Broadening the application of Yarrowia lipolytica synthetic biology tools to explore the potential of Yarrowia clade diversity.

Yeasts have established themselves as prominent microbial cell factories, and the availability of synthetic biology tools has led to breakthroughs in the rapid development of industrial chassis strains. The selection of a suitable microbial host is critical in metabolic engineering applications, but it has been largely limited to a few well-defined strains. However, there is growing consideration for evaluating strain diversity, as a wide range of specific traits and phenotypes have been reported even within a specific yeast genus or species. Moreover, with the advent of synthetic biology tools, non-type strains can now be easily and swiftly reshaped. The yeast Yarrowia lipolytica has been extensively studied for various applications such as fuels, chemicals, and food. Additionally, other members of the Yarrowia clade are currently being evaluated for their industrial potential. In this study, we demonstrate the versatility of synthetic biology tools originally developed for Y. lipolytica by repurposing them for engineering other yeasts belonging to the Yarrowia clade. Leveraging the Golden Gate Y. lipolytica tool kit, we successfully expressed fluorescent proteins as well as the carotenoid pathway in at least five members of the clade, serving as proof of concept. This research lays the foundation for conducting more comprehensive investigations into the uncharacterized strains within the Yarrowia clade and exploring their potential applications in biotechnology.

Phylogenomic and biochemical analysis reassesses temperate marine yeast Yarrowia lipolytica NCIM 3590 to be Yarrowia bubula.

Yarrowia clade contains yeast species morphologically, ecologically, physiologically and genetically diverse in nature. Yarrowia lipolytica NCIM 3590 (NCIM 3590), a biotechnologically important strain, isolated from Scottish sea waters was reinvestigated for its phenotypic, biochemical, molecular and genomic properties as it exhibited characteristics unlike Y. lipolytica, namely, absence of extracellular lipolytic activity, growth at lower temperatures (less than 20 °C) and in high salt concentrations (10% NaCl). Molecular identification using ITS and D1/D2 sequences suggested NCIM 3590 to be 100% identical with reference strain Yarrowia bubula CBS 12934 rather than Y. lipolytica CBS 6124 (87% identity) while phylogenetic analysis revealed that it clustered with Y. bubula under a separate clade. Further, whole genome sequencing of NCIM 3590 was performed using Illumina NextSeq technology and the draft reported here. The overall genome relatedness values obtained by dDDH (94.1%), ANIb/ANIm (99.41/99.42%) and OrthoANI (99.47%) indicated proximity between NCIM 3590 and CBS 12934 as compared to the reference strain Y. lipolytica. No extracellular lipase activity could be detected in NCIM 3590 while LIP2 gene TBLASTN analysis suggests a low 42% identity with e value 2 e-77 and 62% coverage. Hence molecular, phylogenetic, genomics, biochemical and microbial analyses suggests it belongs to Yarrowia bubula.

Yarrowia lipolytica: a multitalented yeast species of ecological significance.

Yarrowia lipolytica is characterized by GRAS (Generally regarded as safe) status, the versatile substrate utilization profile, rapid utilization rates, metabolic diversity and flexibility, the unique abilities to tolerate to extreme environments (acidic, alkaline, hypersaline, heavy metal-pollutions and others) and elevated biosynthesis and secreting capacities. These advantages of Y. lipolytica allow us to consider it as having great ecological significance. Unfortunately, there is still a paucity of relevant review data. This mini-review highlights ecological ubiquity of Y. lipolytica species, their ability to diversify and colonize specialized niches. Different Y. lipolytica strains, native and engineered, are beneficial in degrading many environmental pollutants causing serious ecological problems worldwide. In agriculture has a potential to be a bio-control agent by stimulating plant defense response, and an eco-friendly bio-fertilizer. Engineered strains of Y. lipolytica have become a very promising platform for eco-friendly production of biofuel, commodities, chemicals and secondary metabolites of plant origin, obtaining which by other method were limited or economically infeasible, or were accompanied by stringent environmental problems. Perspectives to use potential of Y. lipolytica's capacities for industrial scale production of valuable compounds in an eco-friendly manner are proposed.

Yeast communities of primary and secondary peat swamp forests in southern Thailand.

Khanthuli peat swamp forest (PSF) is one of a few fertile peat swamp forests that remain in Thailand. It is composed of primary PSF and some areas which have been degraded to secondary PSF due to drought, wildfires and land conversion, which have resulted in a decrease in peat layers and change in the species of the plant community. In this study, diversity of yeasts in peat from both primary and secondary PSF areas of the Khanthuli PSF was determined based on culture-dependent approaches, using dilution plate and enrichment techniques. A total of 66 yeast isolates were identified by the analysis of sequence similarity of the D1/D2 region of the large subunit rRNA gene or the combined analysis of sequence of the D1/D2 region and internal transcribed spacer region and confirmed by phylogenetic analysis of the D1/D2 region to belong to 22 known yeast species and six potential new species in the genera Candida (Kurtzmaniella, Lodderomyces, Ogataea, Pichia and Yamadazyma clades), Clavispora, Cyberlindnera, Galactomyces, Hanseniaspora, Metschnikowia, Saturnispora, Schwanniomyces, Cryptotrichosporon, Pichia, Curvibasidium, Papiliotrema, Rhodotorula, and Saitozyma. The most prevalent yeasts in the primary PSF were Cyberlindnera subsufficiens and Galactomyces candidus, while Saitozyma podzolica was the most frequently found in peat from the secondary PSF. Common yeast species in both, primary and secondary PSF, were Cy. subsufficiens, G. candidus and Rhodotorula mucilaginosa.

Effects of a high-cultivation temperature on the physiology of three different Yarrowia lipolytica strains.

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of non-conventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties, especially with respect to important characteristics of a production strain. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as an agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate the genotypic basis of particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

An endophytic isolate of the fungus Yarrowia lipolytica produces metabolites that ameliorate the negative impact of salt stress on the physiology of maize.

BACKGROUND: To combat salinity, plants need easily accessible, safe and sustainable mechanisms for optimum growth. Recently, endophytes proved to be the promising candidates that helped the host plant to thrive under stress conditions. Therefore, the aim was to discover endophytic strain(s) and their mechanism of action to alleviate salt stress in maize. RESULTS: Keeping the diverse role of endophytes in view, 9 endophytic fungi from the spines of Euphorbia milli L. were isolated. Among the isolated fungal isolates, isolate FH1 was selected for further study on the basis of high antioxidant activity and capability to produce high indole-3-acetic acid (IAA), indole-3-acetamide (IAM), phenol and flavonoid contents. The 18S rDNA sequence homology and phylogenetic analysis of the fungal isolate FH1 revealed to be Yarrowia lipolytica. Furthermore, the inoculation of Y. lipolytica FH1 had significantly promoted plant growth attributes in treated maize as compared to positive (salt stress) and negative (salt stress free) controls. Likewise, differences in chlorophyll, carotenes, electrolyte leakage, leaf relative water, peroxidase, catalase, ABA, IAA and proline contents were observed between treated maize and controls. Interestingly, Y. lipolytica FH1 inoculated plants showed lower endogenous ABA and higher endogenous IAA contents. CONCLUSION: From the results, we have concluded that Y. lipolytica inoculation has promoted the growth of maize plants through controlled metabolism and hormonal secretions (ABA and IAA) under salinity stress. Because of the fact, Y. lipolytica can be tried as an eco-friendly bio-fertilizer to achieve optimum crop productivity under saline conditions.

Morphological response of Yarrowia lipolytica under stress of heavy metals.

The marine dimorphic yeast Yarrowia lipolytica has been proposed as a suitable model for the dimorphism study. In this study, the morphological behaviour of two marine strains of Y. lipolytica (NCIM 3589 and NCIM 3590) was studied under stress of different heavy metals. Scanning electron microscopy was used to investigate the morphological features of yeast cells. This study revealed that the normal ellipsoidal shape of yeast cells was changed into oval, rounded, or elongated in response to different heavy-metal stress. Light microscopy was also used to investigate individual properties of yeast cells. The average cell length and radius of both marine strains was increased with increasing concentrations of heavy-metal ions. In addition, the elongation factor was calculated and was increased in the presence of heavy metals like Pb(II), Co(II), Cr(III), Cr(VI), and Zn(II) under the static conditions.

Yarrowia brassicae f.a., sp. nov., a new yeast species from traditional Chinese sauerkraut.

Two strains of a novel yeast species were isolated from traditional Chinese sauerkraut samples collected in Nanyang, Henan Province, central China. Phylogenetic analysis based on the concatenated sequences of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer (ITS) regions showed that these strains belong to the Yarrowia clade, with seven clones of uncultured Yarrowia as their closest phylogenetic neighbours. They differed from their closest known species, Yarrowia divulgata CBS 11013T, by 3.2 % sequence divergence (14 substitutions and 2 gaps) in the D1/D2 domains and by 5.4 % sequence divergence (12 substitutions and 5 gaps) in the ITS regions. The two strains of novel species reproduced asexually, and no ascospores could be found. The name Yarrowia brassicae f.a., sp. nov. is proposed to accommodate these strains, with NYNU 17218T (=CICC 33263T=CBS 15225T) as the type strain.

Marine yeast Yarrowia lipolytica improves the immune responses in Pacific red snapper (Lutjanus peru) leukocytes.

The climatic conditions in saltern saline environments allows the growth of microorganisms adapted to these peculiar ambient and could represent a promising source of new bioactive compounds that could have applications on as animal food supplements, including aquaculture. In this study, we evaluated the role of Yarrowia lipolytica N-6 isolate, from a hypersaline natural environment (Guerrero Negro, Baja California Sur, Mexico), as immunostimulant of the non-specific immune response of head-kidney and spleen Pacific red snapper (Lutjanus peru) leukocytes after challenge with Vibrio parahaemolyticus. In this study, the presence of Y. lipolytica reduced considerably the V. parahaemolyticus load in spleen leukocytes. In vitro assays using head-kidney and spleen leukocytes showed that the response to V. parahaemolyticus infection reveled that leukocyte pre-incubated with Y. lipolytica N-6 significantly increased the non-specific immune response such as respiratory burst, phagocytic activity, NO and MPO activities follow by an increase in SOD and CAT activities, and at the same time inhibited leukocyte apoptosis caused by V. parahaemolyticus. Moreover, Y. lipolytica N-6 incubation also regulated the transcription of genes related to immunity (IL-1ß) or oxidative stress (MnSOD, icCu/ZnSOD or CAT) in leukocytes. These results strongly support the idea that the extreme yeast Y. lipolytica N-6 isolate can stimulate the non-specific immune parameters and the antioxidant immune mechanism in head-kidney and spleen Pacific red snapper leukocytes and could be used as potential immunostimulant.

A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate.

Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates of Y. lipolytica identified, only a few of the strains have been studied extensively. Furthermore, 12 other yeast species were recently assigned to the Yarrowia clade, and most are not well characterized in terms of cell growth and lipid accumulation, especially in industrially relevant conditions. In the present study, we investigated biomass and lipid production by 57 yeast isolates, representing all 13 species in the Yarrowia clade, on a non-detoxified dilute acid-pretreated switchgrass hydrolysate under highly aerobic conditions. The objective was to compare yeast physiology during growth in an abundant, low-cost biomass feedstock and to expand diversity of genetically tractable, oleaginous yeasts available for lipid research. Screening of 45 Y. lipolytica isolates demonstrated considerable variation within the species in terms of lipid accumulation (min = 0.1 g/L; max = 5.1 g/L; mean = 2.3 g/L); three strains (NRRL YB-420, YB-419, and YB-392) were especially promising for cellulosic biomass conversion with average improvements of 43, 57, and 64%, respectively, in final lipid titer as compared to control strain W29. Subsequently, evaluation of strains from 13 distinct species in the Yarrowia clade identified Candida phangngensis PT1-17 as the top lipid producer with a maximum titer of 9.8 g/L lipid, which was over twofold higher than the second-best species in the clade (Candida hollandica NRRL Y-48254). A small set of the most promising strains from the screenings was further characterized to evaluate inhibitor tolerance, lipid production kinetics, and fatty acid distribution. We expect that the results of this study will pave the way for new biotechnological applications involving previously overlooked and under-characterized strains within the Yarrowia clade.

Comprehensive Analysis of a Yeast Lipase Family in the Yarrowia Clade.

Lipases are currently the subject of intensive studies due to their large range of industrial applications. The Lip2p lipase from the yeast Yarrowia lipolytica (YlLIP2) was recently shown to be a good candidate for different biotechnological applications. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we constructed the evolutionary scenario of the lipase family for six species of the Yarrowia clade. RNA-seq based transcriptome analysis revealed the primary role of LIP2 homologues in the assimilation of different substrates. Once identified, these YlLIP2 homologues were expressed in Y. lipolytica. The lipase Lip2a from Candida phangngensis was shown to naturally present better activity and enantioselectivity than YlLip2. Enantioselectivity was further improved by site-directed mutagenesis targeted to the substrate binding site. The mono-substituted variant V232S displayed enantioselectivity greater than 200 and a 2.5 fold increase in velocity. A double-substituted variant 97A-V232F presented reversed enantioselectivity, with a total preference for the R-enantiomer.

A substrate-based approach for the selection of oil-bearing heterotrophs from nitrogen-deficient soil for lipid production.

In this study, nine heterotrophic yeast isolates were tested for their ability to assimilate crude glycerol and consecutive conversion to triacylglycerides (TGAs). All the organisms were initially screened on crude glycerol-based selection media, and those producing lipid globules were further evaluated for lipid production. Sudan Black B staining of eight isolates showed lipid globules. These strains were further studied at different C/N ratio. The molecular identification revealed that the isolates belonged to the genera of Yarrowia and Candida. Among these isolates, SKY7 (Yarrowia lipolytica) produced up to 42.04 ± 0.11 % of lipid w/w) with a C/N ratio of 100 and fermentation time of 72 h. The other strains produced 5.82 ± 0.4 to 34.57 ± 0.44 % lipid (w/w). The GC-flame ionization detector (FID) lipid profile showed that the lipid produced by the strains had close resemblance with vegetable oil and could serve as a feedstock for biodiesel production. Biolog test of the isolates revealed a wide spectrum of carbon utilization.

Optimization of medium composition for erythritol production from glycerol by Yarrowia lipolytica using response surface methodology.

Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L(-1), respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L(-1) of glycerol the Wratislavia K1 strain produced 46.9 g L(-1) of erythritol, which corresponded to a 0.47 g g(-1) yield and a productivity of 0.85 g L(-1) hr(-1). In the fed-batch mode and medium with the total concentration of glycerol at 300 g L(-1) and C:N ratio at 81:1, 132 g L(-1) of erythritol was produced with 0.44 g g(-1) yield and a productivity of 1.01 g L(-1) hr(-1.)

Mercury bioremoval by Yarrowia strains isolated from sediments of mercury-polluted estuarine water.

Difference in mercuric ion removal by resting and growing cells of two mercury-resistant yeast strains, identified as Yarrowia spp. (strains Idd1 and Idd2), were studied. Resting cells of strain Idd2 exhibited high maximum Hg(2+) removal capacity (59 mg mercury per g dry cell weight [gdw(-1)]) by adsorption than those of resting cells of strain Idd1 (32 mg gdw(-1)). The resting cells of strain Idd2 exhibited a higher Hg(2+) desorption capacity using CaCl2 (68 %) and EDTA (48 %) than strain Idd1, depicting weaker binding of Hg(2+) onto strain Idd2 unlike strain Idd1. The actively growing yeast cells showed opposite Hg removal characteristics to those of the resting cells. Strain Idd1 adsorbed less Hg(2+) from culture medium supplemented with Hg(2+) than strain Idd2. However, the growing strain Idd1 reduced and vaporized 27 % of supplemented Hg(2+) as metallic mercury (Hg(0)), while the growing strains Idd2 vaporized 15 % of the supplemented Hg(2+). These two yeast strains are potential biotechnological tools for the eventual bioremediation of polluted aquatic systems.

Yarrowia porcina sp. nov. and Yarrowia bubula f.a. sp. nov., two yeast species from meat and river sediment.

Eleven yeast strains representing two hitherto undescribed species were isolated from different kinds of meat samples in Hungary and one from the sediment of a tropical freshwater river in Southeastern Brazil. The analysis of the sequences of their large subunit rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) regions placed the two new species in the Yarrowia clade. Some of the seven strains representing the first new species can mate and give rise to asci and form ascospores embedded in capsular material, which qualifies it as the third teleomorph species of the Yarrowia clade. The name Yarrowia porcina sp. nov. (type strain: NCAIM Y.02100(T) = CBS 12935(T) = NRRL Y-63669(T), allotype strain UFMG-RD131(A) = CBS 12932(A)) is proposed for this new yeast species, which, based on physiological characters, is indistinguishable from Yarrowia lipolytica and some other species of the genus. Considerable intraspecific variability was detected among the sequences of the large subunit rRNA gene D1/D2 domains of the seven strains. The variability among the D1/D2 sequences exceeded the divergence observed among the ITS sequences and in some cases more than 1 % substitution among the D1/D2 sequences was detected. The conspecificity of these strains was supported by the low (0-3 substitutions) sequence divergence among their ITS sequences, the result of a parsimony network analysis utilizing the concatenated ITS and D1/D2 sequences and also by the fingerprint patterns generated by microsatellite primed PCR. No ascospore formation was observed in the group of the other five strains representing the second new species. These strains shared identical D1/D2 and ITS sequences. Yarrowia bubula f.a., sp. nov. (type strain: NCAIM Y.01998(T) = CBS 12934(T) = NRRL Y-63668(T)) is proposed to accommodate these strains.

Yarrowia divulgata f.a., sp. nov., a yeast species from animal-related and marine sources.

Five yeast strains, phenotypically indistinguishable from Yarrowia lipolytica and Yarrowia deformans, were recovered from different animal-related samples. One strain was isolated from a bacon processing plant in Denmark, two strains from chicken liver in the USA, one strain from chicken breast in Hungary and one from minced beef in Hungary. Comparisons of the sequences of their large subunit rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) regions revealed that, despite their phenotypic similarity, they represent a novel yeast species of the Yarrowia clade with Y. deformans being the genotypically closest relative (LSU rRNA gene D1/D2 and ITS region similarity of 97.0 and 93.7 %, respectively). Yarrowia divulgata f.a., sp. nov. is proposed to accommodate these strains with F6-17(T) ( = CBS 11013(T) = CCUG 56725(T)) as the type strain. Some D1/D2 sequences of yeasts from marine habitats were found in the GenBank database that were identical to those of the strains of Y. divulgata f.a., sp. nov. Unfortunately, these strains were not available for our study.

Identifying and characterizing Yarrowia keelungensis sp. nov., an oil-degrading yeast isolated from the sea surface microlayer.

Ascomycetous yeast strain SM-22 was isolated from the sea-surface microlayer near the Keelung City off the northern coast of Taiwan. This strain showed a cell surface hydrophobicity higher than 90 %, moderate UV A/B resistance, and it degraded 68 % of the total petroleum hydrocarbon content of an artificial seawater medium containing 1 % (v v(-1)) diesel oil within 15 days at 25 °C. The closest phylogenetic relative of this strain is Candida oslonensis CBS 10146(T), but it differs from strain SM-22 by a 3.7 % divergence (including 18 nucleotide substitutions and 2 gaps) in the D1/D2 domain sequence of the large subunit rRNA gene. This difference clearly suggests that the strain SM-22 represents a distinct species. Strain SM-22 does not produce ascospores on common sporulation media and it can therefore be considered an anamorph of the genus Yarrowia. Thus, the name Yarrowia keelungensis sp. nov. (type strain SM-22(T) = BCRC 23110(T) = JCM 14894(T) = CBS 11062(T)) is proposed as a novel species of genus Yarrowia.

Biomass production by novel strains of Yarrowia lipolytica using raw glycerol, derived from biodiesel production.

This study demonstrated the potential applicability of the isolated strains of Yarrowia lipolytica for the valorization of glycerol waste generated during biodiesel production, throughout biomass production. Twenty-one strains were isolated from different environments and identified as Y. lipolytica. Biomass production from pure glycerol (25 g L(-1)) was performed in the shake-flasks experiment. Eight strains with the best biomass production ability were chosen for studies in bioreactor (pH 3.5). The analysis of technological process parameters and biomass chemical composition demonstrated that S6 strain was the most suitable for biomass production. Its application allowed obtaining 11.7 and 12.3 g L(-1) of the biomass with 1.30 and 1.37 g L(-1) h(-1) productivity, respectively when pure and raw glycerol (25 g L(-1)) was used. In the yeast protein amino acid profile the contents of lysine, threonine and phenylalanine/tyrosine were higher than required by FAO/WHO. According to the EAAI, the nutritional value of the biomass reached up to 72.3%.

Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining.

The ascomycetous yeast Yarrowia lipolytica has been established as model system for studies of several research topics as well as for biotechnological processes in the last two decades. However, frequency of heterologous recombination is high in this yeast species, and so knockouts of genes are laborious to achieve. Therefore, the aim of this study was to check whether a reduction of non-homologous end-joining (NHEJ) of double strand breaks (DSB) results in a strong increase of proportion of homologous recombinants. The Ku70-Ku80 heterodimer is known as an essential protein complex of the NHEJ. We show that deletion of YlKU70 and/or YlKU80 results in an increase of the rate of transformants with homologous recombination (HR) up to 85 % in each case. However, it never reaches near 100 % of HR in any case as described for some other yeast. Furthermore, we demonstrated that growth of Δylku strains was similar to that of the wild-type strain. In addition, no differences were detected between the Δylku strains and the parent strain in respect to sensitivity to the mutagenic agent EMS as well as to the antibiotics hygromycin, bleomycin and nourseothricin. However, Δylku70 and Δylku80 strain showed a slightly higher sensitivity against UV rays. Thus, the new constructed Δylku strains are attractive recipient strains for homologous integration of DNA fragments and a valuable tool for directed knockouts of genes. Nevertheless, our data suggest the existence of another system of non-homologous recombination what may be subject of further investigation.

The teleomorph state of Candida deformans Langeron & Guerra and description of Yarrowia yakushimensis comb. nov.

Yarrowia lipolytica is the only known species in the teleomorph (i.e. sexual) genus Yarrowia and has its anamorph (i.e. asexual state) classified in the genus Candida Berkhout as Candida lipolytica. This species can be found readily in nature, has significant industrial value and is important to the food and medical fields. Candida deformans was first considered a variety of C. lipolytica and was later listed as a synonym of this species. More recent studies based on sequence variation in the nuclear rRNA gene sequences suggested C. deformans to be a separate species with no known teleomorph linked to it. In this study we show that C. deformans strains, obtained from South Africa, can mate with strains present in the CBS yeast collection and produce a Yarrowia teleomorph, described here as Yarrowia deformans. Strains of Candida yakushimensis nom. inval., a species also belonging to the Yarrowia phylogenetic clade, were also studied and described as a novel species of Yarrowia.