Identifying carbohydrate-active enzymes of Cutaneotrichosporon oleaginosus using systems biology

Abstract

Background The oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available. Results In this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide. Conclusions Relative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus.

Value-Added Squalene in Single-Cell Oil Produced with Cutaneotrichosporon oleaginosus for Food Applications

“…In general, it has been shown that the uptake of glucose and xylose shows very similar growth rates and lipid production with C. oleaginosus . Fatty acid biosynthesis in this nonconventional yeast is carried out by the fatty acid synthase complex (FAS) which requires acetyl-CoA .…”

“…In general, it has been shown that the uptake of glucose and xylose shows very similar growth rates and lipid production with C. oleaginosus. 32 Fatty acid biosynthesis in this nonconventional yeast is carried out by the fatty acid synthase complex (FAS) which requires acetyl-CoA. 33 Glucose mainly channels into the glycolysis and pentose phosphate pathways, while xylose can be metabolized both by the phosphoketolase and pentose phosphate pathways.…”

Adaptation of Proteome and Metabolism in Different Haplotypes of Rhodosporidium toruloides during Cu(I) and Cu(II) Stress

“…Proteins were extracted and precipitated with Protein Extraction Reagent Type 4 (Sigma-Aldrich, St. Louis, MO, USA) (1:3, v / v ) and 20% trichloric acid ( v / v ), followed by in-gel digestion with trypsin. Peptides were analyzed using LC-MS/MS analysis with a timsTOF Pro mass spectrometer equipped with a NanoElute LC System (Bruker Daltonik GmbH, Bremen, Germany) on an Aurora column (250 × 0.075 mm, 1.6 μm; IonOpticks, Hanover St., Rozelle, NSW, Australia) [ 21 , 22 ]. For every condition, three biological and two technical replicates were prepared.…”

Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector

“…Its superiority over other oleaginous microorganisms in having high triglyceride (TAG) content, substrate flexibility, and rapid growth rate has made this yeast a prime candidate for oleochemical production [5][6][7]. To date, C. oleaginosus has displayed the highest carbon source flexibility linked with high de-novo oil productivity and yield of any oleaginous yeast strain [7][8][9][10][11][12][13]. Moreover, compared to other yeasts, C. oleaginosus is highly resistant to fermentation inhibitors (including weak organic acids e.g.…”