Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation.

BACKGROUND: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. RESULTS: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on-off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source. CONCLUSIONS: Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.

Cinnamic acid and p-coumaric acid are metabolized to 4-hydroxybenzoic acid by Yarrowia lipolytica.

Yarrowia lipolytica has been explored as a potential production host for flavonoid synthesis due to its high tolerance to aromatic acids and ability to supply malonyl-CoA. However, little is known about its ability to consume the precursors cinnamic and p-coumaric acid. In this study, we demonstrate that Y. lipolytica can consume these precursors through multiple pathways that are partially dependent on the cultivation medium. By monitoring the aromatic acid concentrations over time, we found that cinnamic acid is converted to p-coumaric acid. We identified potential proteins with a trans-cinnamate 4-monooxygenase activity in Y. lipolytica and constructed a collection of 15 knock-out strains to identify the genes responsible for the reaction. We identified YALI1_B28430g as the gene encoding for a protein that converts cinnamic acid to p-coumaric acid (designated as TCM1). By comparing different media compositions we found that complex media components (casamino acids and yeast extract) induce this pathway. Additionally, we discover the conversion of p-coumaric acid to 4-hydroxybenzoic acid. Our findings provide new insight into the metabolic capabilities of Y. lipolytica and hold great potential for the future development of improved strains for flavonoid production.

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates.

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.

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.

Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates.

BACKGROUND: Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. RESULTS: Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. CONCLUSION: We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

Deletion of MHY1 abolishes hyphae formation in Yarrowia lipolytica without negative effects on stress tolerance.

There is a need for development of sustainable production processes for production of fats/oils and lipid derived chemicals. The dimorphic oleaginous yeast Yarrowia lipolytica is a promising organism for conversion of biomass hydrolysate to lipids, but in many such processes hyphae formation will be problematic. We have therefore constructed and compared the performance of strains carrying deletions in several published gene targets suggested to abolish hyphae formation (MHY1, HOY1 and CLA4). The MHY1-deletion was the only of the tested strains which did not exhibit hyphae formation under any of the conditions tested. The MHY1-deletion also had a weak positive effect on lipid accumulation without affecting the total fatty acid composition, irrespective of the nitrogen source used. MHY1 has been suggested to constitute a functional homolog of the stress responsive transcription factors MSN2/4 in Saccharomyces cerevisiae, the deletion of which are highly stress sensitive. However, the deletion of MHY1 displayed only minor difference on survival of a range of acute or long term stress and starvation conditions. We conclude that the deletion of MHY1 in Y.lipolytica is a reliable way of abolishing hyphae formation with few detectable negative side effects regarding growth, stress tolerance and lipid accumulation and composition.

Comparative sequence analysis and mutagenesis of ethylene forming enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs.

BACKGROUND: Ethylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation. RESULTS: We identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast. The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production. Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE. The EFE homolog from P. chrysogenum was engineered at 10 amino acid residues to mimic the P. syringae EFE, but this did not confer ethylene producing capability.Furthermore, we predicted the structure of EFE by homology to known structures of 2-oxoglutarate/Fe(II) dependent dioxygenases. Three of the amino acids correlating with ethylene production are located in the predicted 2-oxoglutarate binding domain. A protein domain specific for the EFE-class was shown to be essential for activity. Based on the structure and alanine substitutions, it is likely that amino acids (H189, D191 and H268) are responsible for binding the Fe(II) ligand. CONCLUSION: We provide further insight into the structure and function of the ethylene forming (EFE) - subclass of 2-oxoglutarate/Fe(II) dependent dioxygenases. We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation. We also demonstrate the use of an alternative EFE gene. The data from this study will provide the basis for directed protein engineering to enhance the ethylene production capability and properties of EFE.

Production of 2-butanol through meso-2,3-butanediol consumption in lactic acid bacteria.

2-Butanol has been an issue of industries in many areas, for example, biofuel production (as an advanced alternate fuel), fermented beverages, and food (as taste-altering component). Thus, its source of production, the biological pathway, and the enzymes involved are of high interest. In this study, 42 different isolates of lactic acid bacteria from nine different species were screened for their capability to consume meso-2,3-butanediol and produce 2-butanol. Lactobacillus brevis was the only species that showed any production of 2-butanol. Five of ten tested isolates of L. brevis were able to convert meso-2,3-butanediol to 2-butanol in a synthetic medium (SM2). However, none of them showed the same capability in a complex medium such as MRS indicating that the ability to produce 2-butanol is subject to some kind of repression mechanism. Furthermore, by evaluating the performance of the enzymes required to convert meso-2,3-butanediol to 2-butanol, that is, the secondary alcohol dehydrogenase and the diol dehydratase, it was shown that the latter needed the presence of a substrate to be expressed.

Ethylene production in relation to nitrogen metabolism in Saccharomyces cerevisiae.

We have previously shown that ethylene production in Saccharomyces cerevisiae expressing the ethylene-forming enzyme (EFE) from Pseudomonas syringae is strongly influenced by variations in the mode of cultivation as well as the choice of nitrogen source. Here, we have studied the influence of nitrogen metabolism on the production of ethylene further. Using ammonium, glutamate, glutamate/arginine, and arginine as nitrogen sources, it was found that glutamate (with or without arginine) correlates with a high ethylene production, most likely linked to an observed increase in 2-oxoglutarate levels. Arginine as a sole nitrogen source caused a reduced ethylene production. A reduction of arginine levels, accomplished using an arginine auxotrophic ARG4-deletion strain in the presence of limiting amounts of arginine or through CAR1 overexpression, did however not correlate with an increased ethylene production. As expected, arginine was necessary for ethylene production as ethylene production in the ARG4-deletion strain ceased at the time when arginine was depleted. In conclusion, our data suggest that high levels of 2-oxoglutarate and a limited amount of arginine are required for successful ethylene production in yeast.

2-Butanol and butanone production in Saccharomyces cerevisiae through combination of a B12 dependent dehydratase and a secondary alcohol dehydrogenase using a TEV-based expression system.

2-Butanol and its chemical precursor butanone (methyl ethyl ketone--MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuteri), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.

Identification of factors for improved ethylene production via the ethylene forming enzyme in chemostat cultures of Saccharomyces cerevisiae.

BACKGROUND: Biotechnological production of the traditional petrochemical ethylene is presently being explored using yeasts as well as bacteria. In this study we quantify the specific ethylene production levels at different conditions in continuous (chemostat) cultivation of Saccharomyces cerevisae expressing the ethylene forming enzyme (EFE) from Pseudomonas syringae. RESULTS: Our study shows that oxygen availability is an important factor for the ethylene formation. Maintaining a high percentage dissolved oxygen in the cultivation was found to be necessary to achieve maximal ethylene productivity. Even at oxygen levels high enough to sustain respiratory metabolism the ethylene formation was restricted. Oxygen was also important for sustaining a high respiratory rate and to re-oxidize the surplus of NADH that accompanies ethylene formation. By employing three different nitrogen sources we further found that the nitrogen source available can both improve and impair the ethylene productivity. Contrary to findings in batch cultures, using glutamate did not give a significant increase in specific ethylene production levels compared to the reference condition with ammonia, whereas a combination of glutamate and arginine resulted in a strongly diminished specific ethylene production. Furthermore, from cultivations at different dilution rates the ethylene formation was found to be coupled to growth rate. CONCLUSION: To optimize the ethylene productivity in S. cerevisiae expressing a bacterial ethylene forming enzyme, controlling the oxygen availability and growth rate as well as employing an ideal nitrogen source is of importance. The effects of these factors as studied here provide a basis for an optimized process for ethylene production in S. cerevisiae.

Physiological adaptations of Saccharomyces cerevisiae evolved for improved butanol tolerance.

BACKGROUND: Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. However, the end product toxicity is one of the main obstacles for developing the production process irrespective of the choice of production organism. The long-term goal of the present project is to produce 2-butanol in Saccharomyces cerevisiae. Therefore, unraveling the toxicity mechanisms of solvents such as butanol and understanding the mechanisms by which tolerant strains of S. cerevisiae adapt to them would be an important contribution to the development of a bio-based butanol production process. RESULTS: A butanol tolerant S. cerevisiae was achieved through a series of sequential batch cultures with gradual increase of 2-butanol concentration. The final mutant (JBA-mut) tolerates all different alcohols tested at higher concentrations compared to the wild type (JBA-wt). Proteomics analysis of the two strains grown under mild butanol-stress revealed 46 proteins changing their expression by more than 1.5-fold in JBA-mut, 34 of which were upregulated. Strikingly, 21 out of the 34 upregulated proteins were predicted constituents of mitochondria. Among the non-mitochondrial up-regulated proteins, the minor isoform of Glycerol-3-phosphatase (Gpp2) was the most notable, since it was the only tested protein whose overexpression was found to confer butanol tolerance. CONCLUSION: The study demonstrates several differences between the butanol tolerant mutant and the wild type. Upregulation of proteins involved in the mitochondrial ATP synthesizing machinery constituents and glycerol biosynthesis seem to be beneficial for a successful adaptation of yeast cells to butanol stress.

A QPCR-based reporter system to study post-transcriptional regulation via the 3' untranslated region of mRNA in Saccharomyces cerevisiae.

Post-transcriptional regulation via the 3' untranslated region (3' UTR) of mRNA is an important factor in governing eukaryotic gene expression. Achieving detailed understanding of these processes requires highly quantitative systems in which comparative studies can be performed. To this end, we have developed a plasmid reporter system for Saccharomyces cerevisiae, in which the 3' UTR can be easily replaced and modified. Accurate quantification of the tandem affinity purification tag (TAP)-reporter protein and of TAP-mRNA is achieved by immuno-QPCR and by RT-QPCR, respectively. We have used our reporter system to evaluate the consequences on gene expression from varying the 3' UTR, a problem often encountered during C-terminal tagging of proteins. It was clear that the choice of 3' UTR was a strong determinant of the reporter expression, in a manner dependent on the growth conditions used. Mutations affecting either decapping (lsm1Delta) or deadenylation (pop2Delta) were also found to affect reporter gene expression in a highly 3' UTR-dependent manner. Our results using this set-up clearly indicate that the common strategy used for C-terminal tagging, with concomitant replacement of the native 3' UTR, will very likely provide incorrect conclusions on gene expression.

Carbon source dependent dynamics of the Ccr4-Not complex in Saccharomyces cerevisiae.

We have investigated the composition of the conserved Ccr4-Not complex during different physiological states of Saccharomyces cerevisiae. Major changes were found, most notably in the expression of the central scaffold protein Not1p, which was strongly reduced in the absence of glucose. The low expression of Not1p was also evident from the inability of Pop2p to co-purify Not1p in cells from cultures lacking glucose. However, Not1p was still essential under conditions of low expression. The downregulation of Not1p indicates that many of the Ccr4-Not complex components are likely to have roles outside of the complex. We suggest that the use of different carbon sources will be a good starting point to unravel these functions.

A complete inventory of all enzymes in the eukaryotic methionine salvage pathway.

The methionine salvage pathway is universally used to regenerate methionine from 5'-methylthioadenosine, a byproduct of certain reactions involving S-adenosylmethionine. We identified and verified the genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system: the yeast Saccharomyces cerevisiae. The genes encoding 5'-methylthioribose-1-phosphate isomerase and 5'-methylthioribulose-1-phosphate dehydratase are herein named MRI1 and MDE1, respectively. The 5'-methylthioadenosine phosphorylase was verified as Meu1p, the 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase as Utr4p and the aci-reductone dioxygenase as Adi1p. The homologue of the enolase/phosphatase gene, YNL010w, was excluded from its candidate role in the cycle. The methodology used involved auxotrophic growth tests and analysis of intracellular 5'-methylthioadenosine in deletion mutants. The last step, a transamination of 4-methylthio-2-oxobutyrate to yield methionine, was found to be a highly redundant step. It was catalysed by amino acid transaminases, mainly coupled with aromatic and branched chain amino acids as amino donors, but also with proline, lysine and glutamate/glutamine. The aromatic amino acid transaminases, Aro8p and Aro9p, and the branched chain amino acid transaminases, Bat1p and Bat2p, seemed to be the main enzymes exhibiting 4-methylthio-2-oxobutyrate transaminase activity. Bat2p was found to be less specific and used proline, lysine, tyrosine and glutamate as amino donors in addition to the branched chain amino acids. Thus, for the first time, all enzymes of the methionine salvage pathway were identified in a eukaryote.

Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification.

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large-scale Western blotting-based quantification of the TAP-tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno-qPCR approach was demonstrated by analysis of osmo-regulated proteins, where the 2000-fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno-qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP-tag and protein A are useful in most model organisms, the immuno-qPCR method is both generic and suitable for large-scale studies.

NADH-reductive stress in Saccharomyces cerevisiae induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (TDH1).

A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2Delta strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2Delta strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2Delta strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.

Dihydroxyacetone kinases in Saccharomyces cerevisiae are involved in detoxification of dihydroxyacetone.

The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting K(m)((DHA)) of 22 and 5 microm and K(m)((ATP)) of 0.5 and 0.1 mm for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1 Delta dak2 Delta), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1 Delta dak2 Delta highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.