Biotechnological approaches for producing natural pigments in yeasts.

Pigments are widely used in the food, cosmetic, textile, pharmaceutical, and materials industries. Demand for natural pigments has been increasing due to concerns regarding potential health problems and environmental pollution from synthetic pigments. Microbial production of natural pigments is a promising alternative to chemical synthesis or extraction from natural sources.

Broadening the application of synthetic biology tools to explore the potential of 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.

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

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 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.

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.

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.

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.

A Strain Engineered for Pyomelanin Production.

The yeast 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.

In this study, we have adopted Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica. To this end, a broad set of destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen elements.

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.

Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth.

Sugar assimilation has been intensively studied in the model yeast S. cerevisiae, and for two decades, it has been clear that the homologous HXT genes, which encode a set of hexose transporters, play a central role in this process. However, in the yeast Yarrowia lipolytica, which is well-known for its biotechnological applications, sugar assimilation is only poorly understood, even though this yeast exhibits peculiar intra-strain differences in fructose uptake: some strains (e.

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production.

Background: Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment.

Overexpression of diacylglycerol acyltransferase in Yarrowia lipolytica affects lipid body size, number and distribution.

In the oleaginous yeast Yarrowia lipolytica, the diacylglycerol acyltransferases (DGATs) are major factors for triacylglycerol (TAG) synthesis. The Q4 strain, in which the four acyltransferases have been deleted, is unable to accumulate lipids and to form lipid bodies (LBs). However, the expression of a single acyltransferase in this strain restores TAG accumulation and LB formation.