There has been a long-standing paradox in oceanic phosphorus (P) cycling in the ocean: high alkaline phosphatase activity (APA) persists in deep waters despite replete dissolved inorganic phosphorus (DIP), and active microbial regulatory mechanisms driving this pattern remain largely untested in the carbon-limited hadal zone. Here, we test the hypothesis that the observed elevated levels of deep-ocean APA is driven by microbial carbon demand, via full-depth water column analyses of dissolved organic phosphorus (DOP) and DIP in the Challenger Deep (Mariana Trench), combined with laboratory-based in situ-simulated high-pressure incubation experiments. We reveal two distinct phosphorus-alkaline phosphatase activity (P-APA) regulatory regimes: P-limitation-driven extreme APA in P-depleted surface waters, and sustained, elevated APA in P-replete, carbon-starved deep waters. Metabolically active alkaline phosphatase (AP)-producing taxa, most notably the SAR11 clade, were detectable throughout the full water column. Path analysis was used to evaluate the consistency of the observed data with a hypothesized causal framework linking active microbial communities, APA kinetics, and coupled phosphorus-carbon (P-C) cycling, with the model explaining 82.3% of the variance in dissolved organic carbon and 75.4% of the variance in DIP in the water column. We propose and validate a “piggyback” strategy whereby deep-sea microbes express AP to acquire carbon from DOP, offering a previously untested, potential mechanistic explanation for the long-standing deep APA paradox, while revealing a microbially mediated P-C coupling pathway that may represent a breakaway of deep-ocean carbon sequestration pathway.

Thraustochytrids are marine protists valued for producing docosahexaenoic acid (DHA) and saturated fatty acids (SFAs), but limited genetic tools have hindered pathway studies and optimization. Here, we developed an optimized DNA transformation protocol for Aurantiochytrium strains to overexpress the Sfp-type phosphopantetheine transferase gene (ppt) and evaluate its role in fatty acid synthesis. Zeocin proved the most effective selection marker, while optimized electroporation parameters (0.50 kV/mm, 1 pulse, 50 μF, 200 Ω) enabled efficient transformation of Aurantiochytrium sp. PKU#Sw8. The overexpression of ppt significantly increased fatty acid yields SFAs rose 2.86-fold, DHA 2.06-fold, and total fatty acids 2.02-fold. Palmitic acid production increased 7.13-fold, shifting the PA/DHA ratio from 0.41 to 1.02. Transcriptomics revealed enhanced precursor supply for FAS and PKS pathways, reduced fatty acid oxidation, and flux redirection toward SFAs via FAS. These results clarify ppt-driven fatty acid enhancement and provide a framework for engineering thraustochytrids for SFAs production.

Salinity is a key environmental factor regulating lipid metabolism in marine oleaginous protists. This study examined the impact of NaCl concentration on growth, glucose utilization, and lipid biosynthesis in Thraustochytrium sp. ATCC 26185. Moderate salinity (20 g/L) enhanced biomass and glucose uptake, while high salinity (45 g/L) induced osmotic stress yet significantly promoted squalene accumulation (17.27 mg/g), a 3.26-fold increase compared with 0 g/L NaCl (5.29 mg/g). Integrated transcriptomic and metabolomic analyses revealed that salinity-dependent activation of glycolysis, the TCA cycle, and the pentose phosphate pathway increased cellular ATP, NADH, and NADPH levels. Under salt stress, the mevalonate (MVA) pathway was transcriptionally upregulated, with key enzymes, including ACAT, HMGR, and IDI, showing marked induction, which supports enhanced carbon flux toward squalene biosynthesis. Despite SQS downregulation, squalene accumulation increased, likely due to elevated precursor availability and reduced flux to downstream sterol pathways. Concurrently, high salinity repressed expression of ACC, FAS-α, and FAS-β, reducing saturated fatty acid levels, while upregulation of PKSB-favored polyunsaturated fatty acid (PUFA) synthesis. These findings suggest that high-salt stress triggers transcriptional reprogramming, redirecting acetyl-CoA from fatty acid synthesis toward squalene and PUFA production. This study offers new insights into the metabolic plasticity of thraustochytrids and highlights salinity modulation as a promising strategy for enhancing high-value lipid yields in marine biotechnology.

BACKGROUND: Marine protists Aurantiochytrium are recognized as promising sources for commercial lipid production, particularly due to their ability to produce high-value natural compounds like docosahexaenoic acid (DHA). However, wild-type strains isolated from natural environments typically fail to meet commercial demands for DHA yields, partly because they are poorly adapted to the decreased pH conditions encountered during fermentation. RESULTS: In this study, we employed a staged acidic adaptive laboratory evolution (ALE) strategy to develop a high DHA-producing strain from Aurantiochytrium sp. PKU#Mn16. By optimizing oxygen and temperature levels under low pH conditions, ALE resulted in a 171.4% increase in DHA concentration in the ALE strain compared to the wild-type strain. Comparative transcriptomics revealed that ALE enhanced the expression of key enzymes in glycolysis and the polyketide synthase (PKS) pathway during both early (metabolic peak) and late (metabolic decline) fermentation stages, promoting growth and polyunsaturated fatty acid synthesis. Additionally, key enzymes in the tricarboxylic acid (TCA) cycle and pentose phosphate (PPP) pathway were upregulated at early and late stages, respectively, suggesting differential ATP/NADPH supply mechanisms that drive DHA accumulation. Notably, the upregulation of glycerol kinase (GK) indicates the potential for using glycerol as an alternative carbon source to further enhance DHA production in our ALE strain. CONCLUSIONS: In this study, Aurantiochytrium sp. PKU#Mn16 was successfully acclimated using a synergistic approach combining high dissolved oxygen, low temperature, and citric acid-induced acidity. This strategy yielded significant increases of 106.3% in biomass, 243.8% in total fatty acid yield, and 171.4% in DHA yield. Transcriptomic analysis revealed extensive rewiring of central carbon and lipid metabolism, including the upregulation of PKS pathway enzymes and enhanced supply of ATP, NADPH, and acetyl-CoA. Additionally, reduced competing secondary metabolic fluxes optimized substrate allocation. This innovative acclimation strategy not only sheds light on the molecular mechanisms driving efficient fatty acid and DHA production but also lays the groundwork for future comparative genomics and genetic editing efforts aimed at further boosting yields of fatty acids and other natural secondary metabolites in thraustochytrids.

DHA (docosahexaenoic acid, C22:6) is a vital omega-3 fatty acid with profound roles in human health, yet sustainable production remains constrained by process inefficiencies. Thraustochytrids are recognized as prolific DHA producers; however, fermentation optimization often relies on empirical adjustments rather than mechanistic insights. Here, we systematically dissected the effects of temperature, dissolved oxygen (DO), and dynamic control strategies on DHA biosynthesis in Aurantiochytrium sp. PKU#Mn16, linking environmental cues to intracellular metabolic states. Biomass accumulation peaked at 65.7 g/L at 32 °C, but 28 °C offered the best balance, yielding 24.5 g/L total fatty acids (TFA) and 9.4 g/L DHA at 96 h. Low temperatures (16-20 °C) enriched DHA to > 50 % of TFA, while high DO (40-50 %) boosted early biomass (68.9 g/L) but caused > 40 % DHA loss post-96 h. Metabolite profiling revealed that low temperature and DO elevated acetyl-CoA, NADPH, and ATP, priming cells for PUFA synthesis. Guided by these insights, we designed a three-stage fed-batch strategy that raised TFA and DHA to 30.3 and 14.7 g/L (1.24- and 1.56-fold improvements), with DHA comprising 48.5 % of TFA. Pilot-scale (50 L) fermentation confirmed reproducibility and scalability, establishing metabolite-informed dynamic control as a robust and industrially relevant strategy for sustainable DHA production.

Nitrogen sources critically influence cellular growth and lipid biosynthesis in thraustochytrids, yet their effects on docosahexaenoic acid (DHA) production in this heterotrophic marine protist remain unclear. This study examines how ammonium chloride, sodium nitrate, and sodium glutamate regulate DHA and fatty acid biosynthesis in Aurantiochytrium sp. PKU#SW8. Sodium glutamate enhanced glycolysis, tricarboxylic acid (TCA) cycle, nitrogen metabolism, and both fatty acid synthase (FAS) and polyketide synthase (PKS) pathways, yielding higher DHA and fatty acid production than inorganic sources. Ammonium chloride stimulated upstream glycolysis, triacylglycerol (TAG) synthesis and pentose phosphate pathway (PPP) activity, while sodium nitrate promoted fatty acid precursor synthesis, TCA energy generation, and nitrogen metabolism. Combining these nitrogen sources could further improve DHA yield. Enzyme annotations confirmed the presence of a complete nitrogen metabolic network in PKU#SW8. These findings offer valuable insights for optimizing nitrogen sources in microbial DHA fermentation and lipid production.

Background:Thraustochytrids are unicellular heterotrophic protists within the Stramenopiles group, widely distributed across marine ecosystems. Understanding the mechanisms underlying their metabolic ecotype evolution is pivotal for revealing how these organisms drive the marine carbon cycle and adapt to diverse environments. Methods:In this study, we report a high-quality genome of Aurantiochytrium sp. TWZ-97 and conduct a comparative genomics analysis of thraustochytrid strains to investigate ecotype-specific differences in genome structure, evolutionary-developmental relationships, and core functional genes. Results:Comparative genomics revealed that "anabolic" strains (TWZ-97, Mn4, SW8) possess larger genomes with lower gene density, whereas "catabolic" strains (S-28, S-429) have smaller, gene-rich genomes with stable repetitive elements. Phylogenetic analyses revealed that the "anabolic" strains diverged relatively recently, around 2.389 million years ago, while the "catabolic" strains evolved independently for over 190.7 million years, reflecting prolonged, lineage-specific adaptation. Functionally, "anabolic" strains were enriched in fatty acid synthase genes, whereas hydrolytic enzyme genes were unique to the "catabolic" strains. Both ecotypes exhibited a significant abundance of fatty acid desaturase (FAD) genes, and polyketide synthase (PKS) genes displayed unique long sequences, multi-domain architectures, and ecotype-specific gene differentiation patterns. Conclusion:Together, this study provides crucial molecular evidence for the genetic basis of metabolic specialization and ecotype diversification in thraustochytrids.

Phenylethylisoquinoline alkaloids (PIAs) are medicinally important natural products derived from the 1-phenylethylisoquinoline precursor. Heterologous production of the PIAs remains challenging due to the incomplete elucidation of biosynthetic pathway and the lack of proper microbial cell factory designed for precursor enhancement. In this work, an artificial pathway composed of eight enzymes from different species was established for de novo 1-phenylethylisoquinoline biosynthesis in engineered Escherichia coli. The yield of the intermediate 4-hydroxydihydrocinnamaldehyde was optimized through screening various NADP+-dependent 2-alkenal reductases, cofactor regeneration and the site-directed mutagenesis of key residues in ChAER1. Subsequently, incorporation of the modified dopamine pathway into an endogenous reductase-deficient E. coli with high tyrosine yield boosted the production of 1-phenylethylisoquinoline, reaching 402.58 mg/L in a 5L fermenter. Our work lays a foundation for the future large-scale production of high value-added 1-phenylethylisoquinoline-related alkaloids.

Pathogens in coastal waters cause infectious diseases and endanger public sanitation safety in humans and animals worldwide. To avoid these risks, timely detection of human-associated pathogens in waters is crucial. In this study, the decay kinetics of the molecular markers for human-associated pathogens, including enteric bacteria (Escherichia coli, Enterococcus, and Bacteroides), non-enteric bacteria (Staphylococcus aureus), crAssphage, and polyomavirus, were monitored over time at different temperatures and background microbes in seawater microcosms. The results indicated that temperature and native marine microbes were the main influential factors in attenuating bacterial pathogens. Remarkably, the effect of native microorganisms was more evidentially striking. Furthermore, Enterococcus was a more reliable and suitable fecal indicator bacterium than E. coli for the marine environment. The decay of crAssphage was like that of polyomavirus, indicating that it may be a good indicator of enterovirus in seawater. More importantly, the 16S amplicon sequencing data highlighted the decay kinetics of multiple bacterial pathogens in parallel with the dynamic changes of the whole bacterial communities. This study provides valuable information for public health risk management and a new approach to understanding the fate of bacteria in the coastal environment.

Transport of organic matter (OM) occurs widely in the form of animal and plant detritus in global oceans, playing a crucial role in global carbon cycling. While wood- and whale-falls have been extensively studied, the in situ process of OM remineralization by microorganisms remains poorly understood particularly in pelagic regions on a global scale. Here, enrichment experiments with animal tissue or plant detritus were carried out in three deep seas for 4-12 months using the deep-sea in situ incubators. We then performed community composition analyses as well as metagenomic and metatranscriptomic analyses. The results revealed strikingly similar microbial assemblages responsible for decomposing animal and plant detritus. Genes encoding peptidases and glucoside hydrolases were highly abundant and actively transcribed in OM enrichments, which confirmed the roles of these enriched microbial assemblages in organic decomposition. Marinifilaceae, Desulfocapsaceae, Spirochaetaceae, and o-Peptostreptococcales were found to potentially contribute to nitrogen fixation. These core bacteria, acting as cosmopolitan anaerobes in decomposing fast-sinking particulate OM, may have been underestimated in terms of their role in deep-sea microbial-mediated biogeochemical cycles during conventional sampling and diversity survey.

Background Most researches on sponge holobionts focus primarily on symbiotic microbes, yet data at the level of the sponge hologenome are still relatively scarce. Understanding of the sponge host and its microbial gene expression profiles and the host-microbes interplay in different niches represents a key aspect of sponge hologenome. Using the Hawaiian demosponge Mycale grandis in different niches as a model, i.e. on rocks, on the surface of coral Porites compressa, under alga Gracilaria salicornia, we compared the bacterial and fungal community structure, functional gene diversity, expression pattern and the host transcriptome by integrating open-format (deep sequencing) and closed-format (GeoChip microarray) high-throughput techniques. Results Little inter-niche variation in bacterial and fungal phylogenetic diversity was detected for M. grandis in different niches, but a clear niche-dependent variability in the functional gene diversity and expression pattern of M. grandis host and its symbiotic microbiota was uncovered by GeoChip microarray and transcriptome analyses. Particularly, sponge host genes related to innate immunity and microbial recognition showed a strong correlation with the microbial symbionts' functional gene diversity and transcriptional richness in different niches. The cross-niche variability with respect to the symbiont functional gene diversity and the transcriptional richness of M. grandis holobiont putatively reflects the interplay of niche-specific selective pressure and the symbiont functional diversity. Conclusions Niche-dependent gene expression profiles of M. grandis hologenome and the host-microbes interplay were suggested though little inter-niche variation in bacterial and fungal diversity was detected, particularly the sponge innate immunity was found to be closely related to the symbiotic microbes. Altogether, these findings provide novel insights into the black box of one sponge holobiont in different niches at the hologenome level.

Global change mediated shifts in ocean temperature and circulation patterns, compounded by human activities, are leading to the expansion of marine oxygen minimum zones (OMZs) with concomitant alterations in nutrient and climate-active trace gas cycling. While many studies have reported distinct bacterial communities within OMZs, much of this research compares across depths rather with oxygen status and does not include eukayrotic microbes. Here, we investigated the Bay of Bengal (BoB) OMZ, where low oxygen conditions are persistent, but trace levels of oxygen remain (< 20 mu M from 200 to 500 m). As other environmental variables are similar between OMZ and non-OMZ (NOZ) stations, we compared the abundance, diversity, and community composition of several microbial groups (bacterioplankton, Labyrinthulomycetes, and fungi) across oxygen levels. While prokaryote abundance decreased with depth, no significant differences existed across oxygen groups. In contrast, Labyrinthulomycetes abundance was significantly higher in non-OMZ stations but did not change significantly with depth, while fungal abundance was patchy without clear depth or oxygen-related trends. Bacterial and fungal diversity was lower in OMZ stations at 500 m, while Labyrinthulomycetes diversity only showed a depthrelated profile, decreasing below the euphotic zone. Surprisingly, previously reported OMZ-associated bacterial taxa were not significantly more abundant at OMZ stations. Furthermore, compared to the bacterioplankton, fewer Labyrinthulomycetes and fungi taxa showed responses to oxygen status. Thus, this research identifies stronger oxygen-level linkages within the bacterioplankton than in the examined microeukaryotes.

With a ubiquitous presence in marine ecosystems, Labyrinthulomycetes protists (LP) play critical ecological roles in oceanic habitats. Recently, some LP strains have been suggested to survive in low-salinity environments, but their distribution in freshwaters was largely unknown. This study investigated LP abundance and diversity dynamics along a fresh-saltwater gradient in 2 seasons. LP were detected in all samples. Although LP abundance in freshwaters (typically 10 4 to 10 5 copies l -1 ) was significantly lower than that in saline waters, their abundance still corresponded to that of previously reported LP in some coastal waters, suggesting their potentially essential roles in riverine ecosystems. High-throughput sequencing analyses identified 110, 54, and 146 LP amplicon sequence variants (ASVs) in fresh, brackish, and saline waters, respectively. Canonical correspondence analysis and variance partitioning analysis further indicated that salinity and temperature were the most significant environmental factors to affect LP community structure. Notably, most of the dominant ASVs in fresh/brackish waters were annotated to a rarely reported Labyrinthulida family, Amphitraemidae, and a newly identified river cluster of the order Thraustochytrida, which were significantly different from those of saline waters. Finally, the metabolic capabilities of the detected LP genera suggest that LP likely play diverse ecological roles in riverine ecosystems.

The ecological and scientific significance of microbial communities in marine ecosystems is underscored by their diverse and dynamic nature [...]

Background Thraustochytrids accumulate lipids with a high content of docosahexaenoic acid (DHA). Although their growth and DHA content are significantly affected by the dissolved oxygen (DO) supply, the role of DO on the transcriptional regulation of metabolism and accumulation of intracellular metabolites remains poorly understood. Here we investigate the effects of three different DO supply conditions (10%, 30%, and 50%) on the fed-batch culture of the Aurantiochytrium PKU#Mn16 strain to mainly reveal the differential gene expressions and metabolite profiles. Results While the supply of 10% DO significantly reduced the rates of biomass and DHA production in the early stages of fermentation, it achieved the highest amounts of biomass (56.7 g/L) and DHA (6.0 g/L) on prolonged fermentation. The transcriptome analyses of the early stage (24 h) of fermentation revealed several genes involved in the central carbon, amino acid, and fatty acid metabolism, which were significantly downregulated at a 10% DO level. The comparative metabolomics results revealed the accumulation of several long-chain fatty acids, amino acids, and other metabolites, supporting the transcriptional regulation under the influence of a low oxygen supply condition. In addition, certain genes involved in antioxidative systems were downregulated under 10% DO level, suggesting a lesser generation of reactive oxygen species that lead to oxidative damage and fatty acid oxidation. Conclusions The findings of this study suggest that despite the slow growth and metabolism in the early stage of fermentation of Aurantiochytrium sp. PKU#Mn16, a constant supply of low dissolved oxygen can yield biomass and DHA content better than that with high oxygen supply conditions. The critical information gained in this study will help to further improve DHA production through bioprocess engineering strategies.

Unicellular heterotrophic eukaryotes are an important component of marine ecosystems. However, their ecological functions and modes of nutrition remain largely unknown.

Fungi have long been known to be dynamic in coastal water columns with multiple trophic modes. However, little is known about their interactions with abiotic and biotic components, contribution to the biological carbon pump (BCP), and organic matter remineralization in the oceanic water column. In this study, we investigated how fungi vary spatially and how their variations relate to that of bacteria in the water column of the South China Sea (SCS). Fungi were about three orders less prevalent than bacteria, and the main factors influencing their distribution were depth, temperature, and distance from the sites of riverine inputs. The decline in the abundance of fungi with depth was less steep than that of bacteria. Correlation tests revealed a strong positive association between the abundance of fungi and bacteria, especially in the twilight (r = 0.62) and aphotic (r = 0.70) zones. However, the co-occurrence network revealed mutual exclusion between certain members of fungi and bacteria. The majority of fungi in the water column were saprotrophs, which indicated that they were generally involved in the degradation of organic matter, particularly in twilight and aphotic zones. Similar to bacteria, the involvement of fungi in the metabolism of carbohydrates, proteins, and lipids was predicted, pointing to their participation in the turnover of organic carbon and the biogeochemical cycling of carbon, nitrogen, and sulfur. These findings suggest that fungi play a role in BCP and support their inclusion in marine microbial ecosystem models.

The consequences of climate change may directly or indirectly impact the marine biosphere. Although ocean stratification has been recognized as one of the crucial consequences of ocean warming, its impacts on several critical aspects of marine microbes remain largely unknown in the Indian Ocean. Here, we investigate the effects of water stratification, in both surface and subsurface layers, on hydrogeographic parameters and bacterioplankton diversity within the equatorial eastern Indian Ocean (EIO). Strong stratification in the upper 200 m of equatorial EIO was detected with evidential low primary productivity. The vertical bacterioplankton diversity of the whole water columns displayed noticeable variation, with lower diversity occurring in the surface layer than in the subsurface layers. Horizontal heterogeneity of bacterioplankton communities was also in the well-mixed layer among different stations. SAR11 and Prochlorococcus displayed uncharacteristic low abundance in the surface water. Some amplicon sequence variants (ASVs) were identified as potential biomarkers for their specific depths in strongly-stratified water columns. Thus, barriers resulting from stratification are proposed to function as an 'ASV filter' to regulate the vertical bacterioplankton community diversity along the water columns. Overall, our results suggest that the effects of stratification on the structure and diversity of bacterioplankton can extend up to the bathypelagic zone in the strongly-stratified waters of the equatorial EIO. This study provides the first insight into the effect of stratification on the subsurface microbial communities in the equatorial eastern Indian Ocean.

The study of heterotrophic protists in the ocean is still in its early stages, compared to other microorganisms such as bacteria, archaea, and photoautotrophic protists. Labyrinthulomycetes protists (LP) are a type of unicellular protists that are widely distributed in global waters and have the potential to produce high-value products. In this study, the abundance, diversity, and community structure of LP in the coastal zone of Hainan Island in the South China Sea were investigated through quantitative PCR and high-throughput sequencing. The results showed that LP abundance varied by location and depth, with the highest levels (37.3 × 103 copies/L) found in the middle layer offshore and the lowest (0.386 × 103 copies/L) in the bottom layer offshore. The middle layer (chlorophyll maximum layer) had higher LP abundance both inshore and offshore than the surface and bottom layers. Interestingly, the highest LP richness and diversity was found in the inshore bottom. There was a significant difference in LP abundance between the offshore surface and bottom layers. The LP community was dominated by the genus Aplanochytrium, and four different ecotypes were identified. Additionally, the genus Aurantiochytrium had different cooperative and competitive strategies with bacteria in different habitats. This study sheds light on the abundance and community structure of LP in the coastal zone of Hainan Island, explores the potential interactions between LP and bacterial populations, and raises questions about the potential differentiation of LP ecotypes.