Daniel Ruben

Akiola Sanya
email: sanyadaniel86@yahoo.com

Djamila Onésime

Volkmar Passoth

Mrinal K Maiti

Atrayee Chattopadhyay

Mahesh B Khot

MINI

Keywords: Oleaginous yeasts, Recombinant proteins, Biofuels, Blastobotrys genus, Lipid feedstock, Metabolic engineering

Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes. Species of this genus are dimorphic, non-pathogenic, thermotolerant, and can assimilate a variety of hydrophilic and hydrophobic substrates. These can constitute a single-cell oil platform in an emerging bio-based economy as oleaginous traits have been discovered recently. However, the regulatory network of lipogenesis in these yeasts is poorly understood. To keep pace with the growing market demands for lipidderived products, it is critical to understand the lipid biosynthesis in these unconventional yeasts to pinpoint what governs the preferential channelling of carbon flux into lipids instead of the competing pathways. This review summarizes information relevant to the regulation of lipid metabolic pathways and prospects of metabolic engineering in Blastobotrys yeasts for their application in food, feed, and beyond, particularly for fatty acid-based fuels and oleochemicals.

Key points

• The production of biolipids by heterotrophic yeasts is reviewed. • Summary of information concerning lipid metabolism regulation is highlighted.

• Special focus on the importance of diacylglycerol acyltransferases encoding genes in improving lipid production is made.

Introduction

Budding ascomycetous yeasts (e.g., Saccharomyces sp., Pichia sp., etc.) belonging to Saccharomycotina subphylum are well known for their applications in basic research, food, healthcare, and agriculture. Oleaginous yeasts (such as Yarrowia lipolytica, Lipomyces sp., Rhodotorula sp.) are explored for more than a decade for the promising role in replacing oleochemicals derived from fossil resources and foodgrade oil plants. This is arising from their lipid production ability coupled to high-density growth on a wide range of low-cost substrates with unique physiological characteristics and availability of constantly evolving genetic tools [START_REF] Juanssilfero | Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi[END_REF][START_REF] Ng | Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil[END_REF]). Blastobotrys adeninivorans is another non-conventional oleaginous yeast belonging to the subphylum Saccharomycotina. The yeast is found in a diverse range of natural habitats. The first strain was isolated from soil and initially been named Trichosporon adeninovorans CBS8244 T (Middelhoven et al. 1984). Further strains have been obtained from wood hydrolysates, chopped maize silage, humus-rich soil, and fermented foods such as Pu-erh tea [START_REF] Wartmann | Comparative biochemical, genetical and immunological studies of glucoamylase producing Arxula adeninivorans yeast strains[END_REF][START_REF] Abe | Characteristic fungi observed in the fermentation process for Puer tea[END_REF][START_REF] Kunze | The complete genome of Blastobotrys (Arxula) adeninivorans LS3 -a yeast of biotechnological interest[END_REF]. Initially described as Trichosporon adeninivorans by yeast taxonomic monograph (Middlehoven et al. 1984), and later as Arxula adeninivorans (Van der Walt 1990), this dimorphic yeast was finally reclassified in the genus Blastobotrys by [START_REF] Kurtzman | Multigene phylogenetic analysis of the Trichomonascus, Wickerhamiella and Zygoascus yeast clades, and the proposal of Sugiyamaella gen. nov. and 14 new species combinations[END_REF], based on a detailed phylogenetic comparison with other related species.

Among the species described in the genus Blastobotrys, B. adeninivorans (e.g., LS3, type strain CBS 8244T and CBS 7766) has been well described [START_REF] Kurtzman | Blastobotrys americana sp. nov., Blastobotrys illinoisensis sp. nov., Blastobotrys malaysiensis sp. nov., Blastobotrys muscicola sp. nov., Blastobotrys peoriensis sp. nov. and Blastobotrys raffinosifermentans sp. nov., novel anamorphic yeast species[END_REF]Kurtzmann and Robnett 2007). Recently, the LS3 strain of B. adeninivorans was reassigned to B. raffinosifermentans [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]). The LS3 strain has been exploited as a microbial biocatalyst and biosensor to detect biomarkers in medicine or pollutants in aquatic samples [START_REF] Malak | Blastobotrys (Arxula) adeninivorans: a promising alternative yeast for biotechnology and basic research[END_REF][START_REF] Pham | The determination of pharmaceuticals in wastewater using a recombinant Arxula adeninivorans whole cell biosensor[END_REF][START_REF] Theron | A broad-range yeast expression system reveals Arxula adeninivorans expressing a fungal self-sufficient cytochrome P450 monooxygenase as an excellent whole-cell biocatalyst[END_REF]Kasprzak et al. 2016b). It exhibits unusual metabolic and physiological characteristics, e.g., the presence of n-butanol degradation pathway; thermo-, halo-, and osmo-tolerance features; and the ability to secrete high-quality proteins including intracellular and extracellular phytases, glucoamylase, cutinases, invertase, xylosidase, cellobiases, and proteases [START_REF] Bischoff | Three new cutinases from the yeast Arxula adeninivorans that are suitable for biotechnological applications[END_REF][START_REF] Wartmann | Genetic transformation and biotechnological application of the yeast Arxula adeninivorans[END_REF][START_REF] Olstorpe | Screening of yeast strains for phytase activity[END_REF][START_REF] Samsonova | A set of genetic markers for the chromosomes of the imperfect yeast Arxula adeninivorans[END_REF]. Post-translational modifications like O-glycosylation and N-glycosylation are reported in this yeast [START_REF] Wartmann | High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans[END_REF]. Blastobotrys species share nitrate assimilation and xerotolerance as common traits [START_REF] Van Der Walt | Arxula gen. nov. (Candidaceae), a new anamorphic, arthroconidial yeast genus[END_REF]). Oleaginous traits have been described for B. adeninivorans CBS 8244T [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF] and recently for the LS3, CBS 7377, and CBS 7766 strains of the yeast [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]. When nitrogen-limitation was imposed, lipid accumulation to above 30% was observed in B. adeninivorans [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF].

The LS3 strain of B. adeninivorans emerged as an attractive host for the expression of recombinant proteins with medical or industrial interest. An increasing number of published articles have reported the LS3 strain for producing recombinant proteins, analyzing its uric acid-reducing gene, adeninereducing gene, guanine-reducing gene, and in improving the p r o d u c t i o n o f p o l y h y d r o x y a l k a n o a t e s ( P H A s ) (polyhydroxybutyrate, PHB-V) or enantiomerically pure 1-(R)-phenyl ethanol (Jankowska et al. 2013b(Jankowska et al. ,2013a[START_REF] Jankowska | A novel enzymatic approach in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine degradative enzymes[END_REF][START_REF] Litwińska | Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in the degradation of pharmaceuticals[END_REF][START_REF] Trautwein-Schult | Arxula adeninivorans recombinant urate oxidase and its application in the production of food with low uric acid content[END_REF][START_REF] Ratledge | The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems[END_REF][START_REF] Rauter | Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol[END_REF][START_REF] Biernacki | Enhancement of poly(3-hydroxybutyrate-co-3hydroxyvalerate) accumulation in Arxula adeninivorans by stabilization of production[END_REF][START_REF] Zhang | Characterization of a novel alkaline Arxula adeninivorans urate oxidase expressed in Escherichia coli and its application in reducing uric acid content of food[END_REF]. [START_REF] Lapeña | Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken by-products[END_REF] confirmed the ability of the LS3 strain to assimilate a wide range of nitrogen-containing carbon sources (e.g., proteins, purines) alongside its capacity to grow well on an enzymatically saccharified sulfite-pulped spruce wood and hydrolysates of by-products from chicken. They also reported the similarity of its amino acid profile to fishmeal and soymeal, especially for essential amino acids, hence suitable for single-cell protein production. B. adeninivorans CBS 8244T described earlier for its amines, purines, and adenines assimilation ability [START_REF] Meesters | Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as the sole source of carbon, nitrogen and energy[END_REF]) has now been recognized for its capacity to hydrolyze maltose, maltose-like substrates (maltulose, melezitose), and sucrose [START_REF] Visnapuu | Characterization of a maltase from an early-diverged non-conventional yeast Blastobotrys adeninivorans[END_REF]. [START_REF] Kurtzman | Advances in yeast systematics and phylogeny and their use as predictors of biotechnologically important metabolic pathways[END_REF] highlighted the growth of both B. adeninivorans and B. raffinosifermentans on high glucose concentration (50%, 60%, and 70%) media. The dimorphic yeast described in this review alongside the CBS 8244T strain also showed promising innate potential in the bioconversion of xylose and glucose as renewable feedstock to various molecules, including lipid, acetic, and citric acid [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF][START_REF] Borelli | Positive selection evidence in xylose-related genes suggests methylglyoxal reductase as a target for the improvement of yeasts' fermentation in industry[END_REF].

Apart from its oleagenicity and prospects in biofuel applications, the Blastobotrys yeasts have been studied for various other biotechnological applications, listed as follows: [START_REF] Katongole | Effect of solid-state fermentation with Arxula adeninivorans or Hypocrea jecorina (anamorph Trichoderma reesei) on hygienic quality and in-vitro digestibility of banana peels by mono-gastric animals[END_REF] assessed the effectiveness of solidstate fermentation with B. raffinosifermentans species to improve the hygienic quality and digestibility of banana peels by mono-gastric animals. B. adeninivorans was employed for single-cell protein (SCP) production from biogas substrate. SCP derived from microbial biomass is usually used as animal feed, particularly in aquaculture (Goldberg 1985). The yeast was suitable for SCP production and its protein content displayed essential amino acids that were similar to those of soymeal and fishmeal [START_REF] Lapeña | Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken by-products[END_REF]. The yeast has also been considered a suitable host for heterologous gene expression [START_REF] Wartmann | Genetic transformation and biotechnological application of the yeast Arxula adeninivorans[END_REF].

Microbial lipases are interesting due to their function in lipid digestion for nutrient acquisition. [START_REF] Stehr | Microbial lipases as virulence factors[END_REF] suggested a potential benefit from these enzymes in utilizing carbohydrate-restricted environments or environments where lipids are the sole carbon source. [START_REF] Kumari | Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts[END_REF] applied B. raffinosifermentans as a host system in shake flask cultures to express the lipase YlLip11 from Y. lipolytica and revealed the production of 2654 U/L lipase. The extracellular lipase Alip1p, encoded by B. raffinosifermentans ALIP1, is a triacylglycerol acylhydrolase that catalyzes the hydrolysis of all ester bonds in between glycerol and medium-chain fatty acids. The genetic elements related to ALIP1 are described in Böer et al. (2005b). The ALIP1 gene regulated by carbon source or lipophilic compounds harbored an open reading frame of 1347 bp and its expressed protein (Alip1p) containing the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) for lipid recognition exhibited a dimeric structure of 100 kDa and an optimum activity at pH 7.5 and temperature 30 °C (Böer et al. 2005b).

There are a growing number of excellent reviews on genetic engineering tools for B. raffinosifermentans, much like the oleaginous yeast species of the genera Yarrowia, Rhodotorula, and Lipomyces [START_REF] Malak | Blastobotrys (Arxula) adeninivorans: a promising alternative yeast for biotechnology and basic research[END_REF][START_REF] Wang | Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica-a review[END_REF][START_REF] Park | The engineering potential of Rhodosporidium toruloides as a workhorse for biotechnological applications[END_REF][START_REF] Sutanto | Lipomyces starkeyi: its current status as a potential oil producer[END_REF]. Nonetheless, owing to recent studies with new approaches and technologies, we have begun to unveil the mechanisms underlying lipid biogenesis processes on a molecular level in this yeast. Additionally, Blastobotrys genus has recently attracted interest as a potential source for biofuels. In the current review, we address the recent applications made with this novel non-conventional oleaginous yeast, review genome engineering strategies, and discuss substrate constraints on lipid production and the state-of-the-art gene targeting tools available for this yeast. Most data in the literature used the name Arxula adeninivorans. In this review, however, we have used Blastobotrys adeninivorans as it remains the current taxonomically valid name.

Taxonomy and genome description

The taxonomic position of Blastobotrys species is summarized in Fig. 1. Generally, non-conventional yeasts under the Saccharomycotina subphylum include species such as Scheffersomyces (Pichia) stipitis, Komagataella phaffii (Pichia pastoris), Ogataea (Hansenula) polymorpha, Kluyveromyces lactis, Y. lipolytica, Kluyveromyces marxianus, Ogataea thermomethanolica [START_REF] Cai | CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications[END_REF], or Trichosporon cutaneum [START_REF] Reiser | ) Nonconventional yeasts in biotechnology: a handbook[END_REF]. Some of them are ubiquitous to all sorts of niches, showing extreme stress tolerance phenotypes. For instance, Trichosporon cutaneum easily accommodates high concentrations of compounds like f o r m i c a c i d , a c e t i c a c i d , l e v u l i n i c a c i d , 5hydroxymethylfurfural, and vanillin syringaldehyde, and Pichia kudriavzevii can sustain high furan aldehyde concentrations [START_REF] Kwon | Effect of lignocellulosic inhibitory compounds on growth and ethanol fermentation of newly-isolated thermotolerant Issatchenkia orientalis[END_REF]. Phylogenetic tree reconstruction placed B. adeninivorans closest to the well-described nonconventional oleaginous yeast Y. lipolytica and coalescencebased phylogenies supported this dimorphic yeast as sister to a clade containing Saprochaete clavata and Geotrichum candidum isolate CLIB 918 (for an overview, see [START_REF] Shen | Reconstructing the backbone of the Saccharomycotina yeast phylogeny using genome-scale data[END_REF]. Yeast species are routinely identified using the internal transcribed spacer (ITS) region and the variable domain of the large subunit (26S) ribosomal DNA or the complete small subunit, D1/D2 domain, coupled with the gene sequences from the nearly complete large subunit (LSU) rRNA gene, mitochondrial small-subunit rRNA gene (MtSm), and cytochrome oxidase II (COXII) [START_REF] Kurtzman | Multigene phylogenetic analysis of the Trichomonascus, Wickerhamiella and Zygoascus yeast clades, and the proposal of Sugiyamaella gen. nov. and 14 new species combinations[END_REF].

B. adeninivorans LS3 is an attractive and amendable microorganism with a small genome (11.8 Mb) organized into four chromosomes-Arad1A, Arad1B, Arad1C, and Arad1D of 1659397, 2016785, 3827910, and 4300524 nucleotides, respectively. The genome size is smaller than that of S. cerevisiae S288c (12.1 Mb) and Debaryomyces hansenii CBS767 (12.2 Mb). The LS3 strain of this species is asexual with a mating-type (MAT) locus containing the transcription factor Matα1 (ARAD1D19294g, MTAL1), also observed in Y. lipolytica. However, no Matα2 (MATL2) has been uncovered in contrast to Y. lipolytica and S. cerevisiae. A genome GC content of around 48.1% resembling that of Y. lipolytica E150 (49.0%), but significantly higher than that of S. cerevisiae S288c (38.3%) and Debaryomyces hansenii CBS767 (36.3%), has been elucidated for this strain. The complete genome sequencing has been performed by Kunze and co-workers and revealed around 6000 genes in just 11.8 Mb. A single transposable element of Ty3/gypsy-type long terminal repeat superfamily of retrotransposons, Taa3 (ARAD1B13860t), has been recognized within the genome alongside genes encoding for enzymes involved in βoxidation and the glyoxylate cycle [START_REF] Kunze | The complete genome of Blastobotrys (Arxula) adeninivorans LS3 -a yeast of biotechnological interest[END_REF].

Morphology and growth conditions

B. adeninivorans is usually cultured at 28-37 °C [START_REF] Knoll | High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors[END_REF][START_REF] Kunze | The complete genome of Blastobotrys (Arxula) adeninivorans LS3 -a yeast of biotechnological interest[END_REF][START_REF] Tsakraklides | Improved gene targeting through cell cycle synchronization[END_REF][START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF][START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. The yeast exhibits temperature-dependent morphology characterized by the formation of budding cells below 42 °C, the appearance of pseudo-hyphae at 42 °C, and hyphae at above 42 °C, as well as the ability to grow at temperatures up to 48 °C without prior adaptation [START_REF] Choudhary | Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass[END_REF]) and a few hours of survival at 55 °C [START_REF] Wartmann | Comparative biochemical, genetical and immunological studies of glucoamylase producing Arxula adeninivorans yeast strains[END_REF]. Its dimorphism is reversible with growth conditions as budding cells are re-established below 42 °C.

Cultivation mode such as shake flask [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF][START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]) and fed-batch cultivation (Bernacki et al. 2017) alongside culture media encompassing selective media [START_REF] Litwińska | Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in the degradation of pharmaceuticals[END_REF][START_REF] Theron | A broad-range yeast expression system reveals Arxula adeninivorans expressing a fungal self-sufficient cytochrome P450 monooxygenase as an excellent whole-cell biocatalyst[END_REF][START_REF] Friedlander | Engineering of a high lipid producing Yarrowia lipolytica strain[END_REF][START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]) and nonselective media [START_REF] Samsonova | A set of genetic markers for the chromosomes of the imperfect yeast Arxula adeninivorans[END_REF]Bernacki et al. 2017) are usually applied to monitor the growth of these yeasts, with the main focus on nitrogen (NH 4 Cl) and carbon sources (glucose) [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF].

Fungi (Kingdom)

Ascomycota (Phylum) SaccharomycoƟna (Subphylum) Hemoascomycetes (Class) Endomycetales (Order) Saccharomycetaceae (Family) Saccharomycetoideae (Sub-family) Blastobotrys (Genus) Blastobotrys adeninivorans (Species)

Oleaginous characteristics

Compared to the conventional procedures of vegetable oil production, microbial lipid production takes advantage of a short production cycle, industrial scalability of bioproducts, zero competition with food crops, independence of arable land, sustainability, and tailored processes. As such, they might circumvent the limitations posed by lipid production from oil crops and plants. For instance, Blomqvist et al. demonstrated the possibility to replace vegetable oil in the feed for Arctic char (Salvelinus alpinus) with microbial oils derived from Lipomyces starkeyi cultivated on lignocellulose hydrolysate from wheat straw [START_REF] Blomqvist | Oleaginous yeast as a component in fish feed[END_REF]. In 2011, nearly 1500 species of ascomycete and basidiomycete yeasts belonging to 149 genera were listed and described (Kurtzman et al. 2011). Less than 30 species were characterized for their oleaginous traits [START_REF] Qin | From low-cost substrates to Single Cell Oils synthesized by oleaginous yeasts[END_REF]). These oleaginous yeast species were categorized under the following classes- As previously reported, lipid accumulation in oleaginous yeast occurs when there is depletion of nutrients in the culture medium, channeling the carbon flux towards cellular lipogenesis [START_REF] Aggelis | Prediction of lipid accumulation-degradation in oleaginous micro-organisms growing on vegetable oils[END_REF]. In the literature, two criteria are mainly described to characterize a yeast as an oleaginous microorganism. Firstly is the ability to store more than 20% lipid in their dry cell weight (DCW) predominantly in the form of triacylglycerol or TAG [START_REF] Sitepu | Oleaginous yeasts for biodiesel: current and future trends in biology and production[END_REF][START_REF] Fakas | Lipid biosynthesis in yeasts: a comparison of the lipid biosynthetic pathway between the model nonoleaginous yeast Saccharomyces cerevisiae and the model oleaginous yeast Yarrowia lipolytica[END_REF]. For instance, the oleaginous red yeast Rhodotorula glutinis produced 70% lipid in their dry weight. Another oleaginous yeast, Lipomyces starkeyi, was reported to have lipid as high as 85.1% DCW [START_REF] Li | Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis[END_REF][START_REF] Juanssilfero | Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi[END_REF]. Secondly is the presence of key enzymes, i.e., ATP citrate Lyse (ACL) and/or malic enzyme (MAE) responsible for the oleaginous trait [START_REF] Ratledge | The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems[END_REF][START_REF] Kamineni | Engineering triacylglycerol production from sugars in oleaginous yeasts[END_REF]. The role of ACL and MAE enzymes as a supplier of acetyl-CoA and NADPH, respectively, appeared to be speciesdependent for fatty acid (FA) synthesis. For instance, Dulermo et al. highlighted that the inactivation of the ACL1 gene in Y. lipolytica decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis. In contrast, inactivation of the MAE1 gene has no effects on FA synthesis, except in a FA over-accumulating strain where it improves FA synthesis by 35% [START_REF] Dulermo | Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis[END_REF].

A lthough cul tivation conditi ons for effici ent B. raffinosifermentans-based processes [START_REF] Stöckmann | Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment[END_REF]) and lipases activity were elucidated a decade ago, it is only recently that the lipid production ability and lipid-related genes have been uncovered for the yeast [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF][START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. The metabolic engineering strategies for improving its lipid production are essentially described in the works of [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. The lipid content of the unmodified wild-type strain (LS3) of B. adeninivorans was 30% DCW on glucose [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]. The lipid profile of B. adeninivorans (e.g., strains CBS 8244 and LS3), at cultivation temperatures ranging from 15 to 37 °C, contained C18:1 (n -7), C18:1 (n -5), C17:1, C18:3 (n -3), and C16 and C18 FAs [START_REF] Olstorpe | Strain-and temperature-dependent changes of fatty acid composition in Wickerhamomyces anomalus and Blastobotrys adeninivorans[END_REF][START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]. Interestingly, Thomas et al. demonstrated variation in FA composition of the yeast at elevated temperatures. For instance, increasing temperature from 28 to 45 °C showed an increase in C18:0 and C18:2 in the CBS 8244 T and LS3, while the relative proportions of C16:0 and C18:1 decreased [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]). In the LS3, CBS 7377, CBS 7766, and CBS 8244 T strains, the dominant FA species in lipid profile, oleic acid (18:1), ranged from 43 to 52%, depending on the strain and medium [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]. These FA species are good candidates for biodiesel quality as palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids are elucidated to be suitable FAs for biodiesel production [START_REF] Knothe | Improving biodiesel fuel properties by modifying fatty ester composition[END_REF]. Of note, [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF] demonstrated the capacity of this yeast to grow efficiently on both hydrophobic (oleic acid) and hydrophilic (glucose, xylose) substrates generating value-added bioproducts such as organic acids (citric acid and acetic acid). A cumulative concentration ranging from 3.4 to 10.1 g/L has been disclosed for both citric and acetic acid [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF]. The anamorphic hemiascomycete yeast of this report contained fatty acid species retrieved from other oleaginous plants or yeasts and therefore can be exploited as renewable resources for industrial raw materials (Supplementary Table S1). It holds excellent promise as non-food feedstocks for the generation of bio-commodities. Some of the key findings regarding Blastobotrys genus are summarized in Fig. 2. In Blastobotrys species, total intracellular lipids were quantified by gas chromatography after lyophilizing the harvested wet cell pellets and transesterifying cellular fatty acid contents to produce fatty acids methyl esters [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF][START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. We compared the lipogenesis ability of B. raffinosifermentans (LS3) on some carbon substrates to that of other well-documented oleaginous yeasts in Table 1. The commonly used strains in research are summarized in Table 2.

Tools and techniques employed to engineer Blastobotrys yeasts

Genetic elements and vector systems developed in Blastobotrys genus

Most of the vector systems have been developed in Blastobotrys genus to integrate a copy of the gene of interest in its genome, for example, integration in an atrp1 ( t r y p t o p h a n ) a u x o t r o p h i c h o s t s t r a i n o f B. raffinosifermentans. Those elements consist of the ATRP1 selection marker fused to truncated ALEU2 promoter of 53 bp or TEF1 (translation-elongation factor 1α) promoter, terminator, expression vector encoding a heterologous enzyme with biotechnological interest. For instance, a high copy number (eight copies) of an amyA expression vector encoding heterologous alpha-amylase from Bacillus amyloliquefaciens instead of a single copy resulted in strains with superior productivity for a secreted recombinant alpha-amylase [START_REF] Steinborn | A novel vector element providing multicopy vector integration in Arxula adeninivorans[END_REF]). The use of green fluorescent protein (GFP)-coding genes from the jellyfish Aequorea victoria as a reporter gene to assess heterologous gene expression was also reported in this yeast [START_REF] Wartmann | High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans[END_REF][START_REF] Terentiev | A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements[END_REF][START_REF] Steinborn | Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts[END_REF]).

The functionality of several endogenous constitutive and inducible promoters (Supplementary Table S2), selection markers, and terminator elements (PHO5 terminator of S. cerevisiae, Kasprzak et al. 2016a;[START_REF] Steinborn | A novel vector element providing multicopy vector integration in Arxula adeninivorans[END_REF]) have been verified in B. raffinosifermentans. For example, the B. raffinosifermentans AHSB4 gene promoter, encoding histone H4, was uncovered to be strongly and constitutively expressed under salt-free and salt-stress conditions (Wartman et al. 2003). On the other hand, a constitutive promoter of TEF1 gene (encoding translation-elongation factor 1α) from B. raffinosifermentans that enables strong constitutive expression in various yeasts alongside genetic elements related to this gene was reported [START_REF] Rösel | Cloning and characterization of a TEF gene for elongation factor 1α from the yeast Arxula adeninivorans[END_REF][START_REF] Steinborn | Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts[END_REF][START_REF] Terentiev | A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements[END_REF]). The [START_REF] Liu | Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species[END_REF] demonstrated an intron in promoter's presence as relevant for improving targeted expression in R. toruloides. Indeed, the native DGA1 gene of R. toruloides was overexpressed using two strong promoters (LDPin and GPD1) and resulted in increased lipid content by 21% in R. toruloides with the LDP intron promoter (LDPin) than with GPD promoter. This is also 55% higher than that reported in the R. toruloides wild-type strain.

The other genetic element employed in the yeast system is the transcription terminator. Eukaryotic terminators are genetically encoded elements post-transcriptionally upregulated by factors associated with cis-elements (e.g., GUUCG/U) within the 3′ untranslated region (UTR) [START_REF] Ito | Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae[END_REF]. Both endogenous and heterologous terminators were either verified or reported functional in B. raffinosifermentans. For example, endogenous terminators ATRP1 have been applied to construct a selective marker module incorporated in the vector/ expression system Xplor®2 or multicopy integration experiments facilitating the insertion of a yeast rDNA integrative expression cassette (YRC) or a yeast integrative expression cassette (YIC) into the genome. It is worth noting that transformants are hardly recovered when transforming B. raffinosifermentans by circular YRC and YIC. Simultaneously, a linearized form of YIC generated even lower transformants than a linearized form of YRC (Böer et al. 2009a(Böer et al. , 2009d)).

Like promoters and terminators, selection markers are also valuable in genome engineering. Some publications charact e r i z e d t h e s u i t a b i l i t y o f i n d i v i d u a l g e n e s o f B. raffinosifermentans as auxotrophic markers. For instance, ILV1, from the LS3 strain, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase, has been shown useful as an auxotrophic selection marker for the transformation of plasmids into this yeast [START_REF] Wartmann | Comparative biochemical, genetical and immunological studies of glucoamylase producing Arxula adeninivorans yeast strains[END_REF][START_REF] Wartmann | AILV1 gene from the yeast Arxula adeninivorans LS3-a new selective transformation marker[END_REF]. Moreover, B. raffinosifermentans ALEU2, encoding Lisopropylmalate dehydrogenase and harboring an open reading frame of 1086 bp, was successfully employed as an auxotrophic marker (Wartmann et al. 2003). In the experiment validating its suitability to complement the auxotrophy of a B. raffinosifermentans aleu2 host, the plasmid pAL-ALEU2m containing ALEU2 as a selection marker and the 25S rDNA was used alongside the GFP gene, encoding an intracellular green fluorescent protein, and the HSA gene, encoding the secreted human serum albumin (Wartmann et al. 2003). The ATRP1 encoding a phosphoribosyl anthranilate isomerase in this yeast was also used as an auxotrophic marker in experiments focused on recombinant protein production [START_REF] Steinborn | Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts[END_REF]. Antibiotic resistance markers like the Escherichia coli-derived hph gene, encoding hygromycin B phosphotransferase have been used in several studies (Wartmann et al. 2003;[START_REF] Rösel | Integrative transformation of the dimorphic yeast Arxula adeninivorans LS3 based on hygromycin B resistance[END_REF].

Transformation methods of Blastobotrys yeast

Preparation of competent cells of B. raffinosifermentans LS3 employed sorbitol, bicine, NaOH, and PEG-1000. Indeed, after harvesting cells cultured in rich media and washing them with water, a suspension on bicine buffer (containing 1 M sorbitol, 10 mM bicine-NaOH/pH 8.35, 3% PEG 1000, 5% DMSO), followed by centrifugation and resuspension in the same buffer generate competent cells [START_REF] Kunze | Arxula adeninivorans[END_REF]. After that, the linearized plasmid DNA of interest is commonly used to transform B. raffinosifermentans LS3 competent cells.

Genome editing toolbox for Blastobotrys yeast

Lipid biogenesis in oleaginous yeasts

The lipogenesis mechanism in oleaginous yeasts is primarily based on the supply of acetyl-CoA and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a provider of the reducing power indispensable for fatty acid synthesis. The role of the pentose phosphate pathway (PPP) has been demonstrated in lipogenesis. Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) in the PPP provide NADPH for lipogenesis in oleaginous microbes (Chen et al. 2015a). The other known NADPH supplier for fatty acid synthesis in oleaginous microbe is the cytosolic MAE (Ratledge 2014). Furthermore, oleaginous yeasts exhibit different mechanisms for the conversion of glucose and acetate during lipogenesis. In the case of glucose as a substrate, depleted elements from the medium such as nitrogen [START_REF] Bandhu | Overproduction of single cell oil from xylose rich sugarcane bagasse hydrolysate by an engineered oleaginous yeast Rhodotorula mucilaginosa IIPL32[END_REF], phosphate (Wang et al. 2018a), or sulfur [START_REF] Wu | Microbial lipid production by Rhodosporidium toruloides under sulfatelimited conditions[END_REF]) usually trigger lipogenesis. [START_REF] Silverman | Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica[END_REF] summarized the biosynthesis of fatty acids in oleaginous yeasts. Nutrient depletion activates the adenosine monophosphate (AMP) deaminase and NADPH supply through the oxidative pentose phosphate pathway (oxPPP) or MAE. Increased AMP deaminase activity reduces the level of mitochondrial AMP, which reduces the activity of AMP-dependent isocitrate dehydrogenase in the TCA cycle, resulting in citrate accumulation. The excess mitochondrial citrate is transported into the cytosol to generate acetyl-CoA, which is the carbon skeleton required for fatty acid biosynthesis leading to the pool of different acyl-CoAs. De novo biosynthesis of TAGs is initiated via the Kennedy pathway, where the transfer of a fatty acyl group from acyl-CoA to glycerol-sn-3-phosphate (G3P) is catalyzed by glycerol-3-phosphate acyltransferase (GPAT) to form 1acylglycerol-sn-3-phosphate (lysophosphatidate or LPA). This step is followed by the acylation of LPA to form 1,2diacylglycerol-sn-3-phosphate (PA), dephosphorylation of PA to form diacylglycerol (DAG). In the final step, transfer of an acyl group on DAG under diacylglycerol acyltransferase (DGAT) activity constitutes TAG. These TAGs are generally stored in lipid bodies, which are cytoplasmic lipid-enriched organelles that originate and are closely related to the endoplasmic reticulum (ER), formed by the channeling of lipids and proteins from the ER toward newly formed lipid droplets [START_REF] Jackson | Lipid droplet biogenesis[END_REF]).

In the case of acetate as substrate, direct uptake through symport at higher pH [START_REF] Casal | Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae[END_REF] or passive diffusion at lower pH [START_REF] Augstein | Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica[END_REF] have been observed in yeasts. After entering the cell, dissociated acetate is converted to acetyl-CoA by the acetyl-CoA synthetase (ACS2), and the acetyl-CoA from the acetyl-CoA pool follows different pathways. It is used (1) for fatty acid synthesis through acetyl-CoA carboxylase (2) employed as a precursor for citrate formation in the mitochondrion and oxidized to carbon dioxide in the tricarboxylic acid (TCA) cycle to make energy (ATP) for the cell or (3) is converted in the glyoxylate cycle to succinate (de la Peña Mattozzi et al. 2010). Further, the ex novo pathway routinely activated in oleaginous yeasts in response to hydrophobic substrates should be noticed. It consisted of (a) the breakdown of hydrophobic compounds into free fatty acids outside the cell-mediated by lipase-catalyzed hydrolysis, (b) the transfer of free fatty acids inside the cell via transporters, and (c) the conversion of free fatty acids and their derivatives into short-chain of acyl-CoAs and acetyl-CoAs by acyl-CoA oxidases through β-oxidation [START_REF] Beopoulos | Yarrowia lipolytica: a model and a tool to understand the mechanisms implicated in lipid accumulation[END_REF].

A communication network between organelles has been noticed during lipid biogenesis. [START_REF] Valm | Applying systems-level spectral imaging and analysis to reveal the organelle interactome[END_REF] indicated that lipid metabolism is dispatched between the ER for lipid synthesis, LDs for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. Emerging evidence revealed beyond organelles' autonomous functions, a direct physical relationship between distinct organelles in eukaryotic cells. For example, the connection between LDs and peroxisomes is described by [START_REF] Joshi | Lipid droplet and peroxisome biogenesis occur at the same ER subdomains[END_REF]. They revealed that the biogenesis of LDs and peroxisome occurred at the same Pex30p subdomain. Wang and colleagues (2018b) also showed that the budding of both LDs and peroxisomes solicited the cooperation between Pex30 and the seipin complex or is inhibited otherwise. The connection between plasma membrane (PM) and ER can be seen through the yeast lipid transfer proteins (LTPs), Osh6p and Osh7p, that channel phosphatidylserine (PS) from the ER to PM via PS/phosphatidylinositol-4-phosphate (PI4P) exchange cycles. Giacomello and Pelligrini (2016) highlighted the motion of ions and lipid between mitochondria and the ER under the mediation of MERCs (Mitochondria-ER contacts). Other contact sites identified to date between cellular organelles are summarized in Fig. 3.

Overview of lipid biogenesis genes in Blastobotrys

The understanding and exploration of lipid metabolism in Blastobotrys species are in their infancy. The lack of knowledge on the regulation of lipid accumulation in these oleaginous yeasts hinders their development as an industrial oil platform. To date, only a few reports explored the role of the significant proteins/enzymes in the lipid biosynthetic pathway of Blastobotrys species, unlike the case of the well-established eukaryotic model for lipid production such as Y. lipolytica and Lipomyces starkeyi. Some of the genes described for the dimorphic B. adeninivorans are summarized in Fig. 4 due to the critical role in their expressed membrane-bound enzymes catalyzing the last step of TAG synthesis. [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF] conferred the oleaginous nature of this yeast by revealing lipid body formation along with the presence of two genes for ATP citrate lyase (ACL), suggesting their promising ability for the production of biofuel and lipids-derived compounds (Thomas 2019). Lipid bodies are essentially neutral lipid or TAG storage organelles having numerous physiological functions of the cell -including energy homeostasis, lipid metabolism, cellular toxins-trapping, and drug resistance [START_REF] Chang | Trapping toxins within lipid droplets is a resistance mechanism in fungi[END_REF]. So far, the lipogenic multienzyme complex consisting of ACL [START_REF] Kamineni | Engineering triacylglycerol production from sugars in oleaginous yeasts[END_REF], glycerol-3-phosphate acyltransferase (SCT1) [START_REF] Tsakraklides | Higholeate yeast oil without polyunsaturated fatty acids[END_REF], malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid (TCA) cycle [START_REF] Kabran | Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica[END_REF]), Dga1p encoded by DGA1 (ARAD1C08250g), and Dga2p encoded by DGA2 (ARAD1D42460g) have been elucidated in LS3 strain [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]). These enzymes have been positioned on Fig. 5, which highlights the key steps in FA and TAG biosynthesis pathways. The genome of B. adeninivorans supposedly contains two different genes for DGAT [START_REF] Thomas | Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars[END_REF][START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. For further insights into the acyltransferases involved in lipid metabolism, we have proposed 3D structures of both Dga1p and Dga2p of B. adeninivorans based on local homology-based structural modeling (Supplementary Fig. 1A and1B). Dga1p shared 13.74% sequence identity with 1-acyl-sn-glycerol-3-phosphate acyltransferase (PDB id 5kym.1) of Thermotoga maritima, which has been used as a template for Swiss-Model. The modeled structure highlighted the presence of α-helices in Dga1p (Supplementary Fig. 1A). For Dga2p, Swiss-Model predicted an α-helix (Supplementary Fig. 1B) sharing 33.82% sequence identity with human diacylglycerol O-acyltransferase 1 (PDB id 6vz1.1) that has been used as a template.

B. raffinosifermentans SCT1 encodes the glycerol-3phosphate acyl-transferase (GPAT) enzyme, which is known to attach the first fatty acid onto the glycerol backbone to produce LPA in the Kennedy pathway. SCT1 also maintains a high level of oleate in the cellular lipid composition in this yeast. When heterologously expressed in Y. lipolytica, it increased oleate level [START_REF] Tsakraklides | Higholeate yeast oil without polyunsaturated fatty acids[END_REF]. On the other hand, the inhibition of NAD + -dependent isocitrate dehydrogenase linked to the increased level of α-ketoglutaric acid, an intermediate in the TCA cycle, was demonstrated in B. adeninivorans VKM Y-2677 cells under thiamine limitation (Komzolova and Morgunov 2016). [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF] studied the diacylglycerol acyltransferases type I and type II of the membrane protein acyl-CoA: diacylglycerol acyltransferases (DGAT) in Blastobotrys species and identified them as essential factors in the last steps of TAG biosynthesis. Four types of diacylglycerol acyltransferases (DGATs) have been described hitherto in eukaryotes: the type 1 (DGAT1) and type 2 (DGAT2) belonging to the membranebound O-acyltransferase (MBOAT) family (Cases et al. 1998) and monoacylgycerol acyltransferase (MGAT) family, respectively, and the dual-specificity DGAT having both wax ester synthase (WS) and DGAT activities (Kalscheuer and Steinbuchel 2003) and a soluble cytosolic DGAT (DGAT3) [START_REF] Saha | Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase[END_REF]. Of note, the Dga1p enzyme encoded by the DGA1 gene belonging to the DGAT2 family plays a significant role in TAG biosynthesis in Y. lipolytica [START_REF] Haïli | Expression of soluble forms of yeast diacylglycerol acyltransferase 2 that integrate a broad range of saturated fatty acids in triacylglycerols[END_REF]. The study of the lipid metabolism pathway of B. raffinosifermentans (LS3 strain) highlighted the prominent role of the DGAT1 family member gene (DGA2). In contrast, the DGAT2 family member gene (DGA1) had little influence on the TAG synthesis [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. To understand the phylogenetic relationships among the DGA1 and DGA2 encoded proteins expressed in B. raffinosifermentans and the other well-known DGAT proteins described in the literature, DGAT proteins employed by Chen et al. (2015b) alongside other DGATs retrieved from the NCBI platform (http://www. ncbi.nlm.nih.gov/gorf/gorf.html) including those from Rhodotorula toruloides and Lipomyces starkeyi have been used to construct a phylogenetic tree. This analysis revealed that the targeted diacylglycerol acyltransferases of B. raffinosifermentans are clustered next to Y. lipolytica and away from Lipomyces starkeyi and Rhotorula turoloides (Fig. 6).

Furthermore, [START_REF] Stoltenburg | Molecular cloning and expression of the ARFC3 gene, a component of the replication factor C from the salt-tolerant, dimorphic yeast Arxula adeninivorans LS3[END_REF] identified the gene ARFC3, encoding one subunit of the replication factor C (RF-C ) c o m p l e x , o n c h r o m o s o m e 1 o f t h e f o u r B. raffinosifermentans chromosomes. B. raffinosifermentans appeared not to be an obligate aerobic oleaginous yeast as Sędzielewska et al. identified and characterized the gene encoding cytosolic fumarate reductase, the enzyme mediating the maintenance of redox balance through the regeneration of reduced cofactors during hypoxic growth conditions, and its promoter and terminator elements [START_REF] Sędzielewska | Role of the AFRD1-encoded fumarate reductase in hypoxia and osmotolerance in Arxula adeninivorans[END_REF].

Strategies for improving lipid production in oleaginous yeasts

Several strategies, including low-cost substrate utilization combined with genetic engineering and mutagenesis, have been exploited to reach a higher yield or titer of lipid production in several species of oleaginous yeasts. The ability to metabolize pentose (xylose) or/and hexose (glucose) sugars derived from lignocellulosic biomass has been widely investigated [START_REF] Poontawee | Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production[END_REF][START_REF] Meesters | Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as the sole source of carbon, nitrogen and energy[END_REF][START_REF] Yamada | Evaluation of lipid production from xylose and glucose/xylose mixed sugar in various oleaginous yeasts and improvement of lipid production by UV mutagenesis[END_REF][START_REF] Chattopadhyay | Efficient xylose utilization leads to highest lipid productivity in Candida tropicalis SY005 among six yeast strains grown in mixed sugar medium[END_REF]. Other examples include Cryptococcus terricola with a lipid content of 61.96% DCW on medium with 5% starch [START_REF] Tanimura | Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing[END_REF], Apiotrichum porosum DSM27194 with 44.3% lipid content on volatile fatty acids from waste organics [START_REF] Qian | Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Apiotrichum porosum DSM27194[END_REF], and Cystobasidium oligophagum with 41.54% lipid on starchy materials without the use of amylolytic enzymes [START_REF] Vyas | Isolation, identification and characterization of Cystobasidium oligophagum JRC1: A cellulase and lipase producing oleaginous yeast[END_REF]. The natural ability of specific yeasts to grow and accumulate lipid utilizing hydrophobic substrates has been of research interest, such as in Cryptococcus curvatus with 69.14% DCW of lipids [START_REF] Patel | A comparative study on de novo and ex novo lipid fermentation by oleaginous yeast using glucose and sonicated waste cooking oil[END_REF]. Studies also reported specific oleaginous yeasts' capacity to simultaneously use several carbon sources [START_REF] Tanimura | Lipid production through simultaneous utilization of glucose, xylose, and L-arabinose by Pseudozyma hubeiensis: a comparative screening study[END_REF][START_REF] Anschau | Effect of feeding strategies on lipid production by Lipomyces starkeyi[END_REF][START_REF] Juanssilfero | Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi[END_REF][START_REF] Yamada | Evaluation of lipid production from xylose and glucose/xylose mixed sugar in various oleaginous yeasts and improvement of lipid production by UV mutagenesis[END_REF], alongside the flocculation capacity [START_REF] Sitepu | Oleaginous yeasts for biodiesel: current and future trends in biology and production[END_REF]. A yeast species' ability to consume sugars withstanding the inhibitory effect of chemicals is also of utmost importance as microbial behavior changes accordingly. For instance, the oleaginous yeast L. starkeyi reaches 68.24% of its DCW in lipids while supporting a mixture of the following growth inhibitors: 5hydroxymethylfurfural, furfural, acetic acid, formic acid, acid levulin, vanillin, and syringaldehyde [START_REF] Juanssilfero | Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi[END_REF].

Researchers have designed constructs mostly to overexpress certain enzymes of the pathway to engineer further the strains for a higher amount of metabolite production or maneuver the carbon flux towards value-added molecule production. Overexpression is commonly carried out by integrating an additional gene copy under a strong promoter [START_REF] Delic | Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion[END_REF]. [START_REF] Kamineni | Engineering triacylglycerol production from sugars in oleaginous yeasts[END_REF] suggested three fundamental strategies to increase TAG biosynthesis in yeast: increase the metabolic flux rate from sugar to TAG, re-design lipid pathway stoichiometry for more efficient conversion of sugar's carbon atoms and electrons into TAG, and eliminate competing by-products. Over the years, researchers have documented that the metabolic engineering for lipid production involve overexpression of enzymes of the fatty acid biosynthesis pathway/TAG biosynthesis pathway, regulation of the pathways associated with the TAG biosynthesis, TAG degradation, lipid droplet formation and lipid droplet degradation, partial blockage of competing for metabolic pathways, and the "multigene" transgenic approach to achieve these objectives [START_REF] Xie | Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production[END_REF][START_REF] Liu | Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species[END_REF][START_REF] Ledesma-Amaro | Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose[END_REF][START_REF] Wang | Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica-a review[END_REF].

As previously mentioned, the DGAT enzyme plays a crucial role in TAG biosynthesis. Hence, the molecular understanding of the genes' regulatory mechanism from the DGAT family appeared to be critical for their genetic manipulation to increase lipid production in oleaginous yeasts [START_REF] Haïli | Expression of soluble forms of yeast diacylglycerol acyltransferase 2 that integrate a broad range of saturated fatty acids in triacylglycerols[END_REF][START_REF] Xue | Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica[END_REF]. Changes in gene expression from the growth phase to the lipid biosynthesis phase in oleaginous yeasts and gene expression analysis of mutants with increased lipid productivity have revealed the expression of genes important for high lipid production. DGAT overexpression has already been performed recently to get insight into the lipid metabolism of B. adeninivorans and B. raffinosifermentans species [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. Overexpression of native DGAT1 in B. raffinosifermentans increased lipid production (26.5% DCW) about 1.6-fold relative to the wild-type strain (LS3, 16.9% DCW) when cultivated on glucose [START_REF] Sanya | The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed[END_REF]. Overexpression or deletions of acetyl-CoA carboxylase, another key enzyme of the fatty acid biosynthetic pathway, has also been attempted [START_REF] Tamano | Increased production of fatty acids and triglycerides in Aspergillus oryzae by enhancing expressions of fatty acid synthesis-related genes[END_REF], along with overexpression of glycerol-3phosphate dehydrogenase [START_REF] Beopoulos | Control of lipid accumulation in the yeast Yarrowia lipolytica[END_REF]. Knowing that palmitoleic acid and oleic acid resulted from Δ9 fatty acid desaturases activities in oleaginous yeasts and they have been disclosed in the lipid profile of the yeasts of Blastobotrys genus, overexpression of this desaturase can be envisioned to enhance the cellular lipid content as it was reported from Rhodosporidium toruloides [START_REF] Tsai | Delta-9 fatty acid desaturase overexpression enhanced lipid production and oleic acid content in Rhodosporidium toruloides for preferable yeast lipid production[END_REF]. Promising approaches to improving intracellular lipid content may also exploit the repertoire of well-established genes involved in glycolysis, β-oxidation cycle, central metabolism, and lipid metabolism that is routinely reported in oleaginous yeasts (e.g., Yarrowia lipolytica, Rhodosporidium toruloides, [START_REF] Beopoulos | Control of lipid accumulation in the yeast Yarrowia lipolytica[END_REF][START_REF] Zhu | A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides[END_REF]. For example, to elucidate the relevance of the metabolic circuits associated with the enhancement of lipids in Rhodosporidium toruloides, Castañeda and colleagues performed the deletion of the genes expressing proteins involved in the central nitrogen metabolism (Glutamate dehydrogenase), the tricarboxylic acid (TCA) cycle (Isocitrate dehydrogenase), and glycolysis (Glucose-6-phosphate isomerase) [START_REF] Castañeda | In silico optimization of lipid production in Rhodosporidium toruloides by gene knockout strategies*. This work was supported by funding projects from Agencia Na-cional de Promocion Científica y Tecnologica (PICT 2014-2394, 2016-2258[END_REF]. On the other hand, [START_REF] Yuzbasheva | Co-expression of glucose-6-phosphate dehydrogenase and acyl-CoA binding protein enhances lipid accumulation in the yeast Yarrowia lipolytica[END_REF] co-expressed the gene encoding NADP + -dependent glucose-6-phosphate dehydrogenase (ZWF1) and that encoding acyl-CoA binding protein (ACBP) to increase the lipid content in Y. lipolytica, while Wang et al. (2018c) improved lipid content in Y. lipolytica by inactivating MHY1 gene encoding Mhy1p that exerts regulatory functions on lipid biosynthesis, amino acid, and nitrogen metabolism and cell cycle. The inactivation of SCT1 as a potent way to boost fatty acyl pool can be envisioned in Blastobotrys genus. A similar experiment in Y. lipolytica contributes to the enhancement of the fatty acid ethyl esters pool identified as a promising renewable alternative for biodiesel [START_REF] Ng | Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil[END_REF]). Breeding using mutagen has been used efficiently for improving lipid production in oleaginous yeasts [START_REF] Liu | An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica[END_REF][START_REF] Yamada | Evaluation of lipid production from xylose and glucose/xylose mixed sugar in various oleaginous yeasts and improvement of lipid production by UV mutagenesis[END_REF]. For example, [START_REF] Takaku | Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation[END_REF] applied ultraviolet treatment to generate Lipomyces starkeyi mutant capable of producing higher lipids compared to the wild-type. They also identified the increased expression of acyl-CoA synthesis-and Kennedy pathwayrelated genes during lipogenesis. On the other hand, [START_REF] Yamazaki | Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol[END_REF] demonstrated that Percoll density gradient centrifugation is an important method recommended for the identification of high-accumulating TAG mutant strains.

Future directions and conclusion

System-level metabolic engineering that highlights the key signaling pathways regulating lipid biosynthesis is necessary to harvest the potential of Blastobotrys strains for developing these yeasts as an industrial platform for lipid production. Albeit the genetic parts uncovered in this review, more sophisticated genome editing methods should be designed and evaluated. Further detailed analyses of the deletion or knockout strategies to validate the role of the genes involved in circuits interfering with the lipid metabolism pathway will provide a novel insight into each gene's relative importance in the lipid biosynthesis pathway. The study of the biochemical role of lipid mediators associated with fluxomics, metabolomics, proteomics, and computational methods can help design a suitable strategy for improving the intracellular lipid content. These strategies could further pave new directions for genetic improvement tailoring fatty acid synthesis with immense food, healthcare, and biofuel industries. A co-cultivation system involving the yeast species of this genus with other oleaginous yeasts can also be envisioned.

Fig. 1

 1 Fig. 1 Taxonomy of Blastobotrys adeninivorans inspired from Gellissen et al. (2005) and Kunze et al. 2014

  S a c c h a r o m y c e t e s , C y s t o b a s i d i o m y c e t e s , M i c r o b o t r y o m y c e t e s , T r e m e l l o m y c e t e s , and Ustilaginomycetes (Sitepu et al. 2014).

Fig. 2

 2 Fig.2Hallmarks of key events concerning Blastobotrys genus. Plasmids (red), other events (darkish), whole genome sequencing (brown), biofuels and industrial potential (yellow), and first species classification (blue)

Fig. 3

 3 Fig.3A schematic diagram showing the names of identified interorganelle contact sites. Junction names are written in green. Single protein or protein complex involved in the junction that tethers two organelles in yeast is written in blue. PM (plasma membrane), ER (endoplasmic reticulum), LD (lipid droplet). vCOuPLE (vacuole-plasma membrane contact), pCLIP (for plasma membrane contact with lipid droplets), PerPECs (for peroxisome plasma membrane contact site), PerVale (for peroxisome vacuole contact), NVJ (nuclear-vacuole junction), MAM (mitochondria-associated ER membranes), vCLAMP (vacuole and

Fig. 4 Fig. 5

 45 Fig. 4 Endogenous genes described for the dimorphic yeast Blastobotrys adeninivorans. Genes involved in the same pathway are highlighted in the same colors. The other colors are retained for the genes acting on different pathways

Table 1

 1 Lipid production by oleaginous yeasts cultivated on various carbon sources in shake flask cultures and their comparison with the present study

	Oleaginous yeast wild-type	Carbon sources	Lipid content	Temperature	References
			(% dry weight)
	Blastobotrys adeninivorans LS3 strain vs.	Glucose	34.70%	28 °C, 37 °C	Thomas et al. (2019)
	other oleaginous yeasts	Xylose	˃20%
		Arabinose	30.10%
		Soluble starch	22.00%
		Glycerol	16.10%
	Cryptococcus terricola	Soluble starch	26.78-36.10%	25 °C	Tanimura et al. (2014)
	Lipomyces starkeyi 2512	Glucose	54.22%	25 °C	Shapaval et al. (2019)
		Xylose	52.19%
	Rhodotorula babjevae DBVPG 8058	Glucose	54.40%	25 °C	Shapaval et al. (2019)
		Xylose	23.61%
	Pseudozyma hubeiensis IPM1-10	Glucose	21.61%	28 °C	Tanimura et al. (2016)
		Xylose	24.59%
		Arabinose	17.26%
	Prunus domestica	Glucose	15.40%	28 °C	Maina et al. (2017)
		Xylose	23.20%
		Arabinose	14.20%
		Galactose	21.40%
	Rhodotorula babjevae Y-SL7	Glucose	39.50%	26 °C	Guerfali et al. (2019)
		Xylose	30.09%
		Arabinose	23.90%
		Glycerol	29.80%
	Cutaneotrichosporon oleaginosus	Glucose	33.78%	28 °C	Awad et al. (2019)
		Xylose	13.48%
		Arabinose	41.66%
		Galactose	19.15%
	Lipomyces starkeyi	Glucose	79.60%	30 °C	Juanssilfero et al. (2018)
		Xylose	36.80%	28 °C	Xavier et al. (2017)
		Glycerol	35.70%	30 °C	Liu et al. (2017)
	Rhodotorula toruloides	Glucose	47.51%	25 °C	Tiukova et al. (2019)
		Xylose	10.70-29.10%	28 °C	Xenopoulos et al. (2020)
		Glycerol	40.03-42.50%	30 °C	Yang et al. (2014)
	Rhodotorula glutinis	Glucose	9.35-47.24%	25 °C	Maza et al. (2020)
		Xylose	12.60-33.10%	28 °C	Xenopoulos et al. (2020)
		Glycerol	10.20-12.7%	28 °C	Kot et al. (2017)
	Yarrowia lipolytica	Glycerol	43.50%	30 °C	Sara et al. (2016)
		Glucose	18.00%	28 °C	Ledesma-amaro et al. (2016)

Acknowledgments Authors acknowledge Hiroko KITAMOTO, Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organisation, Tsukuba, Ibaraki, Japan, and Zongbao Kent Zhao, Laboratory of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023 People's Republic of China and Mahesh Khot from FONDECYT (Fondo Nacional de Desarrollo Científico y Tecnológico/National Fund for Scientific and Technological Development), Government of Chile, Chile, Santiago in Postdoctoral Project Grant context (No. 3180134), for proofreading this review document.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s00253-021-11354-3.

Author contribution DRAS has prepared manuscript, performed literature analysis, and prepared tables and figures. DO, VP, MKM, AC, and MBK have revised the manuscript. All authors read and approved the final manuscript.

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