High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform.

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.

Engineering of Saccharomyces cerevisiae for the accumulation of high amounts of triacylglycerol.

BACKGROUND: Fatty acid-based substances play an important role in many products, from food supplements to pharmaceutical products and biofuels. The production of fatty acids, mainly in their esterified form as triacylglycerol (TAG), has been intensively studied in oleaginous yeasts, whereas much less effort has been invested into non-oleaginous species. In the present work, we engineered the model yeast Saccharomyces cerevisiae, which is commonly regarded as non-oleaginous, for the storage of high amounts of TAG, comparable to the contents achieved in oleaginous yeasts. RESULTS: We investigated the effects of several mutations with regard to increased TAG accumulation and identified six of them as important for this phenotype: a point mutation in the acetyl-CoA carboxylase Acc1p, overexpression of the diacylglycerol acyltransferase Dga1p, deletions of genes coding for enzymes involved in the competing pathways glycogen and steryl ester synthesis and TAG hydrolysis, and a deletion of CKB1, the gene coding for one of the regulatory subunits of casein kinase 2. With the combination of these mutations in a S. cerevisiae strain with a relatively high neutral lipid level already in the non-engineered state, we achieved a TAG content of 65% in the dry biomass. High TAG levels were not only obtained under conditions that favor lipid accumulation, but also in defined standard carbon-limited media. CONCLUSIONS: Baker's yeast, which is usually regarded as inefficient in the storage of TAG, can be converted into a highly oleaginous strain that could be useful in processes aiming at the synthesis of fatty acid-based products. This work emphasizes the importance of strain selection in combination with metabolic engineering to obtain high product levels.

Identification of novel genes involved in neutral lipid storage by quantitative trait loci analysis of Saccharomyces cerevisiae.

BACKGROUND: The accumulation of intracellular fat depots is a polygenic trait. Therefore, the extent of lipid storage in the individuals of a species covers a broad range and is determined by many genetic factors. Quantitative trait loci analysis can be used to identify those genetic differences between two strains of the same species that are responsible for the differences in a given phenotype. We used this method and complementary approaches to identify genes in the yeast Saccharomyces cerevisiae that are involved in neutral lipid storage. RESULTS: We selected two yeast strains, the laboratory strain BY4741 and the wine yeast AWRI1631, with a more than two-fold difference in neutral lipid content. After crossing, sporulation and germination, we used fluorescence activated cell sorting to isolate a subpopulation of cells with the highest neutral lipid content from the pool of segregants. Whole genome sequencing of this subpopulation and of the unsorted pool of segregants implicated several loci that are involved in lipid accumulation. Three of the identified genes, PIG1, PHO23 and RML2, were investigated in more detail. Deletions of these genes and the exchange of the alleles between the two parental strains confirmed that the encoded proteins contribute to neutral lipid storage in S. cerevisiae and that PIG1, PHO23 and RML2 are the major causative genes. Backcrossing of one of the segregants with the parental strains for seven generations revealed additional regions in the genomes of both strains with potential causative genes for the high lipid accumulation phenotype. CONCLUSIONS: We identified several genes that contribute to the phenotype of lipid accumulation in an allele-specific manner. Surprisingly, no allelic variations of genes with known functions in lipid metabolism were found, indicating that the level of storage lipid accumulation is determined by many cellular processes that are not directly related to lipid metabolism.

Tum1 is involved in the metabolism of sterol esters in Saccharomyces cerevisiae.

BACKGROUND: The only hitherto known biological role of yeast Saccharomyces cerevisiae Tum1 protein is in the tRNA thiolation pathway. The mammalian homologue of the yeast TUM1 gene, the thiosulfate sulfurtransferase (a.k.a. rhodanese) Tst, has been proposed as an obesity-resistance and antidiabetic gene. To assess the role of Tum1 in cell metabolism and the putative functional connection between lipid metabolism and tRNA modification, we analysed evolutionary conservation of the rhodanese protein superfamily, investigated the role of Tum1 in lipid metabolism, and examined the phenotype of yeast strains expressing the mouse homologue of Tum1, TST. RESULTS: We analysed evolutionary relationships in the rhodanese superfamily and established that its members are widespread in bacteria, archaea and in all major eukaryotic groups. We found that the amount of sterol esters was significantly higher in the deletion strain tum1Δ than in the wild-type strain. Expression of the mouse TST protein in the deletion strain did not rescue this phenotype. Moreover, although Tum1 deficiency in the thiolation pathway was complemented by re-introducing TUM1, it was not complemented by the introduction of the mouse homologue Tst. We further showed that the tRNA thiolation pathway is not involved in the regulation of sterol ester content in S. cerevisiae, as overexpression of the tEUUC, tKUUU and tQUUG tRNAs did not rescue the lipid phenotype in the tum1Δ deletion strain, and, additionally, deletion of the key gene for the tRNA thiolation pathway, UBA4, did not affect sterol ester content. CONCLUSIONS: The rhodanese superfamily of proteins is widespread in all organisms, and yeast TUM1 is a bona fide orthologue of mammalian Tst thiosulfate sulfurtransferase gene. However, the mouse TST protein cannot functionally replace yeast Tum1 protein, neither in its lipid metabolism-related function, nor in the tRNA thiolation pathway. We show here that Tum1 protein is involved in lipid metabolism by decreasing the sterol ester content in yeast cells, and that this function of Tum1 is not exerted through the tRNA thiolation pathway, but through another, currently unknown pathway.