Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases.

BACKGROUND: Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica. RESULTS: The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y. lipolytica mimicking cocoa butter. This was accomplished by replacing the native Δ9 fatty acid desaturase (Ole1p) with homologs from other species and changing the expression of both Ole1p and the Δ12 fatty acid desaturase (Fad2p). We thereby abolished the palmitoleic acid and reduced the linoleic acid content in TAG, while the oleic acid content was reduced to approximately 40 percent of the total fatty acids. The proportion of fatty acids in TAG changed dramatically over time during growth, and the fatty acid composition of TAG, free fatty acids and phospholipids was found to be very different. CONCLUSIONS: We show that the fatty acid profile in the TAG of Y. lipolytica can be altered to mimic cocoa butter. We also demonstrate that a wide range of fatty acid profiles can be achieved while maintaining good growth and high lipid accumulation, which, together with the ability of Y. lipolytica to utilize a wide variety of carbon sources, opens up the path toward sustainable production of CBE and other food oils.

Expression Profile of Selected Genes Involved in Storage Lipid Synthesis in a Model Oleaginous Yeast Species Yarrowia lipolytica.

Yarrowia lipolytica yeast is a model species of the group of oleaginous microorganisms capable of intracellular lipids accumulation in an amount exceeding 20% of the dry mass. Single cell oil biosynthesis can follow one of two biochemical pathways-de novo accumulation of cellular lipids in medium containing non-lipid carbon sources (including saccharides, glycerol) and ex novo microbial oil synthesis which involves fatty acids uptake from the environment. The mRNA expression of selected genes of de novo and ex novo lipid synthesis pathways was analyzed and correlated with the phenotypically observed features. It was proved that the accumulation yield of storage lipids via ex novo pathway was to some extent dependent on the limitation of the nitrogen source in the medium. It was also proposed that the synthesis of intracellular lipids in lipid-rich medium proceeded mainly via ex novo pathway, although the activity of genes encoding the enzymes of the de novo pathway were not completely inhibited at the stage of transcription by fatty acids present in the medium (e.g., ATP-citrate lyase). Molecular markers of two biosynthesis routes has been outlined and a hypothetical connection point between de novo and ex novo route were indicated.

The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein.

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.

A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

Enhanced ß-carotene production by overexpressing the DID2 gene, a subunit of ESCRT complex, in engineered Yarrowia lipolytica.

OBJECTIVE: ß-Carotene has been widely used in the food and feed industry and has significant commercial value. This study aimed to increase the ß-carotene production in engineered Yarrowia lipolytica by optimizing the host metabolic network. The DID2 gene, a subunit of the endosomal sorting complex required for transport (ESCRT), was integrated into a ß-carotene producing strain. RESULTS: The ß-carotene production was increased by 260%, and the biomass increased by 10% for engineered Y. lipolytica. Meanwhile, DID2 elevated the mRNA level and protein level of the genes in the ß-carotene synthesis pathway, then increased precursors (FPP, Lycopene) utilization. DID2 also increased the mRNA level of the genes in the glucose pathway, pentose phosphate pathway, and tricarboxylic acid cycle and promoted glucose utilization and cofactors consumption. CONCLUSION: The ESCRT protein complex subunit, DID2, improved ß-carotene production in engineered Y. lipolytica and beneficial to glucose utilization and cofactors consumption. This study provided new finding of the DID2 gene's function and it mostly could be used for many other natural product productions.

Systems-level approaches for understanding and engineering of the oleaginous cell factory Yarrowia lipolytica.

Concerns about climate change and the search for renewable energy sources together with the goal of attaining sustainable product manufacturing have boosted the use of microbial platforms to produce fuels and high-value chemicals. In this regard, Yarrowia lipolytica has been known as a promising yeast with potentials in diverse array of biotechnological applications such as being a host for different oleochemicals, organic acid, and recombinant protein production. Having a rapidly increasing number of molecular and genetic tools available, Y. lipolytica has been well studied amongst oleaginous yeasts and metabolic engineering has been used to explore its potentials. More recently, with the advancement in systems biotechnology and the implementation of mathematical modeling and high throughput omics data-driven approaches, in-depth understanding of cellular mechanisms of cell factories have been made possible resulting in enhanced rational strain design. In case of Y. lipolytica, these systems-level studies and the related cutting-edge technologies have recently been initiated which is expected to result in enabling the biotechnology sector to rationally engineer Y. lipolytica-based cell factories with favorable production metrics. In this regard, here, we highlight the current status of systems metabolic engineering research and assess the potential of this yeast for future cell factory design development.

The pH-Responsive Transcription Factors YlRim101 and Mhy1 Regulate Alkaline pH-Induced Filamentation in the Dimorphic Yeast Yarrowia lipolytica.

Environmental pH influences cell growth and differentiation. In the dimorphic yeast Yarrowia lipolytica, neutral-alkaline pH strongly induces the yeast-to-filament transition. However, the regulatory mechanism that governs alkaline pH-induced filamentation has been unclear. Here, we show that the pH-responsive transcription factor Y. lipolytica Rim101 (YlRim101) is a major regulator of alkaline-induced filamentation, since the deletion of YlRIM101 severely impaired filamentation at alkaline pH, whereas the constitutively active YlRIM1011-330 mutant mildly induced filamentation at acidic pH. YlRim101 controls the expression of the majority of alkaline-regulated cell wall protein genes. One of these, the cell surface glycosidase gene YlPHR1, plays a critical role in growth, cell wall function, and filamentation at alkaline pH. This finding suggests that YlRim101 promotes filamentation at alkaline pH via controlling the expression of these genes. We also show that, in addition to YlRim101, the Msn2/Msn4-like transcription factor Mhy1 is highly upregulated at alkaline pH and is essential for filamentation. However, unlike YlRim101, which specifically regulates alkaline-induced filamentation, Mhy1 regulates both alkaline- and glucose-induced filamentation, since the deletion of MHY1 abolished them both, whereas the overexpression of MHY1 induced strong filamentation irrespective of the pH or the presence of glucose. Finally, we show that YlRim101 and Mhy1 positively coregulate seven cell wall protein genes at alkaline pH, including YlPHR1 and five cell surface adhesin-like genes, three of which appear to promote filamentation. Together, these results reveal a conserved role of YlRim101 and a novel role of Mhy1 in the regulation of alkaline-induced filamentation in Y. lipolyticaIMPORTANCE The regulatory mechanism that governs pH-regulated filamentation is not clear in dimorphic fungi except in Candida albicans Here, we investigated the regulation of alkaline pH-induced filamentation in Yarrowia lipolytica, a dimorphic yeast distantly related to C. albicans Our results show that the transcription factor YlRim101 and the Msn2/Msn4-like transcription factor Mhy1 are the major regulators that promote filamentation at alkaline pH. They control the expression of a number of cell wall protein genes important for cell wall organization and filamentation. Our results suggest that the Rim101/PacC homologs play a conserved role in pH-regulated filamentation in dimorphic fungi.

Targeted Integration Through Transformation of Hydroxyurea-Arrested Cells.

Homologous recombination is required to specifically target DNA to a desired genomic locus. Non-homologous end joining is the predominant form of recombination in Yarrowia lipolytica. Transformation of this organism with linear DNA therefore results in random integration of the introduced DNA into the genome. In this protocol, hydroxyurea-mediated cell cycle arrest is applied to significantly increase the rate of homologous recombination during transformation and enhance targeted integration.

Strain Construction for Intracellular Metabolic Pathway Localization in Y. lipolytica.

Pathway localization by fluorophore or epitope tagging can be accomplished through a multi-staged DNA construct and confirmation process, to generate a series of successfully tagged protein targets. Prerequisite conditions for this process in Y. lipolytica are auxotrophic selection (leu2 or ura3), impaired non-homologous end joining by deletion or impairment of ku70, and plasmids or gene pieces for epitope-selection cassette construction. The general approach for gene tagging can work for C- or N-terminal tags. Gene overexpression from an episomal plasmid can be accomplished through transcript amplification and cloning. C-terminal tagging allows expression of a gene-GFP fusion to be regulated from the endogenous promoter. The epitope-selection cassette also includes a constitutive or highly expressed promoter driving the auxotrophic or other selectable marker gene such as one conferring antifungal or antibiotic resistance. Strains for pathway localization utilize overlap PCR, PEG-based transformation, and a fast DNA preparation for rapid colony screening. Successful transformants can be used for pathway localization and condition-specific response analysis.

Engineering Yarrowia lipolytica for Production of Fatty Alcohols with YaliBrick Vectors.

Biosynthesis of fatty alcohol holds great promise as substitute to replace petroleum-derived fatty alcohols to mitigate environmental concerns and reduce earth's carbon footprint. In this protocol, we detail the procedures of how to use the YaliBrick gene assembly platform to achieve modular assembly of fatty alcohol pathway in Y. lipolytica. To limit fatty alcohol oxidation, we will also describe the hydroxyurea-based protocols for the efficient disruption of POX1 gene, encoding the fatty acyl coenzyme A in Y. lipolytica, with the homologous arm about 500 bp. We envision that this chapter would improve our ability to engineer microbial cell factories for oleochemical and fatty alcohol production in oleaginous yeast species.

Methods to Activate and Elucidate Complex Endogenous Sugar Metabolism in Yarrowia lipolytica.

Yarrowia lipolytica has endogenous metabolism to use complex sugars derived from lignocellulosic biomass. However, many of these pathways are cryptic and hence either inactive or inefficient for xylose, arabinose, and cellobiose assimilation. Here we present collective methods to activate and elucidate these endogenous sugar pathways by performing short-term growth adaptation, determining the pathway efficiency, and conducting transcriptomic, enzymatic, and metabolic analyses to identify rate limiting steps for enhanced sugar consumption.

Pathway Engineering for Beta-Carotene and Carotenoid Biosynthesis in Y. lipolytica.

ß-carotene is an increasingly sought-after organic pigment with antioxidant properties and a vitamin precursor. Yarrowia lipolytica, though unable to naturally synthesize carotenoids, can produce high amounts of the precursor acetyl-CoA making it a promising host for metabolic engineering towards novel biotechnological production of carotenoids. Here, we describe a synthetic biology methodology for Y. Lipolytica metabolic engineering based on Golden Gate DNA assembly for the generation of a multigene cassette, subsequent transformation enabling ß-carotene biosynthesis, and quantification of the compound.

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.

Yarrowia lipolytica Bioprocess Development: From Flask to Bioreactor.

Yarrowia lipolytica produces a range of valuable biotechnological products from natural metabolites and enzymes to heterologous proteins. The production of these products is affected by medium composition and various environmental factors. Here we describe bioprocess development for a recombinant laccase production by Y. lipolytica. At first, response surface methodology (RSM), as a statistical technique for design of experiment (DOE), is used for the optimization of medium composition in flask level. Then, results of RSM are applied to increase laccase production in controlled conditions of the bioreactor.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

OBJECTIVE: Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. RESULTS: The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. CONCLUSIONS: A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

A 37-amino acid loop in the Yarrowia lipolytica hexokinase impacts its activity and affinity and modulates gene expression.

The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products. Nonetheless, there are still gaps in our understanding of its central carbon metabolism. Here we present an in-depth study of Y. lipolytica hexokinase (YlHxk1), a structurally unique protein. The greatest peculiarity of YlHxk1 is a 37-amino acid loop region, a structure not found in any other known hexokinases. By combining bioinformatic and experimental methods we showed that the loop in YlHxk1 is essential for activity of this protein and through that on growth of Y. lipolytica on glucose and fructose. We further proved that the loop in YlHxk1 hinders binding with trehalose 6-phosphate (T6P), a glycolysis inhibitor, as hexokinase with partial deletion of this region is 4.7-fold less sensitive to this molecule. We also found that YlHxk1 devoid of the loop causes strong repressive effect on lipase-encoding genes LIP2 and LIP8 and that the hexokinase overexpression in Y. lipolytica changes glycerol over glucose preference when cultivated in media containing both substrates.

A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Biomanufacturing of value-added products from oils or fats: A case study on cellular and fermentation engineering of Yarrowia lipolytica.

The United States produces more than 10 million tons of waste oils and fats each year. This paper aims to establish a new biomanufacturing platform that converts waste oils or fats into a series of value-added products. Our research employs the oleaginous yeast Yarrowia lipolytica as a case study for citric acid (CA) production from waste oils. First, we conducted the computational fluid dynamics (CFD) simulation of the bioreactor system and identified that the extracellular mixing and mass transfer is the first limiting factor of an oil fermentation process due to the insolubility of oil in water. Based on the CFD simulation results, the bioreactor design and operating conditions were optimized and successfully enhanced oil uptake and bioconversion in fed-batch fermentation experiments. After that, we investigated the impacts of cell morphology on oil uptake, intracellular lipid accumulation, and CA formation by overexpressing and deleting the MHY1 gene in the wild type Y. lipolytica ATCC20362. Fairly good linear correlations (R2 > 0.82) were achieved between cell morphology and productivities of biomass, lipid, and CA. Finally, fermentation kinetics with both glucose and oil substrates were compared and the oil fermentation process was carefully evaluated. Our study suggests that waste oils or fats can be economical feedstocks for biomanufacturing of many high-value products.

Plasma membrane H+ pump at a crossroads of acidic and iron stresses in yeast-to-hypha transition.

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.