An Evolved Strain of the Oleaginous Yeast Rhodotorula toruloides, Multi-Tolerant to the Major Inhibitors Present in Lignocellulosic Hydrolysates, Exhibits an Altered Cell Envelope

Abstract

The presence of toxic compounds in lignocellulosic hydrolysates (LCH) is among the main barriers affecting the efficiency of lignocellulose-based fermentation processes, in particular, to produce biofuels, hindering the production of intracellular lipids by oleaginous yeasts. These microbial oils are promising sustainable alternatives to vegetable oils for biodiesel production. In this study, we explored adaptive laboratory evolution (ALE), under methanol- and high glycerol concentration-induced selective pressures, to improve the robustness of a Rhodotorula toruloides strain, previously selected to produce lipids from sugar beet hydrolysates by completely using the major C (carbon) sources present. An evolved strain, multi-tolerant not only to methanol but to four major inhibitors present in LCH (acetic acid, formic acid, hydroxymethylfurfural, and furfural) was isolated and the mechanisms underlying such multi-tolerance were examined, at the cellular envelope level. Results indicate that the evolved multi-tolerant strain has a cell wall that is less susceptible to zymolyase and a decreased permeability, based on the propidium iodide fluorescent probe, in the absence or presence of those inhibitors. The improved performance of this multi-tolerant strain for lipid production from a synthetic lignocellulosic hydrolysate medium, supplemented with those inhibitors, was confirmed.

Cell envelope and stress-responsive pathways underlie an evolved oleaginous Rhodotorula toruloides strain multi-stress tolerance

“…These stress conditions were tested based on the indications obtained from the performed omics analyses. Remarkably, under the conditions used for the susceptibility test assays, either in this study or in the former study [ 15 ], the more marked phenotypes were registered for osmotic-, salt-, and ethanol-induced stresses. These stress conditions are of the same nature as the stress conditions employed during the ALE experiment (methanol and high glycerol concentration).…”

“…The objective was to infer possible functional basal alterations underlying the multi-stress tolerance phenotype in the evolved strain. Interestingly, the multi-stress tolerance phenotypes previously described for the evolved strain (towards methanol, acetic and formic acids, HMF, and furfural) [ 15 ] were extended in this study to hyperosmotic, saline, genotoxic and oxidative stresses, and stresses induced by ethanol, octanoic and butyric acids. These stress conditions were tested based on the indications obtained from the performed omics analyses.…”

“…The dynamic remodelling of the cell wall also facilitates cellular adaptation to fluctuating external osmolarities [ 50 ]. Remarkably, the evolved strain was previously shown to possess a cell wall significantly less susceptible to zymolyase activity compared to the parental strain, suggesting a physiochemical different cell wall [ 15 ]. Furthermore, genomic variants in genes encoding key kinases that occupy a central position in cell wall integrity signalling in S. cerevisiae were identified, highlighting the substantial role of cell surface remodelling as a central adaptive response in the evolved strain.…”

“…Despite the remarkable progress made recently, the available tools for R. toruloides genome engineering are still not highly efficient [ 64 ]. An Adaptive Laboratory Evolution (ALE) strategy was successfully used in our laboratory to enhance R. toruloides IST536 tolerance under selective pressure imposed by increasing inhibitory methanol concentrations added to a minimal medium with 5% (v/v) of glycerol as the sole carbon source [ 15 ]. This evolved strain, IST536 MM15, was found to be more tolerant not only to methanol but also to acetic and formic acids, furfural and 5-hydroxymethylfurfural (HMF), the four major inhibitors expected to be present in lignocellulosic biomass hydrolysates [ 15 ].…”

“…This study was designed to elucidate the underlying mechanisms through comparative functional genomic analyses of R. toruloides IST536 and its evolved counterpart, IST536 MM15. These strains were preliminary characterized in our laboratory and results indicate that the evolved multi-stress tolerant strain has a cell wall less susceptible to zymolyase activity and a decreased permeability, based on the intracellular accumulation of propidium iodide fluorescent probe [ 15 ]. The registered alteration of the cell envelope properties guarantees a robust adaptive response to stress.…”