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Abstract Methanol is a promising feedstock for biomanufacturing, but the efficiency of methanol-based bioprocesses is limited by the low rate of methanol utilization pathways and methanol toxicity. Yeast diversity is an attractive biological resource to develop efficient bioprocesses since any effort with strain improvement is more deserving if applied to innate robust strains with relevant catabolic and biosynthetic potential. The present study is in line with such rational and describes the isolation and molecular identification of seven isolates of the methylotrophic species Candida boidinii from waters derived from the traditional curation of olives, in different years, and from contaminated superficial soil near fuel stations. The yeast microbiota from those habitats was also characterized. The four C. boidinii isolates obtained from the curation of olives’ water exhibited significantly higher maximum specific growth rates (range 0.15–0.19 h−1), compared with the three isolates obtained from the fuel contaminated soils (range 0.05–0.06 h−1) when grown on methanol as the sole C-source (1% (v/v), in shake flasks, at 30°C). The isolates exhibit significant robustness towards methanol toxicity that increases as the cultivation temperature decreases from 30°C to 25°C. The better methanol-based growth performance exhibited by C. boidinii isolates from olives´ soaking waters could not be essentially attributed to higher methanol tolerance. These methanol-efficient catabolizing isolates are proposed as a promising platform to develop methanol-based bioprocesses.

Abstract Background The red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure. Results Comparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses. Conclusions The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.

Abstract Background The improvement of yeast tolerance to acetic, butyric, and octanoic acids is an important step for the implementation of economically and technologically sustainable bioprocesses for the bioconversion of renewable biomass resources and wastes. To guide genome engineering of promising yeast cell factories toward highly robust superior strains, it is instrumental to identify molecular targets and understand the mechanisms underlying tolerance to those monocarboxylic fatty acids. A chemogenomic analysis was performed, complemented with physiological studies, to unveil genetic tolerance determinants in the model yeast and cell factory Saccharomyces cerevisiae exposed to equivalent moderate inhibitory concentrations of acetic, butyric, or octanoic acids. Results Results indicate the existence of multiple shared genetic determinants and pathways underlying tolerance to these short- and medium-chain fatty acids, such as vacuolar acidification, intracellular trafficking, autophagy, and protein synthesis. The number of tolerance genes identified increased with the linear chain length and the datasets for butyric and octanoic acids include the highest number of genes in common suggesting the existence of more similar toxicity and tolerance mechanisms. Results of this analysis, at the systems level, point to a more marked deleterious effect of an equivalent inhibitory concentration of the more lipophilic octanoic acid, followed by butyric acid, on the cell envelope and on cellular membranes function and lipid remodeling. The importance of mitochondrial genome maintenance and functional mitochondria to obtain ATP for energy-dependent detoxification processes also emerged from this chemogenomic analysis, especially for octanoic acid. Conclusions This study provides new biological knowledge of interest to gain further mechanistic insights into toxicity and tolerance to linear-chain monocarboxylic acids of increasing liposolubility and reports the first lists of tolerance genes, at the genome scale, for butyric and octanoic acids. These genes and biological functions are potential targets for synthetic biology approaches applied to promising yeast cell factories, toward more robust superior strains, a highly desirable phenotype to increase the economic viability of bioprocesses based on mixtures of volatiles/medium-chain fatty acids derived from low-cost biodegradable substrates or lignocellulose hydrolysates.

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.

Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. However, sustainable, and economically viable bioprocesses require yeast strains exhibiting: (i) high tolerance to multiple bioprocess-related stresses, including the various chemical inhibitors present in hydrolysates from lignocellulosic biomass and residues; (ii) the ability to efficiently consume all the major carbon sources present; (iii) the capacity to produce lipids with adequate composition in high yields. More than 160 non-conventional (non-Saccharomyces) yeast species are described as oleaginous, but only a smaller group are relatively well characterised, including Lipomyces starkeyi, Yarrowia lipolytica, Rhodotorula toruloides, Rhodotorula glutinis, Cutaneotrichosporonoleaginosus and Cutaneotrichosporon cutaneum. This article provides an overview of lipid production by oleaginous yeasts focusing on yeast diversity, metabolism, and other microbiological issues related to the toxicity and tolerance to multiple challenging stresses limiting bioprocess performance. This is essential knowledge to better understand and guide the rational improvement of yeast performance either by genetic manipulation or by exploring yeast physiology and optimal process conditions. Examples gathered from the literature showing the potential of different oleaginous yeasts/process conditions to produce oils for biodiesel from agro-forestry and industrial organic residues are provided.

Methanol is a promising feedstock for metabolically competent yeast strains-based biorefineries. However, methanol toxicity can limit the productivity of these bioprocesses. Therefore, the identification of genes whose expression is required for maximum methanol tolerance is important for mechanistic insights and rational genomic manipulation to obtain more robust methylotrophic yeast strains. The present chemogenomic analysis was performed with this objective based on the screening of the Euroscarf Saccharomyces cerevisiae haploid deletion mutant collection to search for susceptibility phenotypes in YPD medium supplemented with 8% (v/v) methanol, at 35 °C, compared with an equivalent ethanol concentration (5.5% (v/v)). Around 400 methanol tolerance determinants were identified, 81 showing a marked phenotype. The clustering of the identified tolerance genes indicates an enrichment of functional categories in the methanol dataset not enriched in the ethanol dataset, such as chromatin remodeling, DNA repair and fatty acid biosynthesis. Several genes involved in DNA repair (eight RAD genes), identified as specific for methanol toxicity, were previously reported as tolerance determinants for formaldehyde, a methanol detoxification pathway intermediate. This study provides new valuable information on genes and potential regulatory networks involved in overcoming methanol toxicity. This knowledge is an important starting point for the improvement of methanol tolerance in yeasts capable of catabolizing and copying with methanol concentrations present in promising bioeconomy feedstocks, including industrial residues.