Mechanisms underlying the toxicity of lactone aroma compounds towards the producing yeast cells

AIMS: To study the fundamental mechanisms of toxicity of the fruity aroma compound gamma-decalactone, that lead to alterations in cell viability during its biotechnological production by yeast cells; Yarrowia lipolytica that is able to produce high amounts of this metabolite was used here as a model. METHODS AND RESULTS: Lactone concentrations above 150 mg l-1 inhibited cell growth, depolarized the living cells and increased membrane fluidity. Infrared spectroscopic measurements revealed that the introduction of the lactone into model phospholipid bilayers, decreased the phase transition temperature. Moreover, the H+-ATPase activity in membrane preparations was strongly affected by the presence of the lactone. On the other hand, only a slight decrease in the intracellular pH occurred. CONCLUSIONS: We propose that the toxic effects of gamma-decalactone on yeast may be initially linked to a strong interaction of the compound with cell membrane lipids and components. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings may enable the elaboration of strategies to improve yeast cell viability during the process of lactones bioproduction.     

Cloning, sequencing, and characterization of five genes coding for Acyl-CoA oxidase isozymes in the yeastYarrowia lipolytica

The Acyl-CoA oxidase (AOX) isozymes catalyze the first steps of peroxisomal β-oxidation, which is important for the degradation of fatty acids. Using conserved blocks in previously identified yeastPOX genes encoding AOXs, the authors have shown that fivePOX genes are present in the yeastYarrowia lipolytica. These genes show approx 63% identity among themselves, and 42% identity with thePOX genes from other yeasts. Mono-disruptedY. lipolytica strains were constructed using a variation of the sticky-end polymerase chain reaction method. AOX activity in the mono-disrupted strains revealed that a long-chain oxidase is encoded by thePOX2 gene and a short-chain oxidase by thePOX3 gene.     

Lipase location in Yarrowia lipolytica cells

Lipase production by Yarrowia lipolytica was growth associated and, at the beginning of cultivation, it was mainly cell-bound. Lipase release into the culture medium started when about 50% of the carbon source (olive oil or glucose) was consumed reaching its maximum concentration in the late stationary phase.     

Cell wall composition of the yeast and mycelial forms of Yarrowia lipolytica

The cell walls of the yeast and mycelial forms of Yarrowia lipolytica were isolated and purified. Electron microscopy studies showed no differences between both types of cell walls. Chemical analysis revealed that the yeast cell wall contained 70% neutral carbohydrate, 7% amino sugars, 15% protein, 5% lipids and 0.8% phosphorus. Mycelial cell walls contained 70% carbohydrate, 14% aminosugars, 6% protein, 5% lipids and 0.6% phosphorus. Three polysaccharides: -glucan, mannan and chitin were detected. Proteins were solubilized from both cell wall fractions and separated by polyacrylamide gel electrophoresis. About 50 protein bands were detected, four of them corresponding to glycoproteins. The cell walls of the yeast and mycelial forms of Y. lipolytica were qualitatively similar and only quantitative differences were found.Abbreviations GlcNAc N-acetylglucosamine - FITC-WGA fluorescein isothiocyanate-wheat germ agglutinin - PAS periodic acid Schiff     

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.     

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Combined effects of 13-hydroperoxides and dithiotreitol were optimised by a central composite design and a model was proposed. Finally, 6 mM (600 mg/L) of hexanal was obtained when 119 mM of 13-hydroperoxides (37 g/L) and 50 mM of dithiotreitol were introduced directly in the biocatalytic medium of the yeast Y. lipolytica.     

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p

One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.     

Evaluation of Fourier-transform infrared spectroscopy for data capture in predictive microbiology

Chemical changes in the medium, induced by the fermentative species Lactobacillus plantarum and Lactobacillus brevis and by the enzymatic action of a proteolytic, spoilage species, Yarrowia lipolytica, were analysed using Fourier-transform i.r. spectroscopy (FTIR). Changes in the absorbance data over time could be modelled using one of the more current predictive, mathematical models of microbial growth, such as the Gompertz equation. Moreover, a linear correlation between FTIR data (expressed as absorbance of some selected peaks) and viability data (expressed as log₁₀ c.f.u./g or ml) was observed during the fermentation process, both for L. plantarum and L. brevis.     

Triplicate genes for mitochondrial ADP/ATP carriers in the aerobic yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> are regulated differentially in the absence of oxygen

Yarrowia lipolytica is a strictly aerobic fungus, which differs from the extensively studied model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its physiology, genetics and dimorphic growth habit. We isolated and sequenced cDNA and genomic clones (YlAAC1) from Y. lipolytica that encode a mitochondrial ADP/ATP carrier. The YlAAC1 gene can complement the S. cerevisiae aac2 deletion mutant. Southern hybridization, analysis of Yarrowia clones obtained in the course of the Génolevures project, and further sequencing revealed the existence of two paralogs of the YlAAC1 gene, which were named YlAAC2 and YlAAC3, respectively. Phylogenetic analysis showed that YlAAC1 and YlAAC2 were more closely related to each other than to YlAAC3, and are likely to represent the products of a recent gene duplication. All three Y. lipolytica YlAAC genes group together on the phylogenetic tree, suggesting that YlAAC3 is derived from a more ancient duplication within the Y. lipolytica lineage. A similar branching pattern for the three ScAAC paralogs in the facultative anaerobe S. cerevisiae demonstrates that two rounds of duplication of AAC genes occurred independently at least twice in the evolution of hemiascomycetous yeasts. Surprisingly, in both the aerobic Y. lipolytica and the facultative anaerobe S. cerevisiae, the three paralogs are differentially regulated in the absence of oxygen. Apparently, Y. lipolytica can sense hypoxia and down-regulate target genes in response.     

Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats

Cell growth, lipid accumulation and cellular lipid composition of Yarrowia lipolytica growing on mixtures of industrial fats containing stearic, oleic, linoleic and palmitic acid have been studied. During growth, the strain incorporated oleic and linoleic acids more rapidly than the saturated fatty acids. Relatively high lipid accumulation (up to 0.44 g of lipids per g of dry matter) was observed when stearic acid was included in the culture medium. In contrast, substrates rich in oleic acid did not favor cellular lipid accumulation. The accumulated lipids, mainly composed of triacylglycerols (45-55% w/w), demonstrated a different total fatty acid composition compared with that of the substrate; in all cases, the microorganism showed the unusual capacity to increase its cellular stearic acid level, even if this fatty acid was not found in high concentrations in the substrate. This permitted the synthesis of interesting lipid profiles with high percentages of stearic acid and non-negligible percentages of palmitic and oleic acid, with a composition resembling that of cocoa-butter.     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

发酵产泡性能降低的解脂耶氏酵母菌株的获得及其评价

微生物发酵过程中泡沫的产生是发酵领域遇到的共性问题。在不影响发酵性能的前提下抑制菌株的产泡,对简化操作以及降低发酵成本具有较为重要的意义。解脂耶氏酵母(Yarrowia lipolytica,之前称为Candida lipolytica)是一种常用的合成生物学底盘,也是合成赤藓糖醇等功能糖醇的生产菌株。但在发酵合成赤藓糖醇的过程中会产生大量的泡沫,需要添加消泡剂以消除泡沫。【目的】本研究旨在开发一种产泡能力显著降低的解脂耶氏酵母新菌株,以减少赤藓糖醇发酵过程中消泡剂的添加。【方法】本研究利用解脂耶氏酵母中非同源靶向重组占支配地位的原理,采用一段外源DNA随机插入基因组的手段,随机突变基因组,改变菌株的发酵产泡性能,使突变株在发酵过程中不产泡或者降低其产泡的能力。【结果】通过筛选,获得一株在发酵过程中产泡性能显著降低的工程菌株,该菌株在保留高效合成赤藓糖醇性能的同时,显著降低了泡沫的产生。【结论】所获得的菌株对工业发酵合成赤藓糖醇具有较为重要的意义,也为控制其他微生物发酵过程中泡沫的生成提供了思路。     

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 μmol NADH min⁻¹ mg⁻¹ were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.     

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35^T were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C₄-HL), N-hexanoyl homoserine lactone (C₆-HL), N-octanoyl homoserine lactone (C₈-HL) and N-dodecanoyl homoserine lactone (C₁₂-HL). This study suggests that quorum sensing may also play an important role in extreme environments.     

High-yield production of erythritol from raw glycerol in fed-batch cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

An acetate-negative mutant of Yarrowia lipolytica Wratislavia K1 was selected that, when grown with 300 g raw glycerol l⁻¹ at pH 3, produced 170 g erythritol l⁻¹ after 7 days, corresponding to a 56% yield and a productivity of 1 g l⁻¹ h⁻¹. The Wratislavia K1 strain did not produce citric acid.