The filamentous microalgae Tribonema minus accumulates large amounts of lipids under photoautotrophic condition, while under heterotrophic condition, the lipid content decreased dramatically. Determination of the differences in metabolic pathways between photoautotrophic and heterotrophic growth will provide targets and strategies for improvement of lipid accumulation in heterotrophic cells. The metabolic differences between photoautotrophically and heterotrophically cultivated T. minus cells were studied by comparing the growth, biochemical compositions and transcriptomic and metabolomic profiles of the cells. Based on comparative transcriptomic and metabolomic studies, we generated a global model of the changes in central carbon metabolism and lipid biosynthetic pathways that occur under photoautotrophic and heterotrophic growth conditions. Moreover, the specific effects of supplementation with exogenous key metabolic intermediates on the lipid accumulation in heterotrophic culture were analyzed. Compared to photoautotrophic cultures, heterotrophic cultures exhibited enhanced biomass levels and carbohydrate content, but decreased lipid accumulation. These effects were accompanied by low expression levels of genes involved in glycolysis, de novo fatty acids biosynthesis and lipid biosynthesis, and high levels of genes involved in gluconeogenesis. In addition, the levels of key metabolites involved in glycolysis/gluconeogenesis were elevated in abundance, whereas those of certain fatty acids and citric acid were decreased in heterotrophic cultures. Upon supplementation with exogenous potassium palmitate, the lipid content increased dramatically in heterotrophically cultivated T. minus. An insufficient supply of carbon precursors caused the low levels of lipid accumulation during heterotrophic cultivation. Appropriate carbon metabolite supplementation based on the metabolomic data was shown to promote lipid accumulation. Moreover, gene regulatory metabolic targets were also identified via omics analysis.

Palmitoleic acid, one scarce omega-7 monounsaturated fatty acid, has important applications in the fields of medicine and health products. Tribonema has been considered as a promising candidate for the production of palmitoleic acid due to its high lipid and palmitoleic acid content and remarkable heterotrophic ability. The high-density heterotrophic cultivation of Tribonema minus was conducted in this work, and the highest biomass of 42.9 g L⁻¹ and a relatively low lipid content of 28.7% were observed. To further enhance the lipid and palmitoleic acid accumulation, induction strategies under two regimes of phototrophy and heterotrophy with different conditions were investigated and compared. Results demonstrated encouraging promotions both by heterotrophic and phototrophic ways, and the final lipid contents reached 41.9% and 49.0%, respectively. In consideration of the time cost, however, the induction under heterotrophic conditions was much more advantageous, by which the highest lipid and palmitoleic acid productivities of 1.77 g L⁻¹ d⁻¹ and 924 mg L⁻¹ d⁻¹ were obtained respectively, with the lipid yield on glucose of 0.26 g g⁻¹.

Palmitoleic acid, a valuable functional fatty acid, is notably scarce in traditional oil crops, with the exception of certain wild plants such as macadamia nuts and sea buckthorn. Recently, the lipid from Saccharomyces cerevisiae was found to contain approximately 50% palmitoleic acid. Consequently, S. cerevisiae has the potential to sustainably produce palmitoleic acid through fermentation, provided that the issue of promoting its lipid content is addressed. In this work, based on the previously isolated oleaginous wild strain of S. cerevisiae, the mutagenesis by zeocin combined with ARTP was carried out to generate S. cerevisiae mutants, and then the high lipid content mutants were isolated using the flow-mode Raman-activated cell sorting (FlowRACS) technique, which allowed for the high-throughput selection of these mutants in a label-free and non-invasive manner. The mutant MU2R48 was finally obtained and its lipid content was 40.26%, 30.85% higher than the original type. Transcriptome and targeted metabolome analysis revealed a coordinated interaction of fatty acid precursor biosynthesis, the pentose phosphate pathway, ethanol degradation, and amino acid metabolism, synergistically channeling carbon flux from acetyl-CoA and NADPH into lipid biosynthesis. Additionally, key transcriptional regulators within the lipid metabolism network were implicated in this enhanced lipid accumulation. In this study, a mutant strain of Saccharomyces cerevisiae MU2R48 with 40.26% lipid content was successfully generated through zeocin-ARTP mutagenesis combined with Raman-activated cell sorting. Multi-omics analysis revealed that the enhanced lipid accumulation was driven by coordinated up-regulation of precursor biosynthesis, carbon flux redirection, and key transcriptional regulators, with increased acetyl-CoA and NADPH production fluxes likely serving as the pivotal determinants.