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

Abstract

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.

Production, Biosynthesis, and Commercial Applications of Fatty Acids From Oleaginous Fungi

“…NADPH is taken as the reducing cofactor by FAS, and two NADPH molecules are required in each step of the acyl-CoA chain elongation. The chain is repeatedly extended to the desired length of the synthetic organism, and then some FAs are desaturated to form unsaturated FAs ( 112 – 114 ). The related reactions and enzymes are shown in Figure 2 .…”

Oleaginous yeasts: Time to rethink the definition?

“…On the other hand, strain improvement manipulations can lead to oleaginous phenotypes in otherwise considered non‐oleaginous yeasts. For example, crossing wild‐type S. cerevisiae strains with high lipid accumulation (but not enough to be considered oleaginous), allowed to obtain oleaginous strains able to store lipids between 22% and 27% of their CDW (Arhar et al, 2021). Therefore, oleaginicity is not a property of a species but that of a strain, although this is not always reflected clearly in the scientific literature, where species names instead of strain identities tend to be used (Sitepu et al, 2014).…”

“…On the other hand, strain improvement manipulations can lead to oleaginous phenotypes in otherwise considered non-oleaginous yeasts. For example, crossing wild-type S. cerevisiae strains with high lipid accumulation (but not enough to be considered oleaginous), allowed to obtain oleaginous strains able to store lipids between 22% and 27% of their CDW (Arhar et al, 2021).…”

PHB production from cellobiose with Saccharomyces cerevisiae

“…For example, improved expression of engineered acetyl-CoA synthetase gene ( acsL641P ) and acetaldehyde dehydrogenase gene ( ALD6 ) have shown positive effects on PHB production in S. cerevisiae [ 46 ]. In addition, PHA production in yeast could potentially benefit from the extensive engineering efforts in acetyl-CoA and lipid production, which have resulted already in accumulation of high concentrations of triacylglycerols, 65% and 80% of CDW, in yeasts S. cerevisiae and Y. lipolytica , respectively [ 6 , 47 , 48 ].…”

“…Development of yeast strains as hosts for PHA production is a more recent approach. However, robust yeasts have many advantages for industrial use, e.g., insusceptibility for phase contamination, tolerance for acidic conditions, and ability to grow on inexpensive media [ 6 – 8 ]. We previously engineered a yeast Saccharomyces cerevisiae for PHB synthesis and were able to achieve up to 11% PHB accumulation of CDW from glucose [ 9 ].…”