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- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(author)
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Genomic and transcriptomic analysis of Candida intermedia reveals genes for utilization of biotechnologically important carbon sources
- 2019
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Conference paper (other academic/artistic)abstract
- A future biobased society relies on efficient industrial microorganisms that can convert all sugars from agricultural, forestry and industrial waste streams into fuels, chemicals and materials. To be able to tailor-make such potent cell factories, we need a far better understanding of the proteins responsible for the assimilation of biotechnologically important carbon sources including pentoses, disaccharides and oligomers. The yeast Candida intermedia, known for its superior growth on xylose owing to its efficient uptake and conversion systems, can also utilize a range of other important carbon sources such as cellobiose, galactose and lactose. The aim of this project was to identify the genomic determinants for the utilization of these mono- and disaccharides in our in-house isolated C. intermedia strain CBS 141442. Genome sequencing and transcriptional (RNA seq) data analysis during growth in defined medium supplemented with glucose, xylose, galactose, lactose or cellobiose, revealed numerous distinct clusters of coregulated genes. By scanning the CBS 141442 genome for genes encoding Major Facilitator Superfamily (MFS) sugar transporters, and the RNA-seq dataset for the corresponding transcriptional profiles, we identified several novel genes encoding putative xylose transporters and multiple Lac12-like transporters likely involved in the uptake of disaccharides in C. intermedia. We also found that the yeast possesses no less than three genes encoding aldose reductases with different transcriptional profiles, and heterologous expression of the genes in Saccharomyces cerevisiae showed that the aldose reductases have different substrate and co-factor specificities, suggesting diverse physiological roles. Taken together, the results of this study provide insights into the mechanisms underlying carbohydrate metabolism in C. intermedia, and reveals several genes with potential future applications in cell factory development.
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| 2. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(author)
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Genomic and transcriptomic analysis of Candida intermedia reveals important genes for xylose utilization
- 2018
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Conference paper (other academic/artistic)abstract
- The urgency to reduce carbon emissions and to lower our dependence on oil makes it necessary to strive towards a more sustainable bio-based economy, where energy, chemicals, materials and food are produced from renewable resources. Lignocellulosic biomass constitutes a great source of raw material for such a future bio-based economy since it is widely available, relatively inexpensive and do not compete with food and feed production. The pentose D-xylose, the second most prevalent sugar in lignocellulose after glucose, is an underutilized resource, in large due to the inefficient fermentation of this sugar by the most industrially relevant microorganisms (e.g. Saccharomyces cerevisiae). Thus, development of microorganisms that can ferment all lignocellulosic sugars is of foremost importance for economically viable production processes. Native xylose-utilizing yeasts represent a major source of knowledge and genes for xylose uptake and assimilation that can be transferred to S. cerevisiae. The yeast Candida intermedia is an interesting candidate to characterize further, as it displays a high xylose transport capacity and multiple xylose reductases, of which one appears to prefer NADH over NAPDH. Furthermore, the C. intermedia strain CBS 141442, isolated in the liquid fraction of wheat straw hydrolysate in our laboratory as a contaminant of a xylose fermenting population of S. cerevisiae, is capable of glucose and xylose co-fermentation under certain conditions. The aim of this study was to elucidate the genetic features that are the basis of the xylose utilization capacity of C. intermedia CBS141442. PacBio sequencing and de novo assembly of the genome revealed a haploid yeast with a genome size of 13.2 Mb and a total of 5936 protein-coding genes spread over seven chromosomes. In order to gain insight on the genes involved in the utilization of xylose, we analysed the changes in the transcriptome of C. intermedia CBS141442 during growth in xylose and glucose (as reference condition). Cells were collected in mid-exponential phase at the maximum growth rate when no metabolites were accumulating. The total RNA was extracted and cDNA libraries were prepared after polyA selection. Each sample was sequenced in an Illumina HiSeq2500 system with an average cover of 5-20 million reads. The analysis of the differential expression data lead to the identification of two new genes potentially encoding xylose transporters and no less than three xylose reductases genes with different expression patterns. The xylose reductase genes were heterologously expressed in S. cerevisiae to determine their co-factor preferences and substrate specificities. Whereas two of them are strictly NADPH-dependent, the third can use both co-factors and shows preference for NADH. The heterologous expression of this gene can improve the capacity of S. cerevisiae to ferment xylose, and thus contribute to a more efficient use of lignocellulosic biomass.
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| 3. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(author)
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Lipid accumulation in wild Saccharomyces cerevisiae strains and their application for biodiesel
- 2018
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Conference paper (other academic/artistic)abstract
- Our dependence on fossil fuels has severe consequences for both the environment and national economies worldwide. Considerable effort is being made in Europe and elsewhere to develop cheaper and cleaner energy sources. Biodiesel is one of the main alternatives, being renewable and environment-friendly, and with a wide acceptance in the European transport sector. However, its production is still mostly based on vegetable oils, leading to competition with the food industry and as a consequence to price increases of raw materials. Alternative feedstocks are required to ensure the sustainability of the biodiesel industry. Oils produced by microorganisms are currently the most promising feedstock for sustainable biodiesel production. Yeast is attracting considerable attention, but the low productivity of these microorganisms on low or negative value substrates, such as lignocellulose-enriched residues, still hinders the industrial application of microbial oils. The development of robust yeast strains with increased oil yield and resistance to the different inhibitors present in the substrates is therefore one of the most important steps to improve the feasibility of microbial biodiesel [1]. Bioprospecting of wild environments frequently yields microorganisms with high resistance to a wide range of environmental stresses. Spontaneous fermentations of spirits are often associated with yeast strains adapted to high temperatures and capable of using multiple substrates [2]. In a previous study, yeast strains isolated from spontaneous cachaça fermentation vessels in Brazil exhibited lipid accumulation in the presence of biodiesel-derived glycerol (unpublished results). In this project, wild Saccharomyces cerevisiae strains isolated from the same environment were screened for their potential to produce biodiesel from lignocellulosic residues. The strains that displayed high resistance to common lignocellulosic inhibitors (viz. acetic acid, furfural and 5-hydromethoxyfurfural), were further assessed for lipid production. The strains’ lipid accumulation profiles were evaluated by adding the lipid droplet-specific fluorescent dye BODIPY493/503 to the growth media and measuring growth and fluorescence emission over time using the Biolector. The effects of carbon-to-nitrogen ratio (C/N) and temperature on biomass production and lipid accumulation were also assessed, in order to define the optimal lipid accumulation-inducing conditions for each strain. The best performing strains were further characterized using GC-MS and GC-FID, to identify and quantify the main fatty acids present in the lipids. The best performing S. cerevisiae strains show high potential for application in biodiesel production, presenting high lipid accumulation over a wide range of C/N and temperatures.
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| 4. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(author)
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Split-marker recombination for efficient targeted gene deletions in Candida intermedia
- 2018
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Conference paper (other academic/artistic)abstract
- Candida intermedia is a non-conventional yeast species with a natural ability to produce ethanol from xylose, making it an attractive non-GMO alternative for lignocellulosic biomass conversion in biorefineries and/or gene donor to Saccharomyces cerevisiae to improve its xylose fermentation capacity. We have de novo genome sequenced the C. intermedia strain CBS 141442, previously isolated in our lab, which allows us to study the yeast at a genomic and molecular level. The aim of this project was to develop a molecular toolbox for C. intermedia to enable also targeted genome editing and subsequent mutant phenotyping. C. intermedia is a haploid yeast belonging to the CTG clade of fungal species, and thus requires drug-resistant markers adapted for the alternative codon usage of these organisms. Transformation of linearized plasmid containing the CaNAT1 marker flanked by the TEF1 promoter and terminator from Ashbya gossypii [1] resulted in hundreds of Nourseothricin-resistant transformants. We then constructed an ADE2-deletion cassette, where the CaNAT1 marker was fused to the upstream and downstream sequences (1000bp) of CiADE2. Transformations resulted in less than 1% of ade2 mutants with the characteristic red pigmentation, which indicates that the non-homologous end joining pathway (NHEJ) is dominant over the homologous recombination (HR) pathway in this yeast. Using the cell cycle inhibitor hydroxyurea to arrest cells in the S-phase has been shown to improve the HR/NHEJ ratio in other yeasts [2], and increased the ADE2 deletion efficiency to 4% in C. intermedia. To further improve the targeted deletion rate, we applied the "split-marker” strategy previously developed for Saccharomyces cerevisiae [3]. Here, the selectable marker gene is truncated in two different fragments, and the gene is not functional until homologous recombination takes place between the two overlapping parts of the fragments. The truncated marker gene fragments were flanked by homologous sequences (1000 bp) upstream and downstream of the target gene using fusion PCR, thereby avoiding a tedious cloning step. This approach increased the targeted gene disruption of ADE2 to 56%. As proof of concept, the method was also used to delete KU70, the xylose reductase gene XYL1_2 as well as a large gene cluster in C. intermedia, with allele-specific HR efficiencies between 87 and 100%. The split-marker approach for targeted gene-disruptions will pave the way for high throughput genetic analysis in C. intermedia as well as in other yeasts where NHEJ is the predominant form of recombination.
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| 5. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(author)
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Wild Saccharomyces cerevisiae lipid accumulation in lignocellulosic hydrolysate
- 2019
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Conference paper (other academic/artistic)abstract
- Microbial oils are currently the most promising feedstock for sustainable biodiesel production. Yeast is attracting considerable attention, but its low productivity on cheap crude substrates, such as lignocellulose-enriched residues, still hinders the industrial application of such oils. The development of robust yeast strains with increased oil yield and resistance to the different inhibitors present in the substrates is therefore one of the most important steps to improve the feasibility of microbial biodiesel. Bioprospecting of wild environments frequently yields microorganisms with high resistance to a wide range of environmental stresses. In this project, wild S. cerevisiae strains isolated from spontaneous cachaça fermentation vessels in Brazil were screened for their potential to produce biodiesel from lignocellulosic residues. The strains that displayed high resistance to common lignocellulosic inhibitors were further assessed for lipid production in wheat straw hydrolysate. The strains’ lipid accumulation profiles were evaluated by measuring growth and fluorescence emission, after addition of lipid-specific fluorescent dye BODIPY. The effects of carbon-to-nitrogen ratio (C/N) and temperature on biomass production and lipid accumulation were also assessed, in order to define the optimal lipid accumulation-inducing conditions for each strain. The best performing strains were further characterized in wheat straw hydrolysate and the lipid profile and accumulation assessed using GC-MS and GC-FID, to identify and quantify the main fatty acids present in the lipids.
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| 6. |
- Geijer, Cecilia, 1980, et al.
(author)
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Genomic and transcriptomic analysis of Candida intermedia reveals the genetic determinants for its xylose-converting capacity
- 2020
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 13:1
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Journal article (peer-reviewed)abstract
- Background An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. Results To understand the genetic determinants that underlie the metabolic properties of C. intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. Conclusions In the present study, we performed the first genomic and transcriptomic analysis of C. intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.
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| 7. |
- Lorantfy, Bettina, 1986, et al.
(author)
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Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures
- 2019
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In: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 46:1, s. 33-43
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Journal article (peer-reviewed)abstract
- Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacSgene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.
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