- Genome sequencing, annotation and exploration of the SO 2 -tolerant non- conventional yeast Saccharomycodes ludwigii. - ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome.. - ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. - Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. - Comparative analysis of the predicted S. - Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. - Full list of author information is available at the end of the article. - ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family.. - The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. - Saccharomycodes ludwigii is a budding yeast belonging to the Saccharomycodeacea family [1], a sister family of the better studied Saccharomycetacea family that, among others, includes the paradigmatic species Saccharomyces cerevisiae. - Like other organic acids that are also explored as preservatives, the antimicrobial po- tential of this inorganic acid is dependent on the con- centration of the undissociated form (generally designated as “molecular SO 2. - ludwigii cells excrete high amounts of the SO 2 -sequestering molecule acetaldehyde, how- ever this response does not seem to account for the en- hanced tolerance of this species since similar excretion rates were observed in susceptible S. - cerevisiae relies on the ac- tivity of the sulfite plasma membrane transporter Ssu1, which is believed to promote the extrusion of metabi- sulfite [21, 22]. - However that sequence was scattered across 1360 contigs rendering difficult to have an accur- ate picture of the genomic portrait of this strain and a realiable extraction of biologically relevant information about S. - To improve this, the genome of the UTAD17 strain was resequenced using PacBio, resulting in a genome assembled in only 20 contigs and a pre- dicted ORFeome of 4528 canonical protein-coding genes, closer to what is reported for other members of the Saccharomycodaceae family (e.g. - This work describes the information ex- tracted from this more refined genomic sequence of the UTAD17 strain shedding light into the biology and physiology of the S. - In order to have a suitable portrait of the genomic archi- tecture of the S. - ludwigii strains [30] and is also in line with what is reported for other members of the Sac- charomycodeacea family [31–33]. - Sequencing with Pac- Bio generated 585,118 reads (with a 445.3 coverage) which were de novo assembled into 20 contigs (with sizes ranging from 8.5 kbp to 2.7 Mbp, see supple- mentary Table S2) and an assembled genome of bp, corresponding to approximately 95% of the estimated genome size for UTAD17. - In the end of the run 7 clearly separated bands, presumed to correspond to the 7 chromosomes of S. - The putative CDSs were distributed throghout 17 of the 20 assembled contigs with genes not being detected only in contigs 14, 16 and 19 (supplementary Table S2). - To get a more functional view of the S. - cerevisiae genes clustered in 12 of the 21 functional COG classes surpassed those of S.. - ludwigii UTAD17 by approximately 20% (details provided in supplementary Table S3), an observation that is consist- ent with the later species being pre-whole genome dupli- cation like the other species of the Saccharomycodeacea family . - ludwigii UTAD17 genome revealed traits found in pre-whole gen- ome duplication species such as disassembly of the genes necessary for allantoine metabolism, absence of galactose catabolism genes and the lack of a functional pathway for de novo nicotinic acid biosynthesis [35]. - Furthermore, out of the 555 ohnologue pairs identified in S. - Comparative analysis of the predicted proteomes of S.. - ludiwgii with members of the Saccharomycetaceae and Saccharomycodeacea families. - ludwigii the predicted ORFeome of the UTAD17 strain was. - 2 Functional categorization of the predicted ORFeome of S. - After annotation of the assembled genomic sequence, the validated gene models were clustered according with the biological function they are predicted to be involved in (using COG functional categories) using the eggNOG-mapper tool (black bars). - As a comparison, the distribution of the S. - osmophila, these representing three species of the Saccharomycodeacea family with an avail- able annotated genomic sequence. - This observation was surprising but somehow also in line with the results obtained by phylogenetic analysis of the the ITS sequence of the strains used in this comparative proteomic analysis that also shows a higher divergence of H. - Thus, it is possible that the observed higher similarity of the proteomes of S. - To capture more specific features of the S. - ludwigii UTAD17, how- ever, this was also present in the genome of the other. - N-acetylglucosamine is a main component of the cell wall of bacteria and fungi, also being present in mannoproteins found at the sur- face of yeasts cells [42]. - ludwigii UTAD17 is equipped with all the genes of the main pathways of central metabolism including the pentose phosphate pathway, glycolysis, gluconeogenesis, Krebs cycle and oxidative phosphorylation, besides the already. - 3 a Comparative analysis of the predicted proteome of the Saccharomycodeacea species S. - ludwigii UTAD17 strain was compared with the one of the Hanseniaspora species that also belong to the Saccharomycodeacea family using pair-wise BLASTP alignments. - ludwigii as no robust homologue could be found in any of the other yeast species considered and also the 201 S. - Some of the functions represented in these two protein datasets are highlighted in this picture, with the complete list being provided in supplementary Table S5. - duelbreckii we could only identify one enzyme for each of the different enzymatic steps re- quired for biosynthesis of 3-HMP-PP, similar to what is reported for Kluveromyces lactis, K. - In fact, until thus far the expansion of en- zymes involved in synthesis of 3-HMP-PP has been de- scribed as a specific feature of the Saccharomyces sensu strictu species that harbor 3 enzymes for the synthesis of 3-HMP-P (Thi5, Thi11, Thi12 and Thi13) and two for the synthesis of 3-HMP-PP. - ludwigii was used as an input in the metabolic networks reconstruction tools eggNOG-mapper and KEEG Koala to gather a schematic representation of the metabolic pathways linked to central carbon and nitrogen metabolism active in S. - The picture schematically represents some of the active pathways identified in this in silico analysis, emphasizing in red proteins that were found in S. - cerevisiae [47], it is in line with previous reports of the inability of the UTAD17 strain, and of S. - ludwigii UTAD17 One of the aspects for which S. - In specific, we could identify two alcohol acetyl-transferases in the genome of the UTAD17 strain, SCLUD4.g700, moderately similar to S. - Further investigations will have to be performed to better understand this observation specially focusing whether this trait is specific of the UTAD17 strain (which by some reason could have the activity of alcohol acetyl-transferase enzymes impaired) or whether this re- sulted from the composition of the grape juice used in the fermentations that could be less favourable for pro- duction of ethyl esters by S. - Consistently, three predicted 2,3-butanediol dehydrogenases (SCLUD5.g30, SCLUD7.g344 and SCLUD8.g438) are found in the gen- ome of the UTAD17 strain (Fig. - Enzymes for the production of higher alco- hols, isoamyl alcohols and a putative β-glucosidase were also detected in the genome of the UTAD17 strain (sup- plementary Table S7).. - Notably, mining of the S. - ludwigii UTAD17 strongly suggests that the composition of the cell wall should be quite different from the one found in other Saccharomycodeacea and in S. - ludwigii is observed either in sulfited grape-musts or in stabilized wines indicates that this yeast is equipped with means to survive the harsh enviroment of vinification which in- clude, among others, the high concentration of sugars present in the beginiging of the fermentation or the high concentrations of ethanol obtained in the end, besides the presence of inhibitory concentrations of SO 2 . - guilliiermondii that only harbored 33% of the. - ethanol-tolerance genes and 35% of the “fermentome”. - ludwigii UTAD17 does not encode a clear ortholo- gue for the transcription factors Msn2 and Msn4 [64], responsible for the control of the environmental stress response in S. - Closer mining of the S. - ludwigii genome allowed us to identify one protein (SCLUD3.g330) that shows similar- ity at the level of the C-terminal domain (67% identity) to the C-terminus of ScMsn2 and ScMsn4, the region that comprises the DNA binding domain of these regu- lators [67] (supplementary Figure S4). - cerevisiae has been largely at- tributed to the activity of the sulfite export pump Ssu which was also found to influence tolerance to this preservative in the more tolerant strain Brettano- myces bruxellensis [23]. - bruxellensis strains were found to encode alleles with higher activity of SSU1 than those encoded by less susceptible strains, this being attributed to the existence of point mutations that result in increased activity of the pumps [23]. - In this sense, it will also be interesting to investigate if a similar trait is observed in the case of the SSU1 genes encoded by S. - cerevisiae transcription of SSU1 is largely dependent of the transcriptional activator Fzf1 [70] but we could not identify an orthologue for this regulator in the genome of S. - bruxel- lensis) suggesting that in this species the control of the sulfite efflux pump could be under the control of a dif- ferent regulatory circuit.. - ludwigii, as suggested by mining the genome of the UTAD17 strain. - ludwigii cells, as suggested by mining of the genome of the UTAD17 strain, were selected and are herein highlighted. - It is also hypothesized whether the presumed different structure of the S. - ludwigii cell wall, resulting from this species harboring a set of mannoproteins and a different structure of the β -glucan (compared to the one exhibited by in other Saccharomycodeacea species and by Saccharomycetacea) can contribute for the reported reduced diffusion of SO 2 into the inside of S. - Around 65% of the other SO 2 -resistance genes identified in S. - ludwigii is the different structure of the cell wall which, as discussed above, is likely to be enriched in β-1,2-mannosides due to the presence of β- mannosyltransferases. - In this work we have deepened the genomic sequence and annotation of the wine spoilage species S. - ludwigii UTAD17 shedding light into relevant aspects of the biol- ogy and physiology of this species such as its high resili- ence to the wine preservative SO 2 or its resilience to thrive in the challenging environment of the wine must.. - The autochthonous Saccharomycodes ludwigii UTAD17 strain, part of the collection of wine strains owned by the UTAD laboratory, was isolated in a wine must ob- tained with grapes harvested from the Douro demar- cated region harvested from the experimental vineyard of UTAD (with approved use for research), in Portugal [24, 48]. - Presumptive identification of UTAD17 as a non-Saccharomyces strain was based on the ability of these cells to grow on L-lysine agar selective medium, followed by microscopic examination of the size and morphology of the colonies. - Further confirmation of the identity of UTAD17 as belonging to the S. - ludwigii spe- cies was performed by Sanger sequencing of the con- served D1/D2 ribosomal region using the NL1 (5′- GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) primers which showed more than 99.9% identity with ribosomal se- quences reported from other S. - The completeness of the genomics data was assessed using BUSCO11 (version 4.1.2, run in mode genome and proteome with the lineage dataset:. - The annotation of the 20 curated PacBio contigs was performed in the Geneious software framework (version 2019.2.3). - nidulans) was used to identify putative CDSs in the sequence of the contigs. - Metabolic reconstruction of S.ludwigii UTAD17 was per- formed making use of KEGG BlastKoala annotation tool [44]. - For the comparative ana- lysis of the S. - Additional file 1 Supplementary figures – This file has included a set of 5 supplementary figures providing: a dendrogram comparing the ITS region of the yeast strains used for the comparative proteomic analysis performed (Figure S1), phographs obtained by microscopic imaging of S. - ludwigii UTAD17 cells cultivated in minimal medium having glucose or GlcNAc as the sole carbon source (Figure S2), a functional distribution of the S. - ludwigii UTAD17 ORFeome, as performed by the BLASTKoala Annotation tool (Figure S3), the alignment of the protein sequence of the transcription factors ScMsn2, ScCom2 and the S. - ludwigii predicted regulator SCLUD3.g33 (Figure S4), the alignment of the protein sequence of the efflux pump ScSsu1 and of the four predicted S. - Additional file 3 Original PFGE gel used in the karyotyping of the S.. - MJT performed all the experimental work described in the paper and conducted the ab initio annotation as well as the exploration of the biologically relevant information from the assembled and annotated genomic sequence. - The sequence of the assembled 20 contigs and the annotation performed were submitted at NCBI (Bioproject PRJNA542099. - Oenological impact of the Hanseniaspora/Kloeckera yeast genus on wines — a review. - Genome sequence of the wine yeast Saccharomycodes ludwigi UTAD17.. - Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera. - Genome sequence of the non-conventional wine yeast Hanseniaspora guilliermondii UTAD222 unveils relevant traits of this species and of the Hanseniaspora genus in the context of wine fermentation. - Phylogenetic relationships among yeasts of the. - Peculiarities of the regulation of fermentation and respiration in the Crabtree-negative, xylose-fermenting yeast Pichia stipitis. - Lectin-mediated flocculation of the yeast Saccharomycodes ludwigii NCYC 734. - Characterizing the potential of the non-conventional yeast Saccharomycodes ludwigii UTAD17 in winemaking. - Genome-wide identification of the Fermentome. - Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response
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