Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars

Abstract

Background: In the context of sustainable development, yeast are one class of microorganisms foreseen for the production of oil from diverse renewable feedstocks, in particular those that do not compete with the food supply. However, their use in bulk production, such as for the production of biodiesel, is still not cost effective, partly due to the possible poor use of desired substrates or poor robustness in the practical bioconversion process. We investigated the natural capacity of Blastobotrys adeninivorans, a yeast already used in biotechnology, to store lipids under different conditions.

Results: The genotyping of seven strains showed the species to actually be composed of two different groups, one that (including the well-known strain LS3) could be reassigned to Blastobotrys raffinosifermentans. We showed that, under nitrogen limitation, strains of both species can synthesize lipids to over 20% of their dry-cell weight during shake-flask cultivation in glucose or xylose medium for 96 h. In addition, organic acids were excreted into the medium. LS3, our best lipid-producing strain, could also accumulate lipids from exogenous oleic acid, up to 38.1 ± 1.6% of its dry-cell weight, and synthesize lipids from various sugar substrates, up to 36.6 ± 0.5% when growing in cellobiose. Both species, represented by LS3 and CBS 8244^T, could grow with little filamentation in the lipogenic medium from 28 to 45 °C and reached lipid titers ranging from 1.76 ± 0.28 to 3.08 ± 0.49 g/L in flasks. Under these conditions, the maximum bioconversion yield (Y _FA/S = 0.093 ± 0.017) was obtained with LS3 at 37 °C. The presence of genes for predicted subunits of an ATP citrate lyase in the genome of LS3 reinforces its oleaginous character.

Conclusions: Blastobotrys adeninivorans and B. raffinosifermentans, which are known to be xerotolerant and genetically-tractable, are promising biotechnological yeasts of the Saccharomycotina that could be further developed through genetic engineering for the production of microbial oil. To our knowledge, this is the first report of efficient lipid storage in yeast when cultivated at a temperature above 40 °C. This paves the way to help reducing costs through consolidated bioprocessing.

Keywords: Biotechnology; Lipid metabolism; Microbial oil; Oleaginous yeasts; Saccharomycotina; Thermotolerance.

Fig. 1

Effect of limiting nitrogen on the synthesis and accumulation of lipids in strain LS3. LS3 was cultivated for 72 h in YNB-based medium with carbon source at 30 g/L. The concentrations of 5 g/L and 0.75 g/L NH₄Cl led to a C/N ratio of 90 and 60, respectively, for cultures in glucose (G) and to a C/N ratio of 16 and 106, respectively, for cultures in a mixture of oleic acid and glucose (AO + G). a Cell density is expressed as OD₆₀₀ (white bars) and total lipid content as the percentage of dry-cell weight (black bars). Average values and standard deviations (n = 2) are shown in the histograms. b Relative proportion (%) of main FAs in each FA profile. c BODIPY-stained lipid droplets in cells sampled from low-nitrogen cultures in glucose (left) or in the presence of oleic acid (right)

Fig. 2

Two genotypic groups in strains previously identified as B. adeninivorans and relation with B. raffinosifermentans. a IGS profiles. The right part of the agarose gel shows an example of the PCR product obtained for two strains. The left part shows AluI restriction profiles for the seven strains. Saccharomyces cerevisiae ATCC 42367 was used as a control. The M wells are occupied by the molecular weight standard, a mixture of NEB quick-load 1 kb ladder and pBR322 MspI-digest, for which the size of some bands is indicated in kb. b Single-nucleotide polymorphisms (SNPs) in the ITS-D1D2 (1086 nt) and mitochondrial COXII (598 nt) sequences. The number of SNPs (including one indel in ITS) is indicated for these markers for each comparison between the strain above and the type strains to the right. (–) indicates not determined

Fig. 3

Bioconversion of sugars into lipids during the growth of two strains of B. raffinosifermentans in nitrogen-limited YNB-based media. Glucose and xylose were each used as the C source (30 g/L) at two different C/N ratios. Average values and the standard deviation (n = 3) for OD₆₀₀ (blue line), residual sugar in the medium (g/L, plain green line), cumulative organic acids (g/L, dotted green line), and lipid content (% DCW, orange line) were plotted over time (h)

Fig. 4

Bioconversion of sugars to lipids during growth of two strains of B. adeninivorans in nitrogen-limited YNB-based media. Glucose and xylose were each used as the C source (30 g/L)at two different C/N ratios. Average values and the standard deviation (n = 3) for OD₆₀₀ (blue line), residual sugar in the medium (g/L, plain green line), cumulative organic acids (g/L, dotted green line), and lipid content (% DCW, orange line) were plotted over time (h)

Fig. 5

Bioconversion of various sugars into lipids. LS3 was cultivated for 72 h in nitrogen-limited YNB-based medium (0.75 g/L NH₄Cl) supplemented with the indicated sugars at 30 g/L. a Lipid content (% DCW) is presented as a bar with the average value and mean deviation (n = 2) and a color code indicating the nature of the carbon source: green, C5 monosaccharide; light blue, C6 monosaccharide; blue, disaccharide; gray, polysaccharide; red, polyol. b Microscopic images after BODIPY^® staining of the 72 h-cultivated cells in the relevant medium (same order as in the histograms)

Fig. 6

Shake-flask cultures of strains LS3 and CBS 8244^T at various temperatures. DCW (a), lipid content of the cells (b), and glucose concentration in the medium (c) were measured after 72 h of cultivation in nitrogen-limited YNB-based medium (30 g/L glucose and 0.75 g/L NH₄Cl), and the yields (d) calculated for strains LS3 (black bars) and CBS 8244^T (gray bars). Average values and standard deviations (n = 4) are presented in the histograms