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.

Comparative Analysis of the Alkaline Proteolytic Enzymes of Yarrowia Clade Species and Their Putative Applications.

Proteolytic enzymes are commercially valuable and have multiple applications in various industrial sectors. The most studied proteolytic enzymes produced by Yarrowia lipolytica, extracellular alkaline protease (Aep) and extracellular acid protease (Axp), were shown to be good candidates for different biotechnological applications. In this study, we performed a comprehensive analysis of the alkaline proteolytic enzymes of Yarrowia clade species, including phylogenetic studies, synteny analysis, and protease production and application. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we reconstructed the evolutionary scenario of the XPR2 gene for species of the Yarrowia clade. Furthermore, except for the proteolytic activity of the analyzed Yarrowia clade strains, the brewers' spent grain (BSG) was used as a substrate to obtain protein hydrolysates with antioxidant activity. For each culture, the degree of hydrolysis was calculated. The most efficient protein hydrolysis was observed in the cultures of Y. lipolytica, Y. galli, and Y. alimentaria. In contrast, the best results obtained using the 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) method were observed for the culture medium after the growth of Y. divulgata, Y. galli, and Y. lipolytica on BSG.

High value-added products derived from crude glycerol via microbial fermentation using Yarrowia clade yeast.

BACKGROUND: Contemporary biotechnology focuses on many problems related to the functioning of developed societies. Many of these problems are related to health, especially with the rapidly rising numbers of people suffering from civilization diseases, such as obesity or diabetes. One factor contributing to the development of these diseases is the high consumption of sucrose. A very promising substitute for this sugar has emerged: the polyhydroxy alcohols, characterized by low caloric value and sufficient sweetness to replace table sugar in food production. RESULTS: In the current study, yeast belonging to the Yarrowia clade were tested for erythritol, mannitol and arabitol production using crude glycerol from the biodiesel and soap industries as carbon sources. Out of the 13 tested species, Yarrowia divulgata and Candida oslonensis turned out to be particularly efficient polyol producers. Both species produced large amounts of these compounds from both soap-derived glycerol (59.8-62.7 g dm-3) and biodiesel-derived glycerol (76.8-79.5 g dm-3). However, it is equally important that the protein and lipid content of the biomass (around 30% protein and 12% lipid) obtained after the processes is high enough to use this yeast in the production of animal feed. CONCLUSIONS: The use of waste glycerol for the production of polyols as well as utilization of the biomass obtained after the process for the production of feed are part of the development of modern waste-free technologies.

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.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement.

Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.