RESUMEN
Saccharomyces pastorianus, which is responsible for the production of bottom-fermented lager beer, is a hybrid species that arose from the mating of the top-fermenting ale yeast Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus around the start of the 17th century. Based on detailed analysis of Central European brewing records, we propose that the critical event for the hybridization was the introduction of top-fermenting S. cerevisiae into an environment where S. eubayanus was present, rather than the other way around. Bottom fermentation in parts of Bavaria preceded the proposed hybridization date by a couple of hundred years and we suggest that this was carried out by mixtures of yeasts, which may have included S. eubayanus. A plausible case can be made that the S. cerevisiae parent came either from the Schwarzach wheat brewery or the city of Einbeck, and the formation of S. pastorianus happened in the Munich Hofbräuhaus between 1602 and 1615 when both wheat beer and lager were brewed contemporaneously. We also describe how the distribution of strains from the Munich Spaten brewery, and the development by Hansen and Linder of methods for producing pure starter cultures, facilitated the global spread of the Bavarian S. pastorianus lineages.
Asunto(s)
Hibridación Genética , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fermentación , CervezaRESUMEN
The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.
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Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Cromosomas , Pérdida de HeterocigocidadRESUMEN
Nitrogen is a critical nutrient in beverage fermentations, influencing fermentation performance and formation of compounds that affect organoleptic properties of the product. Traditionally, most commercial wine fermentations rely on Saccharomyces cerevisiae but the potential of alternative yeasts is increasingly recognised because of the possibility to deliver innovative products and process improvements. In this regard, Saccharomyces uvarum is an attractive non-traditional yeast that, while quite closely related to S. cerevisiae, displays a different fermentative and aromatic profile. Although S. uvarum is used in cider-making and in some winemaking, better knowledge of its physiology and metabolism is required if its full potential is to be realised. To address this gap, we performed a comparative analysis of the response of S. uvarum and S. cerevisiae to 13 different sources of nitrogen, assessing key parameters such as growth, fermentation performance, the production of central carbon metabolites and aroma volatile compounds. We observed that the two species differ in the production of acetate, succinate, medium-chain fatty acids, phenylethanol, phenylethyl acetate, and fusel/branched acids in ways that reflect different distribution of fluxes in the metabolic network. The integrated analysis revealed different patterns of yeast performance and activity linked to whether growth was on amino acids metabolised via the Ehrlich pathway or on amino acids and compounds assimilated through the central nitrogen core. This study highlights differences between the two yeasts and the importance that nitrogen metabolism can play in modulating the sensory profile of wine when using S. uvarum as the fermentative yeast.
Asunto(s)
Saccharomyces cerevisiae , Vino , Saccharomyces cerevisiae/metabolismo , Fermentación , Vino/análisis , Nitrógeno/metabolismo , Aminoácidos/metabolismo , Metaboloma , Acetatos/metabolismoRESUMEN
It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as Kluyveromyces marxianus can grow at temperatures of 45 °C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in K. marxianus by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. KLMX_70384 is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose de novo in K. marxianus after the speciation event that separated K. marxianus and K. lactis contribute to some of its unique traits.
Asunto(s)
Kluyveromyces , Termotolerancia , Calor , Temperatura , Termotolerancia/genéticaRESUMEN
There is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.
Asunto(s)
Sistemas CRISPR-Cas , Kluyveromyces , Técnicas de Inactivación de Genes , Kluyveromyces/genética , ARN Guía de KinetoplastidaRESUMEN
The study of nitrogen assimilation in yeast is of interest from genetic, evolutionary, and biotechnological perspectives. Over the course of evolution, yeasts have developed sophisticated control mechanisms to regulate nitrogen metabolism, with domesticated lineages sometimes displaying particular specialisation. The focus of this study was on assimilation of asparagine, which is a significant nutritional source for some alcoholic fermentations. We were particularly interested in ASP3, which encodes a periplasmic asparaginase and that was proposed to have been acquired relatively recently in S. cerevisiae by horizontal gene transfer. We examined 1680 S. cerevisiae genome assemblies to evaluate the distribution and evolutionary trajectory of ASP3. Our findings suggest an alternative hypothesis that ASP3 is an ancient Saccharomyces gene that has generally been lost over the course of evolution but has been retained in certain fermentative environments. As asparagine is the major nitrogen source in apple juice, we explored whether the presence of ASP3 would confer a growth advantage. Interestingly, we found that although ASP3 enhances growth when asparagine is the sole nitrogen source, the same effect is not seen in apple juice. These data indicate that growth in pure culture may not reflect the original selective environment for ASP3+ strains and highlight the role that complex regulation may play in optimising nitrogen assimilation in yeasts.
Asunto(s)
Asparaginasa , Saccharomyces cerevisiae , Asparaginasa/genética , Asparaginasa/metabolismo , Asparagina , Fermentación , Nitrógeno/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Kluyveromyces marxianus is an interesting and important yeast because of particular traits such as thermotolerance and rapid growth, and for applications in food and industrial biotechnology. For both understanding its biology and developing bioprocesses, it is important to understand how K. marxianus responds and adapts to changing environments. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. We report here the development of a ribosome profiling method for K. marxianus, which allows codon resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs. To aid in the analysis and sharing of ribosome profiling data, we added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. We also provide a set of step-by-step protocols for the experimental and bioinformatic methods that we developed.
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Kluyveromyces , Ribosomas , Genoma , Kluyveromyces/genética , Kluyveromyces/metabolismo , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.
Asunto(s)
Proteínas Fúngicas/metabolismo , Kluyveromyces/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pentosas/metabolismo , Arabinosa/metabolismo , Transporte Biológico , Xilosa/metabolismoRESUMEN
Evolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes.
Asunto(s)
Kluyveromyces , Saccharomyces , Zygosaccharomyces , Animales , Hibridación Genética , Kluyveromyces/genética , Estadios del Ciclo de Vida , Zygosaccharomyces/genéticaRESUMEN
Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomyces cerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured by Km, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic glucose uptake kinetics with an intermediate- (Km = 6.5 ± 2.0 mM) and a high-affinity (Km = 0.10 ± 0.01 mM) component, whereas cells grown in 2% glucose displayed monophasic kinetics with an intermediate-affinity (Km of 11.5 ± 1.5 mM). This work contributes to a better characterization of glucose transport in T. delbrueckii, with relevant implications for its exploitation in the food industry.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Torulaspora/genética , Torulaspora/metabolismo , Fermentación , Fructosa/metabolismo , Cinética , Manosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: The yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilise its metabolism as a basis for bio-based production. RESULTS: To illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L-1 2-PE from minimal medium. CONCLUSIONS: The strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform.
Asunto(s)
Vías Biosintéticas , Kluyveromyces/genética , Kluyveromyces/metabolismo , Ingeniería Metabólica/métodos , Alcohol Feniletílico/análisis , Biotecnología , Medios de Cultivo/análisis , Medios de Cultivo/química , Fermentación , Análisis de Flujos Metabólicos , Fenilalanina/metabolismo , Alcohol Feniletílico/metabolismo , Biología Sintética/métodosRESUMEN
Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production.IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains.
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Ácido Láctico/metabolismo , Transcripción Genética/fisiología , Zygosaccharomyces/fisiología , ARN de Hongos/análisis , ARN Mensajero/análisis , Análisis de Secuencia de ARN , Estrés Fisiológico , Zygosaccharomyces/genéticaRESUMEN
While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Edición Génica , Kluyveromyces/genética , ARN Guía de Kinetoplastida/genética , Saccharomycetales/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Expresión Génica , Plásmidos/genéticaRESUMEN
Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Kluyveromyces/genética , Lactosa/metabolismo , Proteínas de Transporte de Membrana/genética , Polimorfismo Genético , Secuencia de Aminoácidos , Mapeo Cromosómico , Cromosomas Fúngicos/química , Medios de Cultivo/química , Fermentación , Proteínas Fúngicas/metabolismo , Dosificación de Gen , Cinética , Kluyveromyces/clasificación , Kluyveromyces/enzimología , Proteínas de Transporte de Membrana/metabolismo , Filogenia , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The ability of Zygosaccharomyces bailii to grow at low pH and in the presence of considerable amounts of weak organic acids, at lethal condition for Saccharomyces cerevisiae, increased the interest in the biotechnological potential of the yeast. To understand the mechanism of tolerance and growth effect of weak acids on Z. bailii, we evaluated the physiological and macromolecular changes of the yeast exposed to sub lethal concentrations of lactic acid. Lactic acid represents one of the important commodity chemical which can be produced by microbial fermentation. We assessed physiological effect of lactic acid by bioreactor fermentation using synthetic media at low pH in the presence of lactic acid. Samples collected from bioreactors were stained with propidium iodide (PI) which revealed that, despite lactic acid negatively influence the growth rate, the number of PI positive cells is similar to that of the control. Moreover, we have performed Fourier Transform Infra-Red (FTIR) microspectroscopy analysis on intact cells of the same samples. This technique has been never applied before to study Z. bailii under this condition. The analyses revealed lactic acid induced macromolecular changes in the overall cellular protein secondary structures, and alterations of cell wall and membrane physico-chemical properties.
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Fermentación , Ácido Láctico/metabolismo , Ácido Láctico/toxicidad , Viabilidad Microbiana/efectos de los fármacos , Estrés Fisiológico , Zygosaccharomyces/efectos de los fármacos , Zygosaccharomyces/fisiología , Anaerobiosis , Reactores Biológicos/microbiología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/fisiología , Fenómenos Químicos , Medios de Cultivo/química , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Propidio/análisis , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Coloración y Etiquetado , Zygosaccharomyces/crecimiento & desarrolloRESUMEN
Kluyveromyces marxianus is emerging as a new platform organism for the production of flavour and fragrance (F&F) compounds. This food-grade yeast has advantageous traits, such as thermotolerance and rapid growth, that make it attractive for cell factory applications. The major impediment to its development has been limited fundamental knowledge of its genetics and physiology, but this is rapidly changing. K. marxianus produces a wide array of volatile molecules and contributes to the flavour of a range of different fermented beverages. Advantage is now being taken of this to develop strains for the production of metabolites such as 2-phenylethanol and ethyl acetate. Strains that were selected from initial screens were used to optimize processes for production of these F&F molecules. Most developments have focused on optimizing growth conditions and the fermentation process, including product removal, with future advancement likely to involve development of new strains through the application of evolutionary or rational engineering strategies. This is being facilitated by new genomic and molecular tools. Furthermore, synthetic biology offers a route to introduce new biosynthetic pathways into this yeast for F&F production. Consumer demand for biologically-synthesized molecules for use in foods and other products creates an opportunity to exploit the unique potential of K. marxianus for this cell factory application.
Asunto(s)
Aromatizantes/metabolismo , Kluyveromyces/metabolismo , Compuestos Orgánicos Volátiles/metabolismo , Fermentación , Microbiología Industrial , Kluyveromyces/genéticaRESUMEN
The yeast Kluyveromyces marxianus produces a range of volatile molecules with applications as fragrances or flavours. The purpose of this study was to establish how nutritional conditions influence the production of these metabolites. Four strains were grown on synthetic media, using a variety of carbon and nitrogen sources and volatile metabolites analysed using gas chromatography-mass spectrometry (GC-MS). The nitrogen source had pronounced effects on metabolite production: levels of the fusel alcohols 2-phenylethanol and isoamyl alcohol were highest when yeast extract was the nitrogen source, and ammonium had a strong repressing effect on production of 2-phenylethyl acetate. In contrast, the nitrogen source did not affect production of isoamyl acetate or ethyl acetate, indicating that more than one alcohol acetyl transferase activity is present in K. marxianus. Production of all acetate esters was low when cells were growing on lactose (as opposed to glucose or fructose), with a lower intracellular pool of acetyl CoA being one explanation for this observation. Bioinformatic and phylogenetic analysis of the known yeast alcohol acetyl transferases ATF1 and ATF2 suggests that the ancestral protein Atf2p may not be involved in synthesis of volatile acetate esters in K. marxianus, and raises interesting questions as to what other genes encode this activity in non-Saccharomyces yeasts. Identification of all the genes involved in ester synthesis will be important for development of the K. marxianus platform for flavour and fragrance production.
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Carbono/metabolismo , Aromatizantes/metabolismo , Kluyveromyces/metabolismo , Nitrógeno/metabolismo , Compuestos Orgánicos Volátiles/metabolismo , Ésteres/metabolismo , Aromatizantes/química , Kluyveromyces/química , Kluyveromyces/clasificación , Filogenia , Compuestos Orgánicos Volátiles/químicaRESUMEN
Two Gram-stain-negative, rod-shaped, orange, catalase- and oxidase-positive, non-motile bacteria, designated W13M1A(T) and W15M10(T), were isolated from the marine sponges Suberites carnosus and Leucosolenia sp., respectively, which were sampled from Lough Hyne, Co. Cork, Ireland. Analysis of the 16S rRNA gene sequences of these isolates revealed that they are members of the genus Maribacter, in the family Flavobacteriaceae of the phylum Bacteroidetes. The type strain most closely related to strain W13M1A(T) is Maribacter forsetii DSM 18668(T) with a gene sequence similarity of 96.5%. The closest related type strain to strain W15M10(T) is Maribacter orientalis DSM 16471(T) with a gene sequence similarity of 98.3%. Phylogenetic inference and phenotypic data combined indicate that the isolates represent two novel species of the genus Maribacter, for which the names Maribacter spongiicola sp. nov. with type strain W15M10(T) ( = NCIMB 14725(T) = DSM 25233(T)) and Maribacter vaceletii sp. nov. with type strain W13M1A(T) ( = NCIMB 14724(T) = DSM 25230(T)), are proposed.
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Flavobacteriaceae/clasificación , Filogenia , Poríferos/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Flavobacteriaceae/genética , Flavobacteriaceae/aislamiento & purificación , Irlanda , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.
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Antibacterianos/farmacología , Antifúngicos/farmacología , Haliclona/microbiología , Streptomyces/metabolismo , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Cromatografía Liquida , Genómica , Irlanda , Espectroscopía de Resonancia Magnética , Metabolómica , Streptomyces/genética , Streptomyces/aislamiento & purificaciónRESUMEN
The morphological effects of an antifungal activity produced by Lactobacillus plantarum 16, a malt-production steep water isolate, on two food-associated fungi were examined microscopically. Spore germination was completely inhibited in Aspergillus fumigatus and Rhizopus stolonifer upon treatment with concentrated cell-free supernatant (cCFS) of strain 16. Furthermore, addition of antifungal cCFS to germ tubes and hyphae halted further development compared to untreated controls. Transcriptome analysis of A. fumigatus Af293 following exposure to antifungal cCFS revealed a number of genes with altered transcription involved in a variety of cellular functions, most notably cell metabolism, suggesting a global metabolic shutdown and subsequent cell death. Increased transcription of the global regulator LaeA was also observed indicating that exposure to the antifungal activity caused a cellular stress response.