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Abstract The biological production of lipids presents a sustainable method for generating fuels and chemicals. Recognized as safe and enhanced by advanced synthetic biology and metabolic engineering tools, yeasts are becoming versatile hosts for industrial applications. However, lipids accumulate predominantly as triacylglycerides in yeasts, which are suboptimal for industrial uses. Thus, there have been efforts to directly produce free fatty acids and their derivatives in yeast, such as fatty alcohols, fatty aldehydes, and fatty acid ethyl esters. This review offers a comprehensive overview of yeast metabolic engineering strategies to produce free fatty acids and their derivatives. This study also explores current challenges and future perspectives for sustainable industrial lipid production, particularly focusing on engineering strategies that enable yeast to utilize alternative carbon sources such as CO2, methanol, and acetate, moving beyond traditional sugars. This review will guide further advancements in employing yeasts for environmentally friendly and economically viable lipid production technologies.

Abstract Background Schizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production. Results Transgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain. Conclusion This study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Abstract Background Microalgae's low tolerance to high CO2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO2 content—an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO2 conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO2) and at various growth phases. Results Cells cultivated with 10% CO2 exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO2 conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO2 conditions, resulting in improved CO2 fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO2 conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae. Conclusions We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.