Isolation and Identification of oleaginous Margarida dos Reis Pereira Pataco1 1Institute for Bioengineering and Biosciences, Instituto Superior Técnico, University of Lisbon Abstract: An alternative for a sustainable biodiesel production is the accumulation of single-cell oils by oleaginous yeasts. Xylose is the second most abundant sugar in plant biomass after glucose, but its metabolization by yeasts is usually not efficient, limiting the utilization of lignocellulosic materials for the production of single-cell oils. The main objective of this work is to find efficient oleaginous yeasts with the ability to metabolize xylose. isolation from several samples was performed and the yeast isolates obtained were taxonomically identified based on the sequencing of D1/D2 and ITS regions from ribosomal DNA, yielding 78 strains from 32 species. The 67 non-pigmented strains isolated and 58 additional strains from the IST Yeast Culture Collection were screened for lipid production using a qualitative method based on Rhodamine B staining. This allowed the selection of 37 potential oleaginous strains grown in glucose and 21 grown in xylose. The species Candida boidinii and Pichia membranifaciens were considered potential oleaginous xylose metabolizing yeasts. A genome mining was performed to identify putative sugar transporters in their genomes, allowing the identification of 33 sugar transporters for Candida boidinii and 10 for Pichia membranifaciens, to which a phylogenetic analysis was done. The phylogenetic trees obtained for Candida boidinii and Pichia membranifaciens sugar transporters showed, respectively, twelve and eight transporters close to known xylose transporters. Future studies will involve the qualitative and quantitative characterization of the lipids produced by these species, as well as the optimization of growth conditions, higher lipid productions and accumulations. Key words: Oleaginous yeast; Intracellular lipids; Bio-oils; Xylose utilization; Lignocellulose utilization; Biodiesel. Introduction enhance the productivity of yeasts in fatty acid production, which is not an easy task, since the Fossil fuel energy is responsible for a substantial part production and composition of lipids by oleaginous of today's demand of industry and economy1. yeasts is subject to change, depending on the However, their exploration and use are not cultivation conditions and stress under growth7. Also, sustainable and are responsible for carbon dioxide one of the main reasons for the high cost of biodiesel emissions to the atmosphere, increasing the production, is the price of the raw material used, since greenhouse effect and contributing to global it makes 70-85% of the total value of this fuel warming1. production8. The solution is to use a cheap fermentation medium, therefore, organic wastes and A small number of microorganisms accumulate lipids agro-food-forest residues are an interesting raw above 20% of their cell mass as a reserve storage material for the synthesis of lipids by oleaginous 2 material . These microorganisms include some yeasts yeasts9. Consequently, when biomass is used for the and fungi and a few algae, and are called oleaginous generation of fuels, the waste is converted into a species, being known for their oil accumulation that resource, making this an efficient process10. may reach values as high as 70% of their cell biomass2. Bio-oils are composed by triglycerides and Xylose is the second most abundant sugar in plant are very similar to vegetable oils, which increases the biomass after glucose, but most of the oleaginous interest in its use for the synthesis of fatty acid methyl yeasts use glucose preferentially to other sugars, due esters into biodiesel3. to carbon catabolite repression, increasing cultivation times when a mixture of xylose and glucose is present, Notwithstanding other microorganisms accumulating consequently reducing lipid productivity11,12. lipids, yeasts show several advantages, not only the Additionally, xylose uptake into cells have been fact that they can accumulate triglycerides, but also highlighted as a significant limitation in the efficient because they include a broader metabolism of feed utilization of lignocellulosic sugars by yeasts and sources and have tolerance to higher ranges of pH, efforts have been made to find specific efficient xylose 4 inhibitors and ionic strength . Additionally, oleaginous transporters13. Therefore, yeasts that successfully co- yeasts are usually non-pathogenic, most are suitable utilize glucose and xylose, would allow an effective for larger fermentations and are easy to manipulate and sustainable process for lipid production from 5,4 genetically . renewable cheap materials11. However, at the moment, biodiesel production is not Nowadays, there are more than 40 known oleaginous competitive with fossil fuels, being necessary to yeast species already identified and distributed among reduce the cost of its production6. It is necessary to

1 several clades and with different inter- and intra- dilutions were plated on agar media (isolation medium specific lipid accumulation potential14,15. Moreover, the with glucose and isolation medium with xylose, with search for new oleaginous species and strains with 20g/L agar) and incubated at 30ºC. After this step, potential for higher lipid accumulation is crucial and is single colonies of the different strains from the in constant actualization15. Therefore, this work has isolation step were selected based on their the purpose of finding effective oleaginous yeast morphology and streaked into new media. strains that also have the ability of assimilating xylose. Soil samples: For the isolation of yeasts from soil Materials and Methods samples, in the primary enrichment, one table spoon of soil (~1g of soil, that usually contains 103 yeast Part of the yeast isolation and identification work here cells) was inoculated in 50 mL of enrichment medium described was done in collaboration with my colleague (with glucose and xylose as carbon sources) and Érica Vieira from the MSc degree in Biotechnology. incubated at 30ºC, 150 rpm, for 48 h. In the case of samples of olive tree soil 1 and garden soil, a half Sampling: Samples were collected from various tablespoon was used for medium inoculation. After, natural sites, in order to proceed with the isolation of the scale-up enrichment consisting of in inoculating 1 yeasts. Samples were collected from soil near two mL from Primary Enrichment in 49 mL of enrichment different olive trees (1-from Póvoa-Cadaval and 2- medium followed by incubation at 30ºC, 150 rpm, for from Ferreira do Alentejo) as well as from soil near an 48 h. This was followed by Differential Enrichment, oak tree, from a vegetable garden, both from Póvoa- also inoculating 1 mL from Scale-up Enrichment into Cadaval, and from surface seawater collected in 49 mL of isolation medium with glucose (glucose as Berlengas and Arrábida. Olives (from the olive tree 2), carbon source) and isolation medium with xylose and olives curing water were also used, as well as (xylose as carbon source) media and incubation at physalis, juniper berries, plums and walnut green 30ºC, 150 rpm for 48 h. All these media were done husk. Finally, sediments from a permafrost lake with ddH2O and Chloramphenicol was added at a located in the Artic provided by Dr. João Canário from concentration of 100 μg/mL after autoclaving. Next, Centro de Química Estrutural of IST and soils the isolation consisted in the steps done for marine contaminated with fuel provided by Dr. Ricardo Santos samples. from Laboratório de análises of IST were also used to isolate yeasts. Fruit samples: To isolate the strains from olives, plums, physalis and juniper berries, demineralized sterile water was added to a tube containing the Marine samples: The Differential Isolation: sample and vortexed. After, 1 mL or 5 mL of the water isolation was based on the method described Zaky et from plums’ sample and 10 mL from physalis and al. (2016)16. This method starts with three enrichment juniper berries (no growth was observed with only cycles. In the primary enrichment, 500 mL, 1 L, 2 L, 1mL) were inoculated in 50 mL of enrichment medium and 3 L of surface seawater were filtered, using a and incubated at 30ºC, 150 rpm, for 48 h. The metallic filtration system (SS Filter Holder 100mL, subsequent steps were made as described for the 47mm; by Mili-Q). The filter was introduced into 100 isolation from marine and soil samples. mL of enrichment medium, which contains glucose and xylose as carbon sources (30 g/L Glucose; 30 g/L Olives-curing water sample: To isolate the strains Xylose ; 3 g/L Malt extract ; 3 g/L yeast extract ; 5 g/L from olives-curing water, the procedure was similar to Peptone; 1 g/L (NH4)2SO4 ; 0.25 g/L KH2PO4 ; pH 5)), the one described for soil samples, but in the first step, and incubated at 30ºC, 150 rpm. In the scale-up 500 μL of this water were inoculated in 50 mL of enrichment, 20 mL from Primary Enrichment were enrichment medium and incubated at 30ºC, 150 rpm, added to 180 mL of enrichment medium and for 48 h. incubated at 30ºC, 150 rpm, for 48 h. In the differential enrichment, 10 mL from Scale-up Enrichment were Direct isolation: Additionally, the strains obtained added to 90 mL of isolation medium with glucose, a from cabbage, nuts and sediments or from a medium with glucose as the carbon source (60 g/L permafrost lake located in the Artic were isolated in a Glucose; 3 g/L yeast extract; 5 g/L Peptone; 1 g/L direct way: demineralized sterile water was added to (NH4)2SO4; 0.25 g/L KH2PO4; pH 5) and isolation a tube containing the cabbage or nuts sample and medium with xylose, a medium with xylose as the vortexed for 2 minutes. The sample of sediments from carbon source (60 g/L Xylose; 3 g/L yeast extract; 5 a permafrost lake from Artic was already suspended g/L Peptone; 1 g/L (NH4)2SO4; 0.25 g/L KH2PO4; pH in the lake water. A total of 100 μL of this suspension 5). All the media used in these steps were prepared was then plated in Wallerstein Nutrient (WLN) agar with filtered seawater, and Chloramphenicol was medium (Yeast extract 4 g/L, Tryptone 5 g/L, Glucose added to a final concentration of 100 μg/mL, after 50 g/L, Potassium dihydrogen phosphate 0.55 g/L, autoclaving. An isolation step was also done, where Potassium chloride 0.425 g/L, Calcium chloride 0.125 ten-fold serial dilutions were performed, in a solution g/L, Magnesium sulphate 0.125 g/L, Ferric chloride with 0.085 g/L of NaCl and 100 mL of the serial 0.0025 g/L, Manganese sulphate 0.0025 g/L,

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Bromocresol green 0.022 g/L, Agar 15 g/L, pH 5.5; Xylose Oleaginous medium (23 g/L xylose; 0.3 g/L cloramphenicol was added at a concentration of 100 peptone; 0.5 g/L yeast extract; 7 g/L KH2PO4 ; 2 g/L μg/mL, after autoclaving). Also, in the samples where Na2HPO4.2H2O; 1.5 g/L MgSO4; 20 g/L agar) and to no yeast growth occurred in the differential isolation YPD agar medium, having a final concentration of 10 (Arrábida sea, juniper berries, physalis, garden soil mg/L. The strains were pre-grown in YPD agar and olive tree soil 1), 100 μL from the Primary medium for 1 day at 30ºC and then inoculated and Enrichment were also inoculated in WLN agar grown for 2 to 4 days at 30ºC in the Glucose medium, and incubated at 30ºC for 48h. The oleaginous medium + Rhodamine B and Xylose subsequent steps were made as described for the oleaginous medium + Rhodamine B. The plates were isolation from marine and soil samples. then observed and the cultures that presented a higher intensity of colour were selected for further For long-term preservation, the isolates were frozen tests. As described by the authors, YPD medium was in YPD and glycerol stocks (15% (w/v)) at -80ºC. used to analyse the red yeasts since the plates with this rich medium look more reddish than the plates Yeast total Identification of the strains: with high C/N medium, due to the presence of peptone gDNA Extraction: Genomic DNA from yeast isolates 17 and yeast extract, thus allowing a better contrast were extracted using phenol:chloroform method . between different colonies under UV light. In this case, Molecular Identification of Yeast Isolates: After to promote lipid accumulation, the incubation period gDNA extraction, a PCR was performed, in order to was superior to 4 days, in order to exhaust the amplify the D1/D2 region of the large 26S rDNA nutrients available. Also, in the case of isolates from subunit, using the primers NL-1 and NL-418 or the the Arctic permafrost samples, strains were incubated internal transcribed spacer (ITS) region (containing in the Glucose oleaginous medium + Rhodamine B at ITS1, 5.8S and ITS2 rRNA) the primers ITS1 and two different temperatures (10ºC and 30ºC), for 14 ITS4 19. Each PCR reaction was performed according days, in order to test lipid production at different to manufacter’s instructions (New England BioLabs) temperatures. and took place in a thermocycler (GTC96S, Cleaver scientific Ltd) using the following program: 1st In silico analysis: Using the D1/D2 region of the denaturation: 98ºC, 30s; 35 cycles of: 2nd denaturation large 26S rDNA subunit sequences, available in the (98ºC, 10s), Annealing (52ºC, 20s), Elongation (72ºC, NCBI repository, a phylogenetic analysis of the species isolated in this work was done, utilizing the 30s); Final elongation: 72ºC, 8min; Storage: 4ºC, Ꚙ; MEGA (Molecular Evolutionary Genetics Analysis) X An electrophoresis of the 50 μL PCR product and of 8 software21. Maximum Likelihood method was used to μL of loading buffer 6X (Takara) and also of the infer the evolutionary history, by using the and NZYladder III was then performed, for 1 h, at 110 V, Tamura-Nei model22. In order to obtain the heuristic 400 Ma, 100 W, in order to confirm the amplification tree, Neighbor-Join and BioNJ algorithms were and the size of the fragment. It was used a 0.8% (w/v) applied to a matrix of pairwise distances estimated agarose gel, containing green safe, in TBE buffer 1X. using the Tamura-Nei model, and then selecting the The DNA was extracted from the gel, using the topology with superior log likelihood value. NZYGelpure purification kit, and the resulting DNA Evolutionary analyses were conducted in MEGA X21. was also quantified in NanoDrop. The DNA samples were sequenced (Sanger sequencing) by STAB VIDA Also, with the aim to identify possible sugar services. The sequences of each PCR DNA fragment transporters, and in particular xylose transporters, were compared to homologous sequences deposited present in the genomes of the species Candida in the NCBI database, using the BLAST tool of NCBI boidinii and Pichia membranifaciens, in silico tools (http://www.ncbi. nlm.nih.gov/BLAST). were used. Since yeast xylose transporters can also uptake glucose, we first identified and retrieved all Screening of oleaginous yeasts: A sugar transporters. The protein sequence of Hxt7, an screening procedure was performed, in order to hexose uniporter from Saccharomyces cerevisie was identify potential oleaginous yeasts. The method used blasted against the genome sequences available in in this work was based on the work by Niehus et al. NCBI repository for Candida boidinii (taxid:5477) and 20 (2018) , which utilized the lipid dye Rhodamine B Pichia membranaefaciens (taxid:4926). The proteins (C28H31N2O3Cl; mol. wt. 479; IUPACname N-[9- were considered homologous when the identity was (ortho-carboxyphenyl)-6-(diethylamino)- 3H-xanthen- superior to 30%23. The homologous proteins resulting 3-ylidene] diethyl ammonium chloride). A Rhodamine from this screening methodology were analysed using B (Merck) solution with a concentration of 1 g/L in the following in silico tools: ScanProsite24,25, Pfam26, ethanol absolute anhydrous was filtered, using a InterProScan27, and TMHMM28, in order to find PURADISC disposable Filter Device, 25 mm diameter functional motifs and do a transmembrane helices’ (Whatman), and added to the Glucose oleaginous analysis. The results from ScanProsite were medium (23 g/L glucose; 0.3 g/L peptone; 0.5 g/L considered when presenting a reliable confidence cut- yeast extract; 7 g/L KH2PO4 ; 2 g/L Na2HPO4.2H2O off (level = 0). In this work, when using Pfam, the (Merck); 1.5 g/L MgSO4 (Merck); 20 g/L agar) or results considered presented a significant match (with

3 an E-value < 0.001). Next, a phylogenetic analysis of Arrábida soil: isolation medium with glucose: the homologous proteins obtained was performed, IST497- Kazachstania viticola; isolation medium with also using MEGA X software. xylose: IST506- Candida membranifaciens; Olives curing water: isolation medium with glucose: IST471- Results and discussion Pichia membranifaciens; IST472- Pichia kluyveri; IST473- Candida boidinii isolation medium with Isolation and identification of yeast xylose: IST509- Candida boidinii; IST493- Pichia membranifaciens; IST496- Pichia kluyveri; Olives: strains from different environments: isolation medium with glucose: IST474- Candida The main objective of this work was the isolation, silvanorum; IST475- Schwanniomyces vanrijae var. identification, and selection of oleaginous yeasts. Vanrijiae/Schwanniomyces polymorphus; isolation Several samples from different environments were medium with xylose: IST492- Candida silvanorum; used to isolate yeasts. Soil samples were used Plums: isolation medium with glucose: IST502 and because several oleaginous yeasts were already IST505, both Meyerozyma guilliermondii; Berlengas isolated from soil samples, such as Rhodosporidium sea water: isolation medium with glucose (IST476, toruloides, Rhodotorula glutinis, Rhodotorula IST477) of Candida palmioleophila. Arrábida sea graminis, Rhodotorula minuta, Rhodotorula water: isolation medium with glucose: IST540- mucilaginosa, Lipomyces lipoferus, Lipomyces Candida carpophila/Meyerozyma starkeyi, Trichosporum cutaneum, Candida guilliermondii/caribbica; IST541- Meyerozyma pulcherrima, Candida tropicalis, Candida utilis, caribbica; isolation medium with xylose: IST542- 29 Trichosporon cutaneum, Cryptococcus curvatus . Meyerozyma caribbica; Cabbage: WLN: IST510- Soil samples were collected underneath two different Ustilaginomycetes sp./Pseudozyma prolifica; IST563- olive trees as well as underneath an oak tree from a Ustilaginomycetes sp; IST511- Pseudozyma aphidis; vegetable garden. Since some of the known IST539- Pseudozyma aphidis; IST512- Rhodotorula oleaginous yeasts are red yeasts, and red yeasts mucilaginosa. Walnut green husk: WLN: five strains belonging to the genera Rhodotorula and from Cystobasidium slooffiae species (IST544- Rhodosporidium, as well as Cryptococcus and IST548), six from Aureobasidium pullulans (IST552- Candida were already isolated from marine IST557); two strains of Rhodotorula babjevae (IST549 30,31 samples , two different surface seawater samples and IST550) species; IST543- Sporobolomyces were also collected. We also used in this work olives ruberrimus, IST551- Sporobolomyces roseus, and olives-curing water, as well as physalis, juniper IST558- Filobasidium magnum and IST559- berries, plums and walnut green husk because Cryptococcus flavescens; Spontaneous 32 oleaginous yeasts were already isolated from fruits . fermentation of malt: IST457- Kluyveromyces Finally, sediments from a permafrost lake located in marxianus; IST461- Rhodotorula mucilaginosa; the Artic and fuel contaminated soils were also used IST458- Pichia kudriavzevii. However, the to isolate yeasts. identification of isolates IST471, IST475, IST505, In this study 78 strains of 32 yeast species, of 16 IST510, IST540, IST563, IST583, IST594 and IST595 different genera were isolated, from glucose and was not conclusive. xylose media. From the 78 strains, 11 were red pigmented yeasts, belonging to the species Rhodotorula mucilaginosa, Rhodotorula babjevae, Ascomycetous yeast isolates: Two species of Cystobasidium slooffiae, Sporobolomyces roseus and the Hansenisapora were isolated in this study: Sporobolomyces ruberrimus. Hanseniaspora guilliermondii and Hanseniaspora osmophila. The genus Hanseniaspora belong to Next, the strains isolated from each sample and their order and isolation media will be listed. family and includes 21 described species33. Olive soil sample: isolation medium with glucose: Hanseniaspora guilliermondii and Hanseniaspora IST499- Hanseniaspora guilliermondii; IST500- Pichia osmophila, as well other species from this genus, are manshurica; IST498- Candida membranifaciens, known abundant species in wine must 33,34 isolation medium with xylose: IST507- Candida Hanseniaspora guilliermondii was already isolated membranifaciens; IST508- Blastobotrys sp.; IST513- from fermented bottled tomatoes, grape juice, grape Pichia manshurica; Oak soil sample: isolation must and even from a bee trachea and a caecum of medium with glucose: IST464- Kluyveromyces lactis baboon. Hanseniaspora osmophila was isolated from and IST465- Kluyveromyces lactis, isolation medium grapes35. In this work, the two species were isolated with xylose: IST494- Meyerozyma caribbica, IST503- from a soil near an olive tree and an oak tree (IST499, Hanseniaspora osmophila and IST504- IST503). Kluyveromyces marxianus; Berlengas soil: isolation medium with glucose: IST466- Moniliella spathulate; Pichia genus is part of Saccharomycetales order and isolation medium with xylose: IST495- Candida Pichiaceae family, being a member of a large clade36. membranifaciens in isolation medium with xylose; The species Pichia manshurica, Pichia membranifaciens, Pichia kluyveri and Pichia

4 kudriavzevii were isolated in this work in samples Meyerozyma guilliermondii was isolated from plums collected from different environments. Pichia (IST502, IST505), Arrábida coastal seawater (IST540) manshurica (IST500, IST513) was isolated from soil and contaminated soils (IST587, IST591, IST595, underneath an olive tree which is consistent with the IST601). This species is widely distributed in nature, fact that this species has been found in rotting plant being isolated from insect frass, flowers, fruits and material36; Pichia membranifaciens is largely other food products, and is also an opportunistic distributed in nature and found in the rots of spoiled pathogen of humans and animals39. fruit and other plant materials as well as in fermented beverages36. This is consistent with the strains found In this work, a species Schwanniomyces vanrijiae or in this work (IST471 and IST493) that were isolated Schwanniomyces polymorphus from from olives-curing water; Pichia kluyveri has been Schwanniomyces genus, Saccharomycetales order isolated from rotted fruits and fleshy parts of plants, and Debaryomycetaceae family, was isolated from natural fermentations of agricultural products such as olives (IST475). Schwanniomyces vanrijiae was coffee beans36. In this work this species (strains previously isolated from soil, exudates of trees and 40 IST472, IST496) was isolated also from olives-curing anthills and Schwanniomyces polymorphus was 40 water; Pichia kudriavzevii was isolated from a isolated from soils and anthills . spontaneous fermentation of malt (IST458), which Kluyveromyces genus belongs to Saccharomycetales goes according to the literature that says that it is within the Saccharomycetaceae family41. Strains from currently found in natural fermentations, as well as in Kluyveromyces marxianus were isolated in this work 36 soil and fruits . from soil underneath an oak tree (IST504) and from a The genus Candida is polyphyletic and is part of spontaneous fermentation of malt (IST457). In the Saccharomycetales order and Saccharomycetaceae literature this species is reported as being isolated family37. Candida membranifaciens has been from foods and beverages, especially dairy products, recovered from sources that include insects and but also occurs in decaying plant tissue and 42 clinical specimens37. In this work, this species (strains associated insects . Kluyveromyces lactis is mostly 42 IST495, IST498, IST506, IST507) was isolated from isolated from dairy products , but in this work it was soil near an olive tree and from soils from Arrábida and isolated from soil near an oak tree (IST464, IST465). Berlengas; Candida boidinii has been isolated from a Torulaspora genus belongs to Saccharomycetales variety of sources, and sometimes it is associated with order and Saccharomycetaceae family43. The species human activity, being found in the sap fluxes of many is broadly distributed in nature 37 tree species around the world . In this work, strains and has been isolated from soil, fermenting grapes from this species (IST473, IST509, IST592, IST599, and other berry juices, agave juice, tea-beer, and tree IST600) were obtained from olives-curing water and bark43. Torulaspora querccum was recently isolated contaminated soils; Candida silvanorum was from an oak tree leaf44. Strains from these species previously isolated from galleries’ materials from tree- 38 were isolated in this work from contaminated soils destroying beetles . In this work, two strains of this (Torulaspora delbrueckii strains IST588, IST593 and species (IST474, IST492) were isolated from olives. Torulaspora querccum IST597). Candida palmioleophila is a sister species to Candida fluviatilis and belongs to a small clade of closely The genus Tetrapisispora belongs to related Candida species that includes Candida Saccharomycetales order, forms a sister clade with globosa. Candida palmioleophila was isolated from Vanderwaltozyma, and is moderately related to soil from sputum and from soil in tropical Saccharomyces45. Tetrapisispora blattae was firstly greenhouse37. Here, it was isolated from Berlengas’ isolated from an oriental cockroach45 and in this work coastal seawater (IST476, IST477). Candida albicans it was found in contaminated soils (IST589, IST594), is currently known for being responsible for human which shows that this species can be widely mycoses and is found in human aliments, but is often distributed. recovered from decaying plant material37. This is consistent with its isolation from soils in this work Debaryomyces genus belongs to Saccharomycetales (strains IST590, IST598). order of the family Saccharomycetaceae46. is a yeast commonly found in Meyerozyma genus is part Saccharomycetales order products like meats, and in various types of cheese or and Debaryomycetaceae family, being closely related in soil46,47. Likewise, in this work a strain of this species to Debaryomyces, Millerozyma, Priceomyces and was isolated from contaminated soil (IST586). Schwanniomyces39. Meyerozyma caribbica has been isolated from soil, corn-derived starch, sugar cane and Kazachstania belongs to Saccharomycetales order, an apparent human infection39. In this work, Saccharomycetaceae family and the species Meyerozyma caribbica was isolated from different Kazachstania viticola was isolated from fermenting environments: Arrábida coastal seawater (IST540, grapes in Kazakhstan48,49. In this work, it was isolated IST541, IST542), soil underneath an oak (IST494) and from Arrábida soil (IST497). fuel contaminated soils (IST595, IST596, IST601).

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The specie Moniliella spathulate, from Moniliella after harvest, fermenting soy sauce and even in the genus, Moniliellales order and Moniliellaceae family50 cerebrospinal fluid of an AIDS patient58. C. was also isolated in this work, from Berlengas soil carnescens was already isolated from muscatel (IST466). Previously, this species was isolated from grapes, seawater, glacial ice, soil from an alluvial Indian buffalo milk51. forest and leaves of Mediterranean plants, being those last two with origin in Portugal58. C. magnum (also The genus Aureobasidium is a member of the order known as Filobasidium magnum) is isolated from Dothideales (Dothideomycetes), Dothioraceae family certain plants, soil, air, and arctic glaciers, being found and Aureobasidium pullulans or Pullularia pullulans in mammals also59,60. was isolated from tropical, temperate and polar areas. It is found in association with diverse plants, in coastal Cystobasidium genus belongs to the order hypersaline water, glacial ice and polar environments, Sporidiobolales, family Sporidiobolaceae61. polluted water, refrigerated, frozen, salt-preserved Cystobasidium sloofiae have been isolated from and dried foods and various indoor habitats, the terrestrial and deep-sea habitats, having also human surface of human skin, aviation fuel tanks and source54,62. Five strains from this species (IST544- plastics52. Several strains from this species were IST548) were also isolated from the walnut green isolated from walnut green husk in this work (IST552- IST557). Screening of oleaginous yeasts: The 67 non- pigmented yeast strains previously isolated and Basidiomycetous yeast isolates: identified, together with other 58 yeast strains Pseudozyma spp. is from the Basidiomycete division. previously deposited in the IST Yeast Culture Two strains of Pseudozyma aphidis (strains IST511 Collection were first screened based on Niehus et al and IST539) were isolated in this work from a (2018)20 protocol for the rapid screening of oleaginous cabbage. This species is included in the yeasts. In this method, the non-pigmented yeast class Ustilaginomycetes and strains were incubated in plates with Glucose order Ustilaginales, being closely related oleaginous medium + Rhodamine B and Xylose to Ustilago spp. Pseudozyma is a known plant oleaginous medium + Rhodamine B, in order to select pathogens that have been implicated occasionally in 53 strains that can assimilate these sugars. Xylose is the human infections . second most abundant sugar in plant biomass11 and, Rhodotorula genus is polyphyletic54. Rhodotorula consequently, in lignocellulosic hydrolysates used as glutinis is the model yeast of the genus and belongs biorefineries feedstock. If yeasts could utilize both to the order Sporidiobolalestinis and Sporidiobolaceae glucose and xylose simultaneously, it would be family, as well as Rhodotorula mucilaginosa, a possible to maximize titers, yields and productivities in 11 species that is present worldwide being terrestrial and biofuels industries where the lipids will be used .The aquatic, including marine habitats. It is also frequently approach used is based on the principle that, when isolated from human beings54. Rhodotorula babjevae growing in this medium, the lipid producer strains was isolated from an agricultural field in Assam, present a pink colour, whereas the ones that do not Northeast India55. In this work, strains of Rhodotorula produce lipids show a white colour. The strains used mucilaginosa were isolated from spontaneous in this study belong to species that have never been fermentation of malt (IST461) and cabbage (IST512) tested for the production of lipids, or to species already and strains of Rhodotorula babjevae were found in a identified as being oleaginous, but due to the Walnut green husk (IST549, IST550). recognized intraspecies variability, also need to be evaluated. Sporobolomyces belong to four lineages, The screening procedure using Glucose Oleaginous Micobotryum, Sporidiobolus, Erythrobasidium and medium + Rhodamine B allowed the selection of 37 Agaricostilbum lineages of the Pucciniomycotina sub- non-pigmented strains as potential oleaginous yeasts, phylum of Basidiomycota, to Sporidiales order and of which 20 were isolated in this work. The most 56 Sporidiobolaveae family . Sporobolomyces roseus is promising strains selected in Glucose oleaginous found in the phyllosphere of many plant species, air medium + Rhodamine B are from Pichia manshurica, and seawater56. Sporobolomyces ruberrimus was Pichia membranifaciens, Pichia apotheca, found in the atmosphere57. In this work, S. roseus Starmerella bacilaris, Candida boidinii, Metschnikowia (IST551) and S. ruberrimus (IST543) were both isolated from a walnut green husk. pulcherrima, Hanseniaspora guilliermondii and Aureobasidium pullulans species. Strains with a lower Cryptococcus genus belongs to Tremellales order and pink intensity include Lipomyces starkeyi, 58 Tremellaceae family . In this work, several strains Metschnikowia pulcherrima, Starmerella bacilaris, from this genus were isolated from a walnut green Cryptococcus flavescens, Pichia membranifaciens, husk: Cryptococcus flavescens (IST559 and IST562), C. carnescens (IST560 and IST561) and C. magnum Nectria bainii, Lipomyces kononenkoae, Pichia (IST558). C. flavescens is known from various Kluyveri, Kazachstania viticola and Aureobasidium sources, such as air, kernels of wheat and corn just pullulans. When the same procedure was performed

6 in Xylose oleaginous medium + Rhodamine B, 21 4785 showed similar lipid production in a range of strains were selected, of which 16 were isolated in this temperatures from -3 to 20ºC65. Exogenous stress work. The most promising strains selected in Xylose factors are responsible for changes in lipids’ Oleaginous medium + Rhodamine are from Candida production, being stablished that, despite the fact that boidinii and Metschnikowia pulcherrima species. Plus, different species have different optimum temperatures strains with a lower pink intensity include for growth, temperature can also influence triacylglycerols accumulation and the type of fatty Cryptococcus flavescens, Pichia membranifaciens, acids produced66,63. A study from Kot et al (2019) with Pichia apotheca and Nectria bainii (Not all results are Rhodotorula yeast species, described an increase in shown, Figure 1). the biosynthesis of intracellular lipids and carotenoids when the strains were submitted to stress temperature conditions67. Therefore, our results suggest that a higher lipid production occurred at 30ºC when compared to 10ºC, indicating that the temperature of 30ºC might be a stressful condition to the three strains isolated from permafrost lake sediments from Artic, in this work.

Figure 1. Growth of yeast strains in Glucose Oleaginous medium + Rhodamine B (left side) and Xylose Oleaginous medium + Rhodamine B (right side) after 4 days of incubation at 30ºC. 1 - Growth of yeast strains in Glucose Oleaginous medium PYCC 4046 - Lipomyces starkeyi; 2 - Saccharomyces cerevisiae Figure 2. S288C; 3 - IST330 - Brettanomyces bruxellensis; 4 - IST335 - + Rhodamine B after 14 days of incubation at 10 ºC (left) and 30ºC Meyerozyma (=Candida) guilliermondii; 5 - IST339 - (right). 1- IST585- Solicoccozyma terricola; 2- IST583 - Debaryomyces hansenii; 6 - IST340 - Debaryomyces hansenii; 7 - Cryptococcus sp; 3- IST584 - Solicoccozyma terricola. IST341 - Candida parapsilosis; 8 - IST348 - Pichia kudriavzevii; 9 - IST350 - Candida boidinii; 10 - IST351 - Pichia apotheca; 11 - From the isolates identified in this work, the most IST353 - Metschnikowia pulcherrima; 12 - IST368 - Pichia promising oleaginous species were Pichia membranifaciens; 13 - IST369 - Meyerozyma guillermondii; membranifaciens (IST471, IST493), Pichia manshurica (IST500, IST513), Candida boidinii The method reported by Niehus was optimized for (IST473, IST509, IST592, IST599, IST600), pigmented red yeasts, where the medium utilized was Hanseniaspora guilliermondii (IST499) and YPD reach medium and the plates were submitted to Aureobasidium pullulans husk (IST552-IST557). UV light. In this work, we also tested pigmented red Nevertheless, the isolated strains from Pichia Kluyveri yeasts using YPD medium, however, no differences (IST496), Kazachstania viticola (IST497), between isolates were detected (Results not shown). Aureobasidium pullulans, Cryptococcus flavescens Usually, the optimum temperatures for lipid (IST559) and Solicoccozyma terricola (IST585) also accumulation in yeasts range from 25 to 30ºC, and showed a not so strong but potential production of lower or higher temperatures may affect the lipids. Moreover, in this work, it was possible to isolate productivity of lipid accumulation63. Considering that lipid producing yeasts strains from different samples, yeasts isolated from permafrost lake sediments from from several places. Therefore, to understand the Artic (strains IST585- Solicoccozyma terricola; IST583 phylogenetic proximity between the species isolated - Cryptococcus sp; IST584 - Solicoccozyma terricola) in this work, a phylogenetic tree was constructed, did grow better at 10ºC than at 30ºC, in order to test using MEGA-X software (Result not shown). In this the lipid production at different temperatures, the three phylogenetic analysis, it was possible to observe that strains isolated from this sample were incubated at the different lipid producing species are distributed in two different temperatures (10ºC and 30ºC), for 14 several branches of the tree, which shows the broad days (Figure 2). A remarkable difference was detected variety existent between oleaginous yeast species in the colours presented by the strains when grown at and highlights the importance of performing a broader different temperatures, being visible a more intense screening approach, like it was done in this study. On pink colour in the strains incubated at 30ºC, despite the other hand, we could observe that three of the the better growth of the strains incubated at 10ºC, most promising species screened in this work (Pichia suggesting that there is a high lipid production at 30ºC. membranifaciens, Pichia manshurica and Candida In previous studies, facultative psychrophilic yeasts, boidinii), are in the same branch of the phylogenetic from the species Rhodotorula laryngis, Cryptococcus tree, indicating that a screening of oleaginous species gilvescens, and Aureobasidium pullulans, presented in near phylogenetic species, may be a good similar lipid yields at 4ºC and 30ºC64 and the approach to find more efficient lipid producing yeasts. psychrophilic strain Rhodotorula glacialis DBVPG

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Identification of putative xylose transporters: of this species (YDR387C, YBR241C, YGL104C, In order to assimilate xylose, yeast cells need to be YDL199C, YFL040W), were also included. A high- able to uptake this sugar inside through sugar affinity glucose transporter (Hgt1) and a lactose transporters5. Therefore, mining and/or engineering permease (Lac12) from Kluyveromyces lactis and xylose specific transporters insensitive to glucose is fructose symporters from Kluyveromyces lactis and essential for economic conversion of mixed sugars Saccharomyces uvarum, respectively (Frt1 e Fsy1) present in the hydrolysates11. were also considered. Additionally, glucose sensors Snf3 and Rgt2 from S. cerevisiae were used. All these Because Candida boidinii isolates (6 strains tested) proteins are part of the Sugar Porter family69. and Pichia membranifaciens solate (4 strains tested) were among the species showing a more intense pink The phylogenetic tree obtained for Candida boidinii colour when grown in Glucose oleaginous medium + putative sugar transporters shows nine homologous Rhodamine and also showed a pink colour in Xylose proteins near Xut transporters (Xut1 and Xut3) from S. oleaginous medium + Rhodamine plates, an in silico stipitis and Gxs1 from C. intermedia. Plus, three analysis of their putative sugar transporters, with proteins are in the cluster of Gxf1, both from C. emphasis on xylose transporters, was done. We first intermedia. These results indicate that those are performed a genome mining to identify putative sugar potential glucose/xylose transporters, being possible transporters responsible for sugar uptake, in each that the nine transporters near Xut1 and Xut3 may species, a BlastP was performed, using as query the have a high xylose affinity. The phylogenetic tree protein sequence of Hxt7, an hexose uniporter from obtained for Pichia membranifaciens shows four Saccharomyces cerevisiae. This protein was blasted homologous proteins near the glucose/xylose against the genomes sequenced available in NCBI facilitator Gxf1, four near Gxs1 and a cluster that repository for Candida boidinii (taxid:5477) and Pichia includes Hxt proteins. One last protein is also closer membranaefaciens (taxid:4926). The proteins were to the fructose facilitator Ffz1 from considered homologous when the identity was Zygosaccharomyces bailii used for comparison. This superior to 30%23. This method allowed the sugar transporter has homology to the DHA1 family of identification of 33 putative sugar transporters for active drug/H+ antiporters, indicating that this Candida boidinii species and 10 for Pichia transporter may have a different mechanism of membranifaciens (Results not shown). transport70. According to the literature, these species may be able of assimilating xylose, but this The resulting homologous proteins were analysed in physiological characteristic is variable between the programs InterPro, ScanProsit (confidence cut-off strains71. level=0) and Pfam (results considered when E-value <0.001) in order to find signatures from sugar Conclusion transporters24,25,27. When analysed in the tool TMHMM the assessment of transmembrane helices was In this work, 78 strains from 32 different species were performed. It was possible to confirm that those identified, from several different samples. A screening sequences are putative transporters that belong to the method using Rhodamine B staining allowed the Major Facilitator Family and most of them have at selection of 37 and 21 potential oleaginous strains least one of the two domain signatures from the sugar grown in glucose and xylose, respectively. Candida transporters. In addition, sugar transporters usually boidinii and Pichia membranifaciens species were have 12 transmembrane helices68, and the proteins considered potential oleaginous xylose metabolizing identified have 10 to 12 transmembrane helices, with yeasts and were utilized in an in silico analysis, where the exception of the hypothetical protein PMKS- putative sugar transporters were found. In the future, 002057 from P. membranifaciens that does not have the qualitative and quantitative characterization of the TMHs (Results not shown). This protein was excluded lipids produced by these species will be done, as well from the next analysis. as the optimization of growth conditions and higher lipid production and accumulation. Plus, the After, a phylogenetic tree of the proteins was characterization of glucose and xylose consumption constructed using MEGAX software (Results not profiles will be done, specifically in the strains IST350, shown). The xylose transporters of Scheffersomyces IST473, IST592, IST599, IST600, IST368, IST471 and stipitis (Xut1, Xut2, Xut3, Xut4) were included, as well IST493, to know if xylose consumption is repressed by as a glucose/xylose facilitator 1 (Gxf1) and a glucose and assess their possible kinetic evaluation. glucose/xylose symporter 1 (Gxs1) from Candida intermedia. Moreover, the Glycerol: H+ symporter Acknowledgements (Stl1), Myoinositol transporters (Itr1 and Itr2), general α-glucoside transporters (Mal11, Mph2, Mph3, Mtt1, This work was performed in the Biological Sciences Mal31 and Mal61), and hexose uniporters (Hxt1, Hxt2, Research Group of the Institute for Bioengineering Hxt3, Hxt4, Hxt5, Hxt6, Hxt7, Hxt8, Hxt9, Hxt10, and Biosciences (iBB), Instituto Superior Técnico, Hxt11, Hxt12, Hxt13, Hxt15, Hxt16, Hxt17, Gal2) from Universidade de Lisboa, under the supervision of Dr. S. cerevisiae, as well as unknown sugar transporters Margarida Palma and Prof. Isabel Sá-Correia.

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