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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↗

DOE OSTI2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short- and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition. Overall design: Sampling of Chlamydomonas CC-4532 cells cultivated photoheterotrophically (TAP) under Fe-starvation condition (0 uM Fe-EDTA). Samples were collected at multiple timepoints from biological duplicate cultures after washing in TAP medium lacking Fe. Two time courses were collected. A short time course with t=0 (pre-wash), 0 (post-wash), 5, 10, 15, 30, 60, 120, and 240 min. A long time course with t= 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 hours. Please note that, for long time course, the GSE44611/PRJNA190650 samples were re-used/re-analyzed together with the short time course data: GSM1087792 C.reinhardtii_Fe_Long_0_hours SRX245324 SAMN01924672 GSM1087793 C.reinhardtii_Fe_Long_0.5_hours SRX245325 SAMN01924673 GSM1087794 C.reinhardtii_Fe_Long_1_hours SRX245326 SAMN01924674 GSM1087795 C.reinhardtii_Fe_Long_2_hours SRX245327 SAMN01924675 GSM1087796 C.reinhardtii_Fe_Long_4_hours SRX245328 SAMN01924676 GSM1087797 C.reinhardtii_Fe_Long_8_hours SRX245329 SAMN01924677 GSM1087798 C.reinhardtii_Fe_Long_12_hours SRX245330 SAMN01924678 GSM1087799 C.reinhardtii_Fe_Long_24_hours SRX245331 SAMN01924679 GSM1087800 C.reinhardtii_Fe_Long_48_hours SRX245332 SAMN01924680

Source record↗

DOE OSTI2023

Tribocorrosion of stainless steel sliding against graphite in FLiNaK molten salt

The molten salt reactor (MSR) concept recently gained renewed interest in developing Generation IV nuclear reactors. One MSR design uses fluoride molten salts to cool tri-structural isotropic particle fuel encapsulated by a carbon matrix into spherical pebbles, which would inevitably contact the reactor's stainless steel container wall during salt circulation. Characterizing this interaction is crucial for reactor safety. Here, in this work, we report the tribocorrosion behavior of graphite sliding against Type 316H stainless steel lubricated by a FLiNaK (LiF:NaF:KF; 46.5:11.5:42 mol %) molten salt in an argon environment. Accelerated wear loss was observed at a higher temperature because of a lower molten salt viscosity and a higher corrosion rate. The graphite had a more rapid material loss at a higher sliding speed than stainless steel because of its higher vulnerability to vibration-induced microfracture. The salt-starved condition caused more material loss than either the no-salt or the salt-flooded condition because neither a graphite transfer film nor stable boundary lubrication could be established at salt starvation. An interesting dual-layer surface film was discovered on the stainless steel worn surface: a chromium-rich top film and a nickel-accumulated but chromium-depleted interlayer. The film composition and structure provide fundamental insights to the mechanochemical interactions between stainless steel and graphite in a molten salt environment.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2024

Meta-omics reveals role of photosynthesis in microbially induced carbonate precipitation at a CO2-rich geyser

Abstract Microbially induced carbonate precipitation (MICP) is a natural process with potential biotechnological applications to address both carbon sequestration and sustainable construction needs. However, our understanding of the microbial processes involved in MICP is limited to a few well-researched pathways such as ureolytic hydrolysis. To expand our knowledge of MICP, we conducted an omics-based study on sedimentary communities from travertine around the CO2-driven Crystal Geyser near Green River, Utah. Using metagenomics and metaproteomics, we identified the community members and potential metabolic pathways involved in MICP. We found variations in microbial community composition between the two sites we sampled, but Rhodobacterales were consistently the most abundant order, including both chemoheterotrophs and anoxygenic phototrophs. We also identified several highly abundant genera of Cyanobacteriales. The dominance of these community members across both sites and the abundant presence of photosynthesis-related proteins suggest that photosynthesis could play a role in MICP at Crystal Geyser. We also found abundant bacterial proteins involved in phosphorous starvation response at both sites suggesting that P-limitation shapes both composition and function of the microbial community driving MICP.

Violette, Marlene J.↗

DOE OSTI2024

Regulation of bacterial stringent response by an evolutionarily conserved ribosomal protein L11 methylation

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics data set from 48 bacterial strains representing six phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Pumping Iron: A Multi-omics Analysis of Two Extremophilic Algae Reveals Iron Economy Management

Marine algae are responsible for half of the world's primary productivity, but this critical carbonsink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, isremarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A relatedspecies, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced highquality genome assemblies and transcriptomes for both species to serve as a foundation for acomparative multi-omics analysis. We identified a host of iron-uptake proteins in both species,including a massive expansion of transferrins and a novel family of siderophore-iron uptakeproteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large ironreservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reducedinvestment in the photosynthetic apparatus coupled with remodeling of antenna proteins bydramatic iron-deficiency induction of TIDI1, a light harvesting complex protein found also in otherchlorophytes. These combinatorial iron scavenging and sparing strategies make Dunaliellaunique among photosynthetic organisms

iron homeostasis, phytoplankton, Iron starvation i↗