains but entire microbial communities have this ability. The functional division within a microbial community on proteo- and sugarlytic links was also reflected in a microbial diet with L-sugars and D-amino acids. Several questions are addressed in this paper. Why and when was this feature developed in a microbial world? Was it a secondary de novo adaptation in a bacterial world? Or is this a piece of genetic information that has been left in modern genomes as an atavism? Is it limited exclusively to prokaryotes, or does this ability also occur in eukaryotes? In this article, we have used a broader approach to study this phenomenon using anaerobic extremophilic strains from our laboratory collection. A series of experiments were performed on physiologically different groups of extremophilic anaerobes (pure and enrichment cultures). The following characteristics were studied: 1) the ability to grow on alternate chirality enantiomers -- L-sugars and D- amino acids; 2) Growth-inhibitory effect of alternate chirality enantiomers; 3) Stickland reaction with alternate chirality amino acids. The results of this research are presented in this paper.

Pikuta, Elena V.↗

DOE OSTI2022

Evolutionary Divergence of Marinobacter Strains in Cryopeg Brines as Revealed by Pangenomics

Marinobacter spp. are cosmopolitan in saline environments, displaying a diverse set of metabolisms that allow them to competitively occupy these environments, some of which can be extreme in both salinity and temperature. Here, we introduce a distinct cluster of Marinobacter genomes, composed of novel isolates and in silico assembled genomes obtained from subzero, hypersaline cryopeg brines, relic seawater-derived liquid habitats within permafrost sampled near Utqiaġvik, Alaska. Using these new genomes and 45 representative publicly available genomes of Marinobacter spp. from other settings, we assembled a pangenome to examine how the new extremophile members fit evolutionarily and ecologically, based on genetic potential and environmental source. This first genus-wide genomic analysis revealed that Marinobacter spp. in general encode metabolic pathways that are thermodynamically favored at low temperature, cover a broad range of organic compounds, and optimize protein usage, e.g., the Entner–Doudoroff pathway, the glyoxylate shunt, and amino acid metabolism. The new isolates contributed to a distinct clade of subzero brine-dwelling Marinobacter spp. that diverged genotypically and phylogenetically from all other Marinobacter members. The subzero brine clade displays genomic characteristics that may explain competitive adaptations to the extreme environments they inhabit, including more abundant membrane transport systems (e.g., for organic substrates, compatible solutes, and ions) and stress-induced transcriptional regulatory mechanisms (e.g., for cold and salt stress) than in the other Marinobacter clades. We also identified more abundant signatures of potential horizontal transfer of genes involved in transcription, the mobilome, and a variety of metabolite exchange systems, which led to considering the importance of this evolutionary mechanism in an extreme environment where adaptation via vertical evolution is physiologically rate limited. Assessing these new extremophile genomes in a pangenomic context has provided a unique view into the ecological and evolutionary history of the genus Marinobacter , particularly with regard to its remarkable diversity and its opportunism in extremely cold and saline environments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Sustainable production of plastic-degrading enzymes in Chlamydomonas pacifica

AbstractThe discovery of a new extremophile alga,Chlamydomonas pacifica, provides an opportunity to expand on heterologous protein expression beyond the traditionalChlamydomonas reinhardtii. C. pacificais a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown thatC. reinhardtiican secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in theC. pacificaalgae biomass.Graphical Abstract

Diaz, Crisandra Jade (ORCID:0000000173429041)↗

NASA NTRS2001

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryo-preserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 T. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗

NASA NTRS2002

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep-sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗

NASA NTRS2002

Life in Extreme Environments

Each recent report of liquid water existing elsewhere in the solar system has reverberated through the international press and excited the imagination of humankind. Why? Because in the last few decades we have come to realize that where there is liquid water on Earth, virtually no matter what the physical conditions, there is life. What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harboring 'extremophiles'. This realization, coupled with new data on the survival of microbes in the space environment and modeling of the potential for transfer of life between celestial bodies, suggests that life could be more common than previously thought. Here we critically examine what it means to be an extremophile, the implications of this for evolution, biotechnology, and especially the search for life in the cosmos.

Rothschild, Lynn↗

NASA NTRS2002

Life in Extreme Environments

Rothschild, Lynn↗

NASA NTRS2003

Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

Bernhardsdotter, E. C. M. J.↗

NASA NTRS2004

New Evidence for the Presence of Indigenous Microfossils in Carbonaceous Chondrites

We present additional evidence for the presence of indigenous microfossils in carbonaceous meteorites scanning electron micrograph studies of freshly fractured interior surfaces of pristine samples of the Murchison CM2 carbonaceous meteorite have revealed forms in-situ that are recognizable as biofilms as well as complex and highly structured forms similar to calcareous and siliceous microfossils. Some of the forms encountered are very well-preserved and exhibit complex associated microstructures similar to bacterial flagella. New images will be presented of forms recently encountered in carbonaceous meteorites and they will be compared with those of known microbial extremophiles. KEYWORDS: carbonaceous chondrites, Murchison, microfossils, extremophiles

Hoover, Richard B.↗

NASA NTRS2007

Microfossils of Cyanobacteria in Carbonaceous Meteorites

During the past decade, Environmental and Field Emission Scanning Electron Microscopes have been used at the NASA/Marshall Space Flight Center to investigate freshly fractured interior surfaces of a large number of different types of meteorites. Large, complex, microfossils with clearly recognizable biological affinities have been found embedded in several carbonaceous meteorites. Similar forms were notably absent in all stony and nickel-iron meteorites investigated. The forms encountered are consistent in size and morphology with morphotypes of known genera of Cyanobacteria and microorganisms that are typically encountered in associated benthic prokaryotic mats. Even though many coccoidal and isodiametric filamentous cyanobacteria have a strong morphological convergence with some other spherical and filamentous bacteria and algae, many genera of heteropolar cyanobacteria have distinctive apical and basal regions and cellular differentiation that makes it possible to unambiguously recognize the forms based entirely upon cellular dimensions, filament size and distinctive morphological characteristics. For almost two centuries, these morphological characteristics have historically provided the basis for the systematics and taxonomy of cyanobacteria. This paper presents ESEM and FESEM images of embedded filaments and thick mats found in-situ in the Murchison CM2 and Orgueil cn carbonaceous meteorites. Comparative images are also provided for known genera and species of cyanobacteria and other microbial extremophiles. Energy Dispersive X-ray Spectroscopy (EDS) studies indicate that the meteorite filaments typically exhibit dramatic chemical differentiation with distinctive difference between the possible microfossil and the meteorite matrix in the immediate proximity. Chemical differentiation is also observed within these microstructures with many of the permineralized filaments enveloped within electron transparent carbonaceous sheaths. Elemental distributions of these embedded filaments are not consistent with recent cyanobacteria or other living or preserved microbial extremophiles that have been investigated during this research. The meteorite filaments often have nitrogen content below the sensitivity level of the EDS detector. Carbon, Sulphur, Iron or Silicon are often highly enriched and hence anomalous C/N and CIS ratios when compared with modem cyanobacteria. The meteorite forms that are unambiguously recognizable as biological filaments are interpreted as indigenous microfossils analogous to several known genera of modem cyanobacteria and associated trichomic filamentous prokaryotes.

Hoover, Richard B.↗

NASA NTRS2007

Comets, Carbonaceous Meteorites, and the Origin of the Biosphere

Evidence for indigenous microfossils in carbonaceous meteorites suggests that the paradigm of the endogenous origin of life on Earth should be reconsidered. It is now widely accepted that comets and carbonaceous meteorites played an important role in the delivery of water, organics and life critical biogenic elements to the early Earth and facilitated the origin and evolution of the Earth's Biosphere. However; the detection of embedded microfossils and mats in carbonaceous meteorites implies that comets and meteorites may have played a direct role in the delivery of intact microorganisms and that the Biosphere may extend far into the Cosmos. Recent space observations have found the nuclei of comets to have very low albedos (approx.0.03) and. these jet-black surfaces become very hot (T approx. 400 K) near perihelion. This paper reviews recent observational data-on comets and suggests that liquid water pools could exist in cavities and fissures between the internal ices and rocks and the exterior carbonaceous crust. The presence of light and liquid water near the surface of the nucleus enhances the possibility that comets could harbor prokaryotic extremophiles (e.g., cyanobacteria) capable of growth over a wide range of temperatures. The hypothesis that comets are the parent bodies of the CI1 and the CM2 carbonaceous meteorites is advanced. Electron microscopy images will be presented showing forms interpreted as indigenous-microfossils embedded' in freshly. fractured interior surfaces of the Orgueil (CI1) and Murchison (CM2) meteorites. These forms are consistent in size and morphologies with known morphotypes of all five orders of Cyanobacteriaceae: Energy Dispersive X-ray Spectroscopy (EDS) elemental data shows that the meteoritic forms have anomalous C/O; C/N; and C/S as compared with modern extremophiles and cyanobacteria. These images and spectral data indicate that the clearly biogenic and embedded remains cannot be interpreted as recent biological contaminants and'therefore are indigenous'microfossils in the meteorites.

Hoover, Richard B.↗

NASA NTRS2009

Panel Discussion: Life in the Cosmos

Water appears to be essential to all life on Earth. For this reason, "Follow the Water" has been adopted as a mantra for the search for Life in the Cosmos. Expeditions have helped to establish the limits and biodiversity of life in the most extreme environments on Earth. Microbial extremophiles inhabit acidic streams; hypersaline and hyperalkaline lakes and pools; the cold deep sea floor, permafrost, rocks, glaciers, and perennially ice-covered lakes of the polar environments; geysers, volcanic fumaroles, hydrothermal vents and hot rocks deep within the Earth's crust. The ESA Venus Express Spacecraft entered Venusian Orbit in 2006 and continues to produce exciting results. The Visible and Infrared Thermal Imaging Spectrometer (VIRTIS) instrument made the first detection of hydroxyl in the atmosphere of Venus, indicating it is much more similar to Earth and Mars than previously thought. Huge hurricane-like vortices have been found above the poles of the planet and as yet unidentified UV absorbers that form mysterious dark bands in the upper atmosphere. At 70 km and below, water vapor and sulfur dioxide combine to form sulfuric acid droplets that create a haze above the cloud tops. Thermophilic acidophiles, such as have recently been discovered on Earth, could possibly survive in the hot sulfuric acid droplets that exist in the upper atmosphere of Venus. In order to understand how to search for life elsewhere in the Solar System, over 40 VIRTIS images of Earth from Venus have been obtained to search for evidence of life on Earth. The signatures of water and molecular Oxygen were detected in the Earth s atmosphere, but the atmosphere of Venus also exhibits these signatures. The water and water ice are far more abundant on comet, the polar caps and permafrost of Mars and the icy moons of Jupiter and Saturn. These "frozen worlds" of our Solar System, are much more promising regimes where extant or extinct microbial life may exist. The ESA Mars Advanced Radar for Subsurface and Ionospheric Sounding (MARSIS) probe has found that both the North and South Polar Caps of Mars are approximately 3.5 km thick and are composed almost entirely of water ice. In winter, a thin dry ice layer covers the caps, but it sublimates directly to CO2 in the spring. The ESA Mars Express Orbiter images reveal Rupes Tenuis to be a vast snow-laden region on the southern edge of the Martian North Polar Cap. The Mars Exploration Rover Spirit found alkaline volcanic rocks in the Gusev Crater and the Phoenix Mars Lander has shown that the soil of Mars is much more alkaline than previously expected. The Phoenix Mars Lander has also made direct observations of frozen and liquid water on Mars. It is known that microorganisms from Alaska, Siberia and Antarctica can remain alive frozen in permafrost or ice for long periods of time. These discoveries increase the possibility that the Labeled Release Experiment may have discovered life on Mars during the Viking Mission and provide strong impetus for the return of life detection experiments to Mars. Changes in the spin rate of Saturn's moon Titan indicate that it may also harbor a 300 km thick liquid water ocean beneath its icy crust. The NASA/ESA/Italian Space Agency Cassini Spacecraft has imaged geysers containing water vapor, methane, carbon dioxide and organics erupting from the "tiger stripe" regions near the South Pole of Saturn's moon Enceladus. The high temperatures observed, the water vapor and large number of ice particles expelled suggest that a liquid water lake may exist beneath the "tiger stripe" ice cracks of Enceladus. The NASA Deep Impact probe found the surface temperature of comet 9P/Temple 1 at 1.5 AY was slightly above the ice/water phase change temperature (273 K). This suggests melting of water ice near the comet surface. A spectrometer the spacecraft detected a mixture of clay and carbonate minerals (that form in the presence of liquid water) streaming off the comet after the collision with the pactor. The study of chemical and mineral biomarkers, chiral amino acids and possible indigenous microfossils in SNC and carbonaceous meteorites continues. These results suggest that comets should be considered prime targets in the search for Life in the Cosmos. The ESA Rosetta mission is on track to rendezvous with comet 67P/Churyumov-Gerasimenko. The recent space observations combined with new information about the ability of microbial extremophiles to thrive in polar environments suggest that life may be far more widely distributed in the Cosmos than previously thought possible. The Panelists will review recent discoveries and provide their own insights about Life in the Cosmos -- followed by a question and answer session with the audience.

Hoover, Richard B.↗

NASA NTRS2016

Space Biology Meets Astrobiology: Critical Synergies and Concerns

The broad fields of space biology and astrobiology share much in common in terms of science questions, approaches, and goals. However, historical circumstances and funding agency practices have frequently resulted in a wide separation between the two related areas. Is this a good thing? We believe that it is not, and that much is to be gained in each field from sharing ideas, resources, and perhaps projects between investigators traditionally working in one discipline or the other. Some of the strengths that the Space Biology community offers include sophistication and experience in flying experiments on space missions. In turn, Astrobiology has focused heavily on ground-based and field research. Challenging physical and chemical conditions experienced in space and on other planets partially overlap, and much can be gleaned from the body of work of each community along these topical lines. A combination of these areas of expertise and experience could result in major advances to all involved. When possible, avoiding having to reinvent methods or approaches already used by a sister community can result in greater efficiencies of resource use. We will discuss some case studies where we believe there are significant overlaps including adaptation to a variety of environmental stresses, extremophiles as potential flight organisms, microfluidics as applied to planetary environment simulations, and others.

Extremophiles↗

NASA NTRS2020

Environmental factors driving spatial heterogeneity in desert halophile microbial communities

Spatial heterogeneity in microbial communities is observed in all-natural ecosystems and can stem from both adaptations to local environmental conditions as well as stochastic processes. Extremophile microbial communities inhabiting evaporitic halite nodules (salt rocks) in the Atacama Desert, Chile, are a good model ecosystem for investigating factors leading to microbiome heterogeneity, due to their diverse taxonomic composition and the spatial segregation of individual nodules. We investigated the abiotic factors governing microbiome composition across different spatial scales, allowing for insight into the factors that govern halite colonization from regional desert-wide scales to micro-scales within individual nodules. We found that water availability and community drift account for microbiome assembly differently at different distance scales, with higher rates of cell dispersion at the smaller scales resulting in a more homogenous composition. This trend likely applies to other endoliths, and to non desert communities, where dispersion between communities is limited. At the intra-nodule scales, a light availability gradient was most important in determining the distribution of microbial taxa despite intermixing by water displacement via capillary action.

microbiome extremophile, desert, heterogeneity, Me↗

DOE OSTI2022

Climate warming causes photobiont degradation and carbon starvation in a boreal climate sentinel lichen

The long-term potential for acclimation by lichens to changing climates is poorly known, despite their prominent roles in forested ecosystems. Although often considered “extremophiles,” lichens may not readily acclimate to novel climates well beyond historical norms. In a previous study, Evernia mesomorpha transplants in a whole-ecosystem climate change experiment showed drastic mass loss after 1 yr of warming and drying; however, the causes of this mass loss were not addressed. We examined the causes of this warming-induced mass loss by measuring physiological, functional, and reproductive attributes of lichen transplants. Severe loss of mass and physiological function occurred above +2°C of experimental warming. Loss of algal symbionts (“bleaching”) and turnover in algal community compositions increased with temperature and were the clearest impacts of experimental warming. Enhanced CO 2 had no significant physiological or symbiont composition effects. The functional loss of algal photobionts led to significant loss of mass and specific thallus mass (STM), which in turn reduced water-holding capacity (WHC). Although algal genotypes remained detectable in thalli exposed to higher stress, within-thallus photobiont communities shifted in composition toward greater diversity. The strong negative impacts of warming and/or lower humidity on Evernia mesomorpha were driven by a loss of photobiont activity. Analogous to the effects of climate change on corals, the balance of symbiont carbon metabolism in lichens is central to their resilience to changing conditions.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Oleaginous Yeast Biology Elucidated With Comparative Transcriptomics

ABSTRACT Extremophilic yeasts have favorable metabolic and tolerance traits for biomanufacturing‐ like lipid biosynthesis, flavinogenesis, and halotolerance – yet the connection between these favorable phenotypes and strain genotype is not well understood. To this end, this study compares the phenotypes and gene expression patterns of biotechnologically relevant yeasts Yarrowia lipolytica , Debaryomyces hansenii , and Debaryomyces subglobosus grown under nitrogen starvation, iron starvation, and salt stress. To analyze the large data set across species and conditions, two approaches were used: a “network‐first” approach where a generalized metabolic network serves as a scaffold for mapping genes and a “cluster‐first” approach where unsupervised machine learning co‐expression analysis clusters genes. Both approaches provide insight into strain behavior. The network‐first approach corroborates that Yarrowia upregulates lipid biosynthesis during nitrogen starvation and provides new evidence that riboflavin overproduction in Debaryomyces yeasts is overflow metabolism that is routed to flavin cofactor production under salt stress. The cluster‐first approach does not rely on annotation; therefore, the coexpression analysis can identify known and novel genes involved in stress responses, mainly transcription factors and transporters. Therefore, this work links the genotype to the phenotype of biotechnologically relevant yeasts and demonstrates the utility of complementary computational approaches to gain insight from transcriptomics data across species and conditions.

Weintraub, Sarah J. [Department of Bioinformatics ↗

DOE OSTI2025

Identification and overexpression of endogenous transcription factors to enhance lipid accumulation in the biotechnologically relevant species Chlamydomonas pacifica

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wild-type. However, CpaPSR1 increased lipid accumulation compared to wild-type in normal media and did not increase further under nitrogen deprivation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild-type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production.

Biofuels↗