Screening of xylose utilizing and high lipid producing yeast strains as a potential candidate for industrial application

Abstract

Background Sustainable production of oil for food, feed, fuels and other lipid-based chemicals is essential to meet the demand of the increasing human population. Consequently, novel and sustainable resources such as lignocellulosic hydrolysates and processes involving these must be explored. In this paper we screened for naturally-occurring xylose utilizing oleaginous yeasts as cell factories for lipid production, since pentose sugar catabolism plays a major role in efficient utilization of lignocellulosic feedstocks. Glycerol utilization, which is also beneficial in yeast-based oil production as glycerol is a common by-product of biodiesel production, was investigated as well. Natural yeast isolates were studied for lipid accumulation on a variety of substrates, and the highest lipid accumulating strains were further investigated in shake flask cultivations and fermenter studies on xylose and hydrolysate. Results By collecting leaves from exotic plants in greenhouses and selective cultivation on xylose, a high frequency of oleaginous yeasts was obtained (> 40%). Different cultivation conditions lead to differences in fatty acid contents and compositions, resulting in a set of strains that can be used to select candidate production strains for different purposes. In this study, the most prominent strains were identified as Pseudozyma hubeiensis BOT-O and Rhodosporidium toruloides BOT-A2. The fatty acid levels per cell dry weight after cultivation in a nitrogen limited medium with either glucose, xylose or glycerol as carbon source, respectively, were 46.8, 43.2 and 38.9% for P. hubeiensis BOT-O, and 40.4, 27.3 and 42.1% for BOT-A2. Furthermore, BOT-A2 accumulated 45.1% fatty acids per cell dry weight in a natural plant hydrolysate, and P. hubeiensis BOT-O showed simultaneous glucose and xylose consumption with similar growth rates on both carbon sources. The fatty acid analysis demonstrated both long chain and poly-unsaturated fatty acids, depending on strain and medium. Conclusions We found various natural yeast isolates with high lipid production capabilities and the ability to grow not only on glucose, but also xylose, glycerol and natural plant hydrolysate. R. toruloides BOT-A2 and P. hubeiensis BOT-O specifically showed great potential as production strains with high levels of storage lipids and comparable growth to that on glucose on various other substrates, especially compared to currently used lipid production strains. In BOT-O, glucose repression was not detected, making it particularly desirable for utilization of plant waste hydrolysates. Furthermore, the isolated strains were shown to produce oils with fatty acid profiles similar to that of various plant oils, making them interesting for future applications in fuel, food or feed production.

Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation

“…The natural yeast isolate Rhodotorula toruloides BOT-A2 [ 43 ] and its derived strains were used in all experiments and cultivated at 30 °C. A list of all strains can be found in Table 3 .…”

“…The KOH/ethanol extraction method [ 60 ] was used to extract fatty acids from the cultivations for RNA sequencing, with slight changes as previously described [ 43 ]. In short, after freeze-drying of the samples, biomass was determined using a micro scale.…”

“…Several R. toruloides transcriptome datasets are available from a variety of different culture conditions, including nitrogen limitation [ 14 , 39 ], phosphate limitation [ 5 ], stress response during growth on hydrolysates [ 11 , 40 , 41 ] and growth on different carbon sources [ 10 , 30 , 33 , 39 , 42 ], but no study has yet compared the effect of hexose and pentose sugars during nitrogen starvation. We here generated a highly contiguous, functionally annotated genome assembly from our recently isolated Rhodotorula toruloides strain BOT-A2 (previously classified as Rhodosporidium toruloides ) [ 43 ] and performed RNA sequencing (RNAseq) on samples cultivated in nitrogen-limited media with either glucose or xylose as the sole carbon source. Differential expression analysis of samples taken during the exponential growth phase and at nitrogen depletion was used to identify putative promoter candidate sequences, which were further evaluated using reporter gene expression.…”