Jean-Marc Nicaud

Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowialipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y.lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y.lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y.lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.

Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeasts metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.

Involvement of the G3P shuttle and β-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica can accumulate up to 38% of its dry weight (DW) as lipids. Factors involved in lipid accumulation, particularly triglycerides, are not well identified. Using different mutations in the glycerol-3-phosphate (G3P) shuttle pathway (Δgut2 affecting the anabolic dehydrogenase or overexpressing GPD1 affecting the catabolic dehydrogenase), we were able to modulate G3P concentration. We show that in a Po1d genetic background, GPD1 overexpression, GUT2 inactivation or both mutations together result in 1.5, 2.9, and 5.6-fold respective increases in the level of G3P leading to an increase of triacylglyceride (TAG) accumulation. Moreover, our results indicate that each strain with an increased concentration of G3P, also presented a decreased concentration of glycerol. Analysis of the different genes involved in glycerol metabolism indicated that Y. lipolytica does not possess a gene for glycerol-3-phosphatase. These findings suggest that Y. lipolytica has a modified and unique metabolism of glycerol that is dedicated to G3P synthesis (and also to TAG synthesis) which may contribute to its oleaginous character. Furthermore, coupling the G3P shuttle disorders to a deficient β-oxidation pathway (by inactiving POX1-6 or MFE1 genes) increased TAG and free fatty acids content. Finally, we obtained strains that accumulated up to 65-75% of their DW as lipid. Transcriptional analysis in these strains, revealed that the high levels of lipids resulted from over-expression of genes involved in TAG synthesis (SCT1, encoding a sn-1 acyltransferase; and DGA1, encoding an acylCoA diacylglycerol acyltransferase) and the repression of genes involved in the degradation of TAG (TGL3 and TGL4, encoding triacylglycerol lipases). These findings indicate that TAG synthesis is limited by the availability of G3P and fatty acids, and that the expression of genes involved in TAG homeostasis is regulated by the G3P shuttle and the β-oxidation pathway. Finally, the synergistic contribution of acyltransferase gene expression to G3P synthesis is required for high levels of TAG synthesis and lipid accumulation in Y. lipolytica.

Hexokinase-A limiting factor in lipid production from fructose in Yarrowia lipolytica

Microbial biolipid production has become an important part of making biofuel production economically feasible. Genetic engineering has been used to improve the ability of Yarrowia lipolytica, an oleaginous yeast, to produce lipids using glucose-based media. However, few studies have examined lipid accumulation by Y. lipolyticas ability to utilize other hexose sugars, and as of yet, the rate-limiting steps in this process are unidentified. In this study, we investigated the de novo accumulation of lipids by Y. lipolytica when grown in glucose, fructose, and sucrose. Three Y. lipolytica wild-type (WT) strains of varied origin differed significantly in their lipid production, growth, and fructose utilization. Hexokinase (ylHXK1p) activity partially explained these differences. Overexpression of the ylHXK1 gene led to increased hexokinase activity (6.5-12 times higher) in the mutants versus the WT strains; a pronounced reduction in cell filamentation in mutants grown in fructose-based media; and improved biomass production, particularly in the mutant whose parent had shown the lowest growth capacity in fructose (French strain W29). All mutants showed improved lipid yield and production when grown on fructose, although the effect was strain dependent (23-55% improvement). Finally, we overexpressed ylHXK1 in a highly modified strain of Y. lipolytica W29 engineered to optimize oil production. This modification was combined with Saccharomyces cerevisiae invertase gene expression to evaluate the resulting mutants ability to produce lipids using cheap industrial substrates, namely sucrose (a major component of molasses). Sucrose turned out to be a better substrate than either of its building blocks, glucose or fructose. Over its 96 h of growth in the bioreactors, this highly modified strain produced 9.15 g L(-1) of lipids, yielding 0.262 g g(-1) of biomass.

Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved

Lip2 lipase from Yarrowia lipolytica is a very promising lipase with many potential applications (e.g. resolution of racemic mixtures, production of fine chemicals). Unfortunately this potential is impeded by a very low thermostability for temperatures higher than 40°C. Error-prone PCR and screening of the library in a high-performance yeast expression system (Y. lipolytica) enabled a thermostable variant to be identified. This variant presents only one mutation, the free cysteine 244 is changed into an alanine. At 60°C, the half-life time of the purified variant was 127-fold increased compared to the WT enzyme (from 1.5 min to 3 h). Saturation mutagenesis experiment at position 244 demonstrated that the presence of a cysteine at this position was responsible for the thermal denaturation. It was demonstrated that WT Lip2 and the thermostable variant are both inactivated through aggregation mechanisms, but that the kinetics and the nature of the aggregation were different. For the WT enzyme, rapid intermolecular disulphide bridge interchanges triggered by the free cysteine 244 mediates aggregation. For the variant C244A, aggregation still occurred but much slower than for the WT lipase and was mainly driven by hydrophobic forces.

Co-expression of heterologous desaturase genes in Yarrowia lipolytica.

The hybrid promoter (hp4d) expression cassette, one of the efficient tools of Yarrowia lipolytica expression system, has been applied to produce or secrete a variety of recombinant proteins. This cassette directs a strong gene expression, because the hp4d promoter exhibits high level quasi-constitutive activity. The objective of this study is to test whether two expression cassettes inserted into a vector could function efficiently and simultaneously. Taking advantage of the well-known biosynthesis pathway of gamma-linolenic acid (GLA), we examined the performance of Y. lipolytica, transformed with two expression cassettes containing previously cloned Delta12-desaturase and Delta6-desaturase genes, by monitoring fatty acid composition of cellular lipids. Our results confirmed that each individual desaturase gene was expressed efficiently by the expression cassette. When two cassettes with respective desaturase genes, carried on the same vector, were integrated into yeast genome, a significant level of GLA was synthesized from endogenous linoleic acid (LA) and oleic acid (OA). Besides, both expression cassettes functioned effectively without influence from each other. These findings indicated that co-expression of two desaturase genes by this dual cassette vector was effective and simultaneous. Results from the present study provide an alternative approach for both the production of several proteins at the same time, and the development of single cell oil containing high-valued polyunsaturated fatty acids (PUFAs).

SOA genes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeast Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene (LIP2) was monitored using a LIP2-beta-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol.

First complexomic study of alkane-binding protein complexes in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica uses hydrophobic substrates, such as alkanes, fatty acids and oils, for its growth. It has developed a strategy for the use of such substrates, involving the production of hydrophobic binding structures called protrusions on the cell surface. These protrusions are resemble channels connecting the cell wall to the inside of the cell, and are probably involved in transport mechanisms that we do not yet fully understand. The complete genome of the haploid Y. lipolytica strain E150 (CLIB99) was sequenced in 2004 by the Génolevures Consortium. The availability of a complete genome sequence for this species has made it possible to carry out proteomic and other investigations, leading to the characterization of lipid bodies (LB) in terms of (i) their lipid composition, (ii) the major LB proteins, as identified by mass spectrometry, and (iii) differences in protein or lipid composition as a function of the carbon source used. Functional analyses would provide insight into the biological processes associated with these bodies and 2D BN/SDS-PAGE is a highly suitable method for the analysis of protein complexes. This report provides a first description of the analysis and identification of hydrophobic binding protein complexes in Y. lipolytica. For this purpose, we used 2D BN/SDS-PAGE for the separation of protein complexes and HPLC-chip-MS for protein identification. We separated and identified 40 protein complexes (11 heteromultimeric and 29 homomultimeric), providing insight into their function. This study represents a major step forward, as most previous studies identified proteins either on the basis of sequence similarity to proteins from other organisms (44% of the proteins identified in this study) or by prediction (50% of proteins identified in this study) alone.

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

BackgroundDroplet-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.ResultsFive hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-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-β-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.

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.