Integrating omics analysis and evolutionary engineering for the optimisation of an industrial thraustochytrid strain

Abstract

The rapidly expanding global markets for aquaculture feed and human Omega-3 supplements currently rely predominantly on fish oil as their primary source of Omega-3 polyunsaturated fatty acids (ω-3-PUFAs). However, this dependence presents significant sustainability challenges, as approximately one-third of global wild fish stocks are currently overfished, with remaining stocks already exploited at maximum sustainable capacity. This concerning depletion of marine resources, coupled with growing concerns about bioaccumulation of toxic pollutants in fish-derived oil, has promoted the search for alternative ω-3-PUFA sources. Marine microalgae, as the primary producers of these valuable fatty acids in the marine food chain, could serve as a direct source of these essential nutrients for both human consumption and animal nutrition.

Among marine microorganisms, thraustochytrids, heterotrophic unicellular marine protists, have emerged as particularly promising candidates for commercial ω-3-PUFA production, especially due to high accumulation of Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA). Additionally, they are valuable sources of carotenoids, including astaxanthin, cantaxanthin, and β-carotene, which are commercially important natural pigments with potent antioxidant properties used in nutraceutical, pharmaceutical, and feed industries. These organisms offer distinct advantages over other oleaginous microorganisms, including fast heterotrophic growth rates, competency for large-scale industrial fermentation, and production of toxin-free oils. Thraustochytrids accumulate substantial lipid content as a stress response mechanism, capable of producing biomass containing over 50% lipids by cell dry weight, with ω-3-PUFAs constituting more than 40% of this lipid fraction. Despite these favorable characteristics, further phenotypic improvements are necessary to achieve full economic viability and environmental sustainability in commercial applications.

Previous studies have demonstrated that dissolved oxygen levels are critical in optimizing biomass production and DHA synthesis in thraustochytrid cultivation. Paradoxically, elevated oxygen levels, while beneficial for growth, create conditions where polyunsaturated fatty acids become prone to oxidative degradation, potentially compromising production efficiency. The challenges of lipid peroxidation and limitations in cellular antioxidant defense systems are often overlooked in fermentation process development. Furthermore, despite advances in fermentation techniques, the high genetic variation among thraustochytrid strains and the scarcity of comprehensive genomic references continue to restrict our understanding of the molecular mechanisms governing stress adaptation and metabolite production in these organisms.

This study presents a comprehensive investigation of a novel industrial thraustochytrid Thrausto_test strain, combining genomic characterization, adaptive laboratory evolution (ALE), and transcriptomic analysis to enhance its biotechnological potential. Our dual objectives were to increase the oxidative stress resistance of the strain using the ALE approaches, focusing on PUFA and carotenoid production, and to generate high-quality genomic resources for this and further functional genomics studies of thraustochytrid species.

We first generated Thrausto_test de novo genome assembly using a hybrid approach combining Nanopore long-read sequencing with Illumina short-read polishing resulting in approximately 450x coverage. The resulting 36.7 Mb assembly demonstrated exceptional quality and completeness with only 33 contigs, an N50 of 1988 Kb, and 91.09% of BUSCO conserved genes, surpassing many existing thraustochytrid genome assemblies. We complemented this with a comprehensive de novo transcriptome assembly by integrating RNA-sequencing data from three distinct growth phases, capturing a complete transcriptional profile of Thrausto_test strain. This transcriptome comprised 9,038 sequences averaging 3.54 kb in length, with an N50 of 4.93 kb and 80.39% BUSCO completeness. This assembly, alongside our comprehensive transcriptome, addresses a critical gap in thraustochytrid genomic resources and establishes a robust foundation for functional studies.

Adaptive laboratory evolution experiments were performed using two strategies: UV-C mutagenesis with high oxygenation (ALE_1), and UV-C mutagenesis with tert-butyl hydroperoxide (tBuOOH) selection (ALE_2). tBuOOH was strategically selected as an oxidative stressor due to its primary mechanism of causing oxidative stress by triggering lipid peroxidation, particularly relevant for the lipid-rich Thrausto_test strain. The ALE_1 experiment resulted in ALE_UV strain with increased DHA content under high oxygenation conditions, indicating that UV-induced mutations combined with selective pressure can drive improvements in PUFA production under high oxygenation conditions. The ALE_2 experiment produced final strain with robust oxidative stress adaptation, exhibiting significant improvements in cell survival from just 3.6% in the first cycle to 60% by the end of the experiment, accompanied by a remarkable 23.69% increase in total carotenoid content and significant increases in individual carotenoids including astaxanthin (39.39%), cantaxanthin (21.81%), β-carotene (33.91%), and lutein (28.41%).

Comparative transcriptomic analysis revealed distinct patterns of stress response between the evolved strains. Particularly, the final ALE_2_cycle_10 strain demonstrated comprehensive transcriptional reprogramming characterized by complex multi-level defense strategy. This included significant upregulation of key antioxidant enzymes (SOD, APX, PRDX-6), increased heat shock protein expression and activation of stress signalling regulatory network through AP-1 transcription factors and MAPK pathways. The oxidative stress adaptation of ALE2_cycle_10 was further supported by significant upregulation of central carbon metabolism, enhanced NADPH regeneration pathways, and strategic reallocation of resources between FAS and PKS pathways. Notably, key carotenoid biosynthesis enzymes, particularly β-carotene synthase and geranylgeranyl pyrophosphate (GGPP) genes, were significantly upregulated in the final ALE2_cycle_10 strain, identifying promising targets for future strain improvement. In contrast, ALE_1 and ALE_UV strains showed more moderate adaptations primarily focused on maintaining PUFA synthesis under stress conditions, with selective upregulation of key glycolysis enzymes and alternative NADPH generation through malic enzyme.

These transcriptional adaptations were found to be correlated with significant morphological changes in the ALE2_cycle_10 strain, including significantly increased cell size and altered lipid body distribution. The strategic shift in fatty acid metabolism, evidenced by increased SFA ratio (45.03%) and decreased PUFA percentage (51.99%) in total fatty acids, appeared to be operated through differential regulation of FAS and PKS pathways, correlating with transcriptional upregulation of genes involved in NADPH generation and malonyl-CoA production.

The evolved strains with enhanced oxidative stress resistance represent a significant advancement for industrial biotechnology, offering solutions for sustainable production of ω-3-PUFAs and natural pigments for both human nutrition and aquaculture feed production. This research establishes a robust foundation for future studies by integrating genomic characterization, adaptive laboratory evolution, biochemical analysis, and transcriptomics to elucidate stress resistance mechanisms while exploring their connections to targeted metabolite biosynthetic pathways. The identification of key regulatory elements and metabolic pathways provides valuable targets for future strain improvement efforts, and for developing more targeted optimization strategies for thraustochytrids and related oleaginous microorganisms. These approaches have not only generated improved strains but also provided insights into the complex molecular interplay between oxidative stress, lipid and carotenoid metabolism, and antioxidant defence in thraustochytrids. The value of this work extends beyond solely research boundaries, addressing global sustainability challenges in both human and animal feed production. Importantly, our non-GMO approach to strain improvement aligns with consumer preferences for natural production systems and regulatory frameworks in many countries.