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.

Purification of Natural Pigments Violacein and Deoxyviolacein Produced by Fermentation Using Yarrowia lipolytica.

Violacein and deoxyviolacein are bis-indole pigments synthesized by a number of microorganisms. The present study describes the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis, the subsequent extraction of the intracellular pigments, and ultimately their purification using column chromatography. The results show that the optimal separation between the pigments occurs using an ethyl acetate/cyclohexane mixture with different ratios, first 65:35 until both pigments were clearly visible and distinguishable, then 40:60 to create a noticeable separation between them and recover the deoxyviolacein, and finally 80:20, which allows the recovery of the violacein. The purified pigments were then analyzed by thin-layer chromatography and nuclear magnetic resonance.

A yeast-based tool for screening mammalian diacylglycerol acyltransferase inhibitors.

Dysregulation of lipid metabolism is associated with obesity and metabolic diseases but there is also increasing evidence of a relationship between lipid body excess and cancer. Lipid body synthesis requires diacylglycerol acyltransferases (DGATs) which catalyze the last step of triacylglycerol synthesis from diacylglycerol and acyl-coenzyme A. The DGATs and in particular DGAT2, are therefore considered potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGAT activities, to evaluate the functionality of the enzymes in this heterologous host and DGAT activity inhibitors. This work provides evidence that mammalian DGATs expressed in Y. lipolytica are a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest.

Golden Gate Multigene Assembly Method for Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica has emerged as a powerful alternative for biolipid production due to its high capacity for lipid accumulation. Genetic engineering and synthetic biology are promoted forward to improve production and reroute metabolism for high-value compound synthesis. In this context, efficient, modular, and high-throughput compatible cloning and expression system are required to speed up and rationalize research in this field. Here, we present the fast and modular Golden Gate cloning strategy for the construction of multigene expression vectors and their transformation into Y. lipolytica. As an example, we used the heterologous expression of the carotenoid pathway by cloning three genes involved in this pathway in only one vector allowing reaching production of ß-carotene after a single transformation.

Bioproduction of 2-Phenylethanol through Yeast Fermentation on Synthetic Media and on Agro-Industrial Waste and By-Products: A Review.

Due to its pleasant rosy scent, the aromatic alcohol 2-phenylethanol (2-PE) has a huge market demand. Since this valuable compound is used in food, cosmetics and pharmaceuticals, consumers and safety regulations tend to prefer natural methods for its production rather than the synthetic ones. Natural 2-PE can be either produced through the extraction of essential oils from various flowers, including roses, hyacinths and jasmine, or through biotechnological routes. In fact, the rarity of natural 2-PE in flowers has led to the inability to satisfy the large market demand and to a high selling price. Hence, there is a need to develop a more efficient, economic, and environmentally friendly biotechnological approach as an alternative to the conventional industrial one. The most promising method is through microbial fermentation, particularly using yeasts. Numerous yeasts have the ability to produce 2-PE using l-Phe as precursor. Some agro-industrial waste and by-products have the particularity of a high nutritional value, making them suitable media for microbial growth, including the production of 2-PE through yeast fermentation. This review summarizes the biotechnological production of 2-PE through the fermentation of different yeasts on synthetic media and on various agro-industrial waste and by-products.

Droplet-Based Microfluidic High-Throughput Screening of Enzyme Mutant Libraries Secreted by Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost.Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.

A Yarrowia lipolytica Strain Engineered for Pyomelanin Production.

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway.

Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast's potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback-insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l-1 in minimal growth medium), which represented a 93-fold increase compared to the wild-type strain (0.0015 g l-1 ). Production was further boosted to 0.48 g l-1 when glycerol, a low-cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l-1 ). Among these metabolites, 2-phenylethanol is of particular interest due to its rose-like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan. We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio-production of high-value aromatic compounds, and we have greatly improved our understanding of the potential feedback-based regulation of the shikimate pathway in this yeast.

Ethylzingerone, a Novel Compound with Antifungal Activity.

Preservatives increase the shelf life of cosmetic products by preventing growth of contaminating microbes, including bacteria and fungi. In recent years, the Scientific Committee on Consumer Safety (SCCS) has recommended the ban or restricted use of a number of preservatives due to safety concerns. Here, we characterize the antifungal activity of ethylzingerone (hydroxyethoxyphenyl butanone [HEPB]), an SCCS-approved new preservative for use in rinse-off, oral care, and leave-on cosmetic products. We show that HEPB significantly inhibits growth of Candida albicans, Candida glabrata, and Saccharomyces cerevisiae, acting fungicidally against C. albicans Using transcript profiling experiments, we found that the C. albicans transcriptome responded to HEPB exposure by increasing the expression of genes involved in amino acid biosynthesis while activating pathways involved in chemical detoxification/oxidative stress response. Comparative analyses revealed that C. albicans phenotypic and transcriptomic responses to HEPB treatment were distinguishable from those of two widely used preservatives, triclosan and methylparaben. Chemogenomic analyses, using a barcoded S. cerevisiae nonessential mutant library, revealed that HEPB antifungal activity strongly interfered with the biosynthesis of aromatic amino acids. The trp1Δ mutants in S. cerevisiae and C. albicans were particularly sensitive to HEPB treatment, a phenotype rescued by exogenous addition of tryptophan to the growth medium, providing a direct link between HEPB mode of action and tryptophan availability. Collectively, our study sheds light on the antifungal activity of HEPB, a new molecule with safe properties for use as a preservative in the cosmetic industry, and exemplifies the powerful use of functional genomics to illuminate the mode of action of antimicrobial agents.

Fermentation process for producing CFAs using Yarrowia lipolytica.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43 mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06 g L-1) after 72 h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56 g L-1). After 165 h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3 mg L-1 h-1) was maximal at 72.5 h of cultivation.

Transforming Candida hispaniensis, a promising oleaginous and flavogenic yeast.

Candida hispaniensis is an oleaginous yeast with a great potential for production of single cell oil according to its naturally high lipid accumulation capacity. Its unusual small genome size trait is also attractive for fundamental research on genome evolution. Our physiological study suggests a great potential for lipid production, reaching 224 mg/g of cell dry weight in glucose minimum medium. C. hispaniensis is also able to secrete up to 34.6 mg/L of riboflavin promising further riboflavin production improvements by cultivation optimization and genetic engineering. However, while its genome sequence has been released very recently, no genetic tools have been described up to now for this yeast limiting its use for fundamental research and for exploitation in an industrial biotechnology. We report here the first genetic modification of C. hispaniensis by introducing a heterologous invertase allowing the growth on sucrose using a biolistic transformation approach using a dedicated vector. The first genetic tool and transformation method developed here appear as a proof of concept, and while it would benefit from further optimization, heterogeneous expression of invertase allows for metabolism of an additional sugar and shows heterologous enzyme production capacity.

A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity.

OBJECTIVES: The construction and validation of a set of Yarrowia lipolytica CRISPR/Cas9 vectors containing six different markers that allows virtually any genetic background to be edited, including those of wild-type strains. RESULTS: Using the Golden Gate method, we assembled a set of six CRISPR/Cas9 vectors, each containing a different selection marker, to be used for editing the genome of the industrial yeast Y. lipolytica. This vector set is available via Addgene. Any guide RNA (gRNA) sequence can be easily and rapidly introduced in any of these vectors using Golden Gate assembly. We successfully edited six different genes in a variety of genetic backgrounds, including those of wild-type strains, with five of the six vectors. Use of these vectors strongly improved homologous recombination and cassette integration at a specific locus. CONCLUSIONS: We have created a versatile and modular set of CRISPR/Cas9 vectors that will allow any Y. lipolytica strain to be rapidly edited; this tool will facilitate experimentation with any prototroph wild-type strains displaying interesting features.

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology.

The oleaginous yeast Yarrowia lipolytica is an established host for the bio-based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one-step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine-tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic-resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three-gene pathway allowing xylose utilization by Y. lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. lipolytica strains and is available to the community through Addgene.

Selection of Heterologous Protein-Producing Strains in Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via a thermal cycler-based microassay.

Optimization of cyclopropane fatty acids production in Yarrowia lipolytica.

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long-term resistance to oxidization and low-temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large-scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed-batch fermentation set-up enable us to reach a specific productivity of 0.032 g·L-1 ·hr-1 with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.

Engineering the architecture of erythritol-inducible promoters for regulated and enhanced gene expression in Yarrowia lipolytica.

The non-conventional model yeast Yarrowia lipolytica is of increasing interest as a cell factory for producing recombinant proteins or biomolecules with biotechnological or pharmaceutical applications. To further develop the yeast's efficiency and construct inducible promoters, it is crucial to better understand and engineer promoter architecture. Four conserved cis-regulatory modules (CRMs) were identified via phylogenetic footprinting within the promoter regions of EYD1 and EYK1, two genes that have recently been shown to be involved in erythritol catabolism. Using CRM mutagenesis and hybrid promoter construction, we identified four upstream activation sequences (UASs) that are involved in promoter induction by erythritol. Using RedStarII fluorescence as a reporter, the strength of the promoters and the degree of erythritol-based inducibility were determined in two genetic backgrounds: the EYK1 wild type and the eyk1Δ mutant. We successfully developed inducible promoters with variable strengths, which ranged from 0.1 SFU/h to 457.5 SFU/h. Erythritol-based induction increased 2.2 to 32.3 fold in the EYK1 + wild type and 2.9 to 896.1 fold in the eyk1Δ mutant. This set of erythritol-inducible hybrid promoters could allow the modulation and fine-tuning of gene expression levels. These promoters have direct applications in protein production, metabolic engineering and synthetic biology.

Generating genomic platforms to study Candida albicans pathogenesis.

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

Overexpression screen reveals transcription factors involved in lipid accumulation in Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional oleaginous yeast that displays high lipid titers and yields; its production capacity holds significant promise for industrial biolipid applications. While its lipid metabolism has been widely studied, little is known about its transcriptional regulatory network. Deciphering the role of transcriptional regulators is crucial for understanding lipid accumulation, a complex phenomenon. To identify the transcription factors involved in lipid metabolism, we developed a systematic overexpression approach for 148 putative transcription factors. Analyses of overexpressing transformants revealed that 38 had an impact on lipid accumulation under at least one of the growth conditions tested. For most of these factors, our results provide the first experimentally determined functional annotation. Our data suggest that the regulation network differs depending on the carbon source, which is critical information when carrying out industrial bioprocesses. These results will therefore help guide further rational metabolic engineering for improving biolipid production by Y. lipolytica. Moreover, this work has created the largest collection of Y. lipolytica overexpressing strains to date, which will be useful in phenotype screening.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

BACKGROUND: Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. RESULTS: Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-ß-1,4-xylanase B and C; 1,4-ß-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-ß-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). CONCLUSIONS: Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.