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1. A global metagenomic map of urban microbiomes and antimicrobial resistance.

Author

Danko, David, Bezdan, Daniela, Afshin, Evan E, Ahsanuddin, Sofia, Bhattacharya, Chandrima, Butler, Daniel J, Chng, Kern Rei, Donnellan, Daisy, Hecht, Jochen, Jackson, Katelyn, Kuchin, Katerina, Karasikov, Mikhail, Lyons, Abigail, Mak, Lauren, Meleshko, Dmitry, Mustafa, Harun, Mutai, Beth, Neches, Russell Y, Ng, Amanda, Nikolayeva, Olga, Nikolayeva, Tatyana, Png, Eileen, Ryon, Krista A, Sanchez, Jorge L, Shaaban, Heba, Sierra, Maria A, Thomas, Dominique, Young, Ben, Abudayyeh, Omar O, Alicea, Josue, Bhattacharyya, Malay, Blekhman, Ran, Castro-Nallar, Eduardo, Cañas, Ana M, Chatziefthimiou, Aspassia D, Crawford, Robert W, De Filippis, Francesca, Deng, Youping, Desnues, Christelle, Dias-Neto, Emmanuel, Dybwad, Marius, Elhaik, Eran, Ercolini, Danilo, Frolova, Alina, Gankin, Dennis, Gootenberg, Jonathan S, Graf, Alexandra B, Green, David C, Hajirasouliha, Iman, Hastings, Jaden JA, Hernandez, Mark, Iraola, Gregorio, Jang, Soojin, Kahles, Andre, Kelly, Frank J, Knights, Kaymisha, Kyrpides, Nikos C, Łabaj, Paweł P, Lee, Patrick KH, Leung, Marcus HY, Ljungdahl, Per O, Mason-Buck, Gabriella, McGrath, Ken, Meydan, Cem, Mongodin, Emmanuel F, Moraes, Milton Ozorio, Nagarajan, Niranjan, Nieto-Caballero, Marina, Noushmehr, Houtan, Oliveira, Manuela, Ossowski, Stephan, Osuolale, Olayinka O, Özcan, Orhan, Paez-Espino, David, Rascovan, Nicolás, Richard, Hugues, Rätsch, Gunnar, Schriml, Lynn M, Semmler, Torsten, Sezerman, Osman U, Shi, Leming, Shi, Tieliu, Siam, Rania, Song, Le Huu, Suzuki, Haruo, Court, Denise Syndercombe, Tighe, Scott W, Tong, Xinzhao, Udekwu, Klas I, Ugalde, Juan A, Valentine, Brandon, Vassilev, Dimitar I, Vayndorf, Elena M, Velavan, Thirumalaisamy P, Wu, Jun, Zambrano, María M, Zhu, Jifeng, Zhu, Sibo, Mason, Christopher E, and International MetaSUB Consortium

Subjects
International MetaSUB Consortium, AMR, BGC, NGS, antimicrobial resistance, built Environment, de novo assembly, global health, metagenome, microbiome, shotgun sequencing, Antimicrobial Resistance, Biotechnology, Human Genome, Genetics, Infection, Developmental Biology, Biological Sciences, Medical and Health Sciences
Abstract

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Published
2021

4. The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis

Author

Baumann, Kristin, Dato, Laura, Graf, Alexandra B, Frascotti, Gianni, Dragosits, Martin, Porro, Danilo, Mattanovich, Diethard, Ferrer, Pau, and Branduardi, Paola

Abstract

Abstract Background Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains. In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. Results The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. Conclusions The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Published
2011

5. Customizing amino acid metabolism of Pichia pastoris for recombinant protein production

Author

Rußmayer, Hannes, Buchetics, Markus, Mattanovich, Matthias, Neubauer, Stefan, Steiger, Matthias, Graf, Alexandra B., Koellensperger, Gunda, Hann, Stephan, Sauer, Michael, Gasser, Brigitte, Mattanovich, Diethard, Rußmayer, Hannes, Buchetics, Markus, Mattanovich, Matthias, Neubauer, Stefan, Steiger, Matthias, Graf, Alexandra B., Koellensperger, Gunda, Hann, Stephan, Sauer, Michael, Gasser, Brigitte, and Mattanovich, Diethard

Abstract

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.

Published
2023

10. Additional file 4 of Tamock: simulation of habitat-specific benchmark data in metagenomics

Author

Gerner, Samuel M., Graf, Alexandra B., and Rattei, Thomas

Abstract

Additional file 4: Supplementary Figure 4. The total length of aligned contigs for genome bin 36 compared to the top five Bifidobacterium strains classified in theiHMP2 stool sample J00827 (Benchmark data set) are shown. The total aligned length of all contigs sums up to 1875 kbps, constituting for 89.73% genome coverage for GCF_000010425.1 (Bifidobacterium adolescentis ATCC 15703). The figure is produced with MetaQUAST v5.0.2 (Mikheenko et al. 2016).

Published
2021
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12. Additional file 3 of The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis

Author

Baumann, Kristin, Dato, Laura, Graf, Alexandra B, Frascotti, Gianni, Dragosits, Martin, Porro, Danilo, Mattanovich, Diethard, Ferrer, Pau, and Branduardi, Paola

Abstract

Additional file 3:MetaCyc data Pichia pastoris. Regulated P. pastoris pathways in hypoxia vs. normoxia. Individual P. pastoris (recombinant strain) pathways that were transcriptionally regulated (i e. exceeding the log2 FC threshold of 0.59) in the comparison hypoxic vs. normoxic conditions, as resulting from the MetaCyc analysis presented in Figure 3 http://pathway.yeastgenome.org . Pathway numbers in the first column are referred to Figure 3. Pathway diagrams show all the intermediates of each pathways; reaction lines and the corresponding genes are colour-coded (three colour bins) according to the fold change threshold: red for upregulated, yellow for downregulated and blue for unregulated; log2 FC for each gene are also shown in colour. Last column contains the extended enzyme names corresponding to each gene of the pathway. (DOC 949 KB)

Published
2020
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17. Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment

Author

Chng, Kern Rei, Li, Chenhao, Bertrand, Denis, Ng, Amanda Hui Qi, Kwah, Junmei Samantha, Low, Hwee Meng, Tong, Chengxuan, Natrajan, Maanasa, Zhang, Michael Hongjie, Xu, Licheng, Ko, Karrie Kwan Ki, Ho, Eliza Xin Pei, Av-Shalom, Tamar, Teo, Jeanette Woon Pei, Khor, Chiea Chuen, Chen, Swaine L., Mason, Christopher E., Ng, Oon Tek, Marimuthu, Kalisvar, Ang, Brenda, Nagarajan, Niranjan, Danko, David, Bezdan, Daniela, Afshinnekoo, Ebrahim, Ahsanuddin, Sofia, Bhattacharya, Chandrima, Butler, Daniel J., De Filippis, Francesca, Hecht, Jochen, Kahles, Andre, Karasikov, Mikhail, Kyrpides, Nikos C., Leung, Marcus H. Y., Meleshko, Dmitry, Mustafa, Harun, Mutai, Beth, Neches, Russell Y., Ng, Amanda, Nieto-Caballero, Marina, Nikolayeva, Olga, Nikolayeva, Tatyana, Png, Eileen, Sanchez, Jorge L., Shaaban, Heba, Sierra, Maria A., Tong, Xinzhao, Young, Ben, Alicea, Josue, Bhattacharyya, Malay, Blekhman, Ran, Castro-Nallar, Eduardo, Canas, Ana M., Chatziefthimiou, Aspassia D., Crawford, Robert W., Deng, Youping, Desnues, Christelle, Dias-Neto, Emmanuel, Donnellan, Daisy, Dybwad, Marius, Elhaik, Eran, Ercolini, Danilo, Frolova, Alina, Graf, Alexandra B., Green, David C., Hajirasouliha, Iman, Hernandez, Mark, Iraola, Gregorio, Jang, Soojin, Jones, Angela, Kelly, Frank J., Knights, Kaymisha, Labaj, Pawel P., Lee, Patrick K. H., Shawn, Levy, Ljungdahl, Per, Lyons, Abigail, Mason-Buck, Gabriella, McGrath, Ken, Mongodin, Emmanuel F., Moraes, Milton Ozorio, Noushmehr, Houtan, Oliveira, Manuela, Ossowski, Stephan, Osuolale, Olayinka O., Ozcan, Orhan, Paez-Espino, David, Rascovan, Nicolas, Richard, Hugues, Raetsch, Gunnar, Schriml, Lynn M., Semmler, Torsten, Sezerman, Osman U., Shi, Leming, Song, Le Huu, Suzuki, Haruo, Court, Denise Syndercombe, Thomas, Dominique, Tighe, Scott W., Udekwu, Klas, Ugalde, Juan A., Valentine, Brandon, Vassilev, Dimitar, Vayndorf, Elena, Velavan, Thirumalaisamy P., Zambrano, Maria M., Zhu, Jifeng, Zhu, Sibo, Chng, Kern Rei, Li, Chenhao, Bertrand, Denis, Ng, Amanda Hui Qi, Kwah, Junmei Samantha, Low, Hwee Meng, Tong, Chengxuan, Natrajan, Maanasa, Zhang, Michael Hongjie, Xu, Licheng, Ko, Karrie Kwan Ki, Ho, Eliza Xin Pei, Av-Shalom, Tamar, Teo, Jeanette Woon Pei, Khor, Chiea Chuen, Chen, Swaine L., Mason, Christopher E., Ng, Oon Tek, Marimuthu, Kalisvar, Ang, Brenda, Nagarajan, Niranjan, Danko, David, Bezdan, Daniela, Afshinnekoo, Ebrahim, Ahsanuddin, Sofia, Bhattacharya, Chandrima, Butler, Daniel J., De Filippis, Francesca, Hecht, Jochen, Kahles, Andre, Karasikov, Mikhail, Kyrpides, Nikos C., Leung, Marcus H. Y., Meleshko, Dmitry, Mustafa, Harun, Mutai, Beth, Neches, Russell Y., Ng, Amanda, Nieto-Caballero, Marina, Nikolayeva, Olga, Nikolayeva, Tatyana, Png, Eileen, Sanchez, Jorge L., Shaaban, Heba, Sierra, Maria A., Tong, Xinzhao, Young, Ben, Alicea, Josue, Bhattacharyya, Malay, Blekhman, Ran, Castro-Nallar, Eduardo, Canas, Ana M., Chatziefthimiou, Aspassia D., Crawford, Robert W., Deng, Youping, Desnues, Christelle, Dias-Neto, Emmanuel, Donnellan, Daisy, Dybwad, Marius, Elhaik, Eran, Ercolini, Danilo, Frolova, Alina, Graf, Alexandra B., Green, David C., Hajirasouliha, Iman, Hernandez, Mark, Iraola, Gregorio, Jang, Soojin, Jones, Angela, Kelly, Frank J., Knights, Kaymisha, Labaj, Pawel P., Lee, Patrick K. H., Shawn, Levy, Ljungdahl, Per, Lyons, Abigail, Mason-Buck, Gabriella, McGrath, Ken, Mongodin, Emmanuel F., Moraes, Milton Ozorio, Noushmehr, Houtan, Oliveira, Manuela, Ossowski, Stephan, Osuolale, Olayinka O., Ozcan, Orhan, Paez-Espino, David, Rascovan, Nicolas, Richard, Hugues, Raetsch, Gunnar, Schriml, Lynn M., Semmler, Torsten, Sezerman, Osman U., Shi, Leming, Song, Le Huu, Suzuki, Haruo, Court, Denise Syndercombe, Thomas, Dominique, Tighe, Scott W., Udekwu, Klas, Ugalde, Juan A., Valentine, Brandon, Vassilev, Dimitar, Vayndorf, Elena, Velavan, Thirumalaisamy P., Zambrano, Maria M., Zhu, Jifeng, and Zhu, Sibo

Abstract

Although disinfection is key to infection control, the colonization patterns and resistomes of hospital-environment microbes remain underexplored. We report the first extensive genomic characterization of microbiomes, pathogens and antibiotic resistance cassettes in a tertiary-care hospital, from repeated sampling (up to 1.5 years apart) of 179 sites associated with 45 beds. Deep shotgun metagenomics unveiled distinct ecological niches of microbes and antibiotic resistance genes characterized by biofilm-forming and human-microbiome-influenced environments with corresponding patterns of spatiotemporal divergence. Quasi-metagenomics with nanopore sequencing provided thousands of high-contiguity genomes, phage and plasmid sequences (>60% novel), enabling characterization of resistome and mobilome diversity and dynamic architectures in hospital environments. Phylogenetics identified multidrug-resistant strains as being widely distributed and stably colonizing across sites. Comparisons with clinical isolates indicated that such microbes can persist in hospitals for extended periods (>8 years), to opportunistically infect patients. These findings highlight the importance of characterizing antibiotic resistance reservoirs in hospitals and establish the feasibility of systematic surveys to target resources for preventing infections. Spatiotemporal characterization of microbial diversity and antibiotic resistance in a tertiary-care hospital reveals broad distribution and persistence of antibiotic-resistant organisms that could cause opportunistic infections in a healthcare setting.

Published
2020
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19. A subcellular proteome atlas of the yeast Komagataella phaffii

Author

Valli, Minoska, primary, Grillitsch, Karlheinz, additional, Grünwald-Gruber, Clemens, additional, Tatto, Nadine E, additional, Hrobath, Bernhard, additional, Klug, Lisa, additional, Ivashov, Vasyl, additional, Hauzmayer, Sandra, additional, Koller, Martina, additional, Tir, Nora, additional, Leisch, Friedrich, additional, Gasser, Brigitte, additional, Graf, Alexandra B, additional, Altmann, Friedrich, additional, Daum, Günther, additional, and Mattanovich, Diethard, additional

Published
2020
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21. Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure

Author

Hann Stephan, Köllensperger Gunda, Maurer Michael, Pfeffer Martin, Graf Alexandra B, and Mattanovich Diethard

Subjects
Microbiology, QR1-502
Abstract

Abstract Background The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive 34S labeled sodium sulfate meets both demands. Results We used a novel labeling method with the stable sulfur isotope 34S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g-1 h-1 protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells. Conclusions A novel 34S labeling procedure that enables in vivo quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion.

Published
2011
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22. The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis

Author

Porro Danilo, Dragosits Martin, Frascotti Gianni, Graf Alexandra B, Dato Laura, Baumann Kristin, Mattanovich Diethard, Ferrer Pau, and Branduardi Paola

Subjects
Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains. In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. Results The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. Conclusions The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Published
2011
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23. The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium inaugural meeting report

Author

Afshinnekoo, Ebrahim, Ahsanuddin, Sofia, Ghedin, Elodie, Read, Timothy, Fraser, Claire, Dudley, Joel, Bowler, Christopher, Mason, Christopher E., Chernomoretz, Ariel, Stolovitzky, Gustavo, Łabaj, Paweł P., Graf, Alexandra B., Darling, Aaron, Burke, Catherine, Noushmehr, Houtan, Dias-Neto, Emmanuel, Guo, Yongli, Xie, Zhi, Lee, Patrick K. H., Shi, Leming, Ruiz-Perez, Carlos A., Zambrano, Maria Mercedes, Siam, Rania, Ouf, Amged, Richard, Hugues, Lafontaine, Ingrid, Wieler, Lothar H., Semmler, Torsten, Prithiviraj, Bharath, Nedunuri, Narasimha, Mehr, Shaadi, Banihashemi, Kambiz, Lista, Florigio, Anselmo, Anna, Suzuki, Haruo, Kuroda, Makoto, Yamashita, Riu, Sato, Yukoto, Kaminuma, Eli, Aranda, Celia M. Alpuche, Martinez, Jesus, Dada, Christopher, Dybwad, Marius, Oliveira, Manuela, Schuster, Stephan, Siwo, Geoffrey H., Jang, Soojin, Seo, Sung Chul, Hwang, Sung Ho, Ossowski, Stephan, Bezdan, Daniela, Chaker, Salama, Chatziefthimiou, Aspassia D., Udekwu, Klas, Liungdahl, Per, Sezerman, Ugur, Meydan, Cem, Elhaik, Eran, Gonnet, Gaston, Schriml, Lynn M., Mongodin, Emmanuel, Huttenhower, Curtis, Gilbert, Jack, Eisen, Jonathan, Hirschberg, David, Hernandez, Mark, McGrath, Ken, McGrath, Leanne, Gray, Andrew, Osuolale, Olayinka, Segata, Nicola, Fillo, Silvia, Iraola, Gregorio, Zhou, Yiming, Chang, Yujun, Li, Yang, Zhend, Yuanting, Hou, Wanwan, Ramirez, Adan, Cepeda, Martha, Desnues, Christelle, Rascovan, Nicolas, Baron, Colin, Nagarajan, Niranjan, Ercolini, Danilo, Menary, Wayne, Tighe, Scott, Donia, Mohamed, Levy, Shawn, Benito, Joseph, Jones, Angela, Kasarskis, Andrew, Maritz, Julia, Jorgensen, Ellen, Neches, Russell, Livelli, Tom, Barnetche, Jesus Martinez, Pasolli, Edoardo, Greenfield, Nick, Hasan, Nur, Brownstein, John, Nozick, Linda, Michels, Harold, Schriml, Lynn, Brownstein, Catherine, Garbarino, Jeanne, Lyons, Abby, Zhu, Jeff, Genome Center [UC Davis], University of California [Davis] (UC Davis), University of California (UC)-University of California (UC), American University in Cairo, Biologie Computationnelle et Quantitative = Laboratory of Computational and Quantitative Biology (LCQB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Robert Koch Institute [Berlin] (RKI), Centre for Infection Medicine, Institute of Microbiology and Epizootics, Health Corps Italian Army, Department of Molecular Biology, Immunology and Experimental Medicine, Army Medical and Veterinary Research Center, Integrative Biology Unit, Parco Tecnologico Padano, Department of Environmental Science and Engineering, Graduate School of Science and Engineering, Department of Population Medicine and Diagnostic Sciences, Cornell University [New York], Dynamique cellulaire et moléculaire de la muqueuse respiratoire, Université de Reims Champagne-Ardenne (URCA)-IFR53-Institut National de la Santé et de la Recherche Médicale (INSERM), Biostatistics Department, Harvard School of Public Health, Institute for genomic and systems biology, Argonne National Laboratory [Lemont] (ANL), Department of Ecology and Evolution [Chicago], University of Chicago, Alfred P. Sloan Foundation [2015-13964], National Institutes of Health [F31GM111053, R01NS076465, R25EB020393], Promega, CosmosID, Illumina, Copan, QIAGEN, University of California-University of California, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Acibadem University Dspace, Afshinnekoo, Ebrahim, Ahsanuddin, Sofia, Ghedin, Elodie, Read, Timothy, Fraser, Claire, Dudley, Joel, Bowler, Christopher, Mason, Christopher E., Chernomoretz, Ariel, Stolovitzky, Gustavo, Łabaj, Paweł P., Graf, Alexandra B., Darling, Aaron, Burke, Catherine, Noushmehr, Houtan, Dias-Neto, Emmanuel, Guo, Yongli, Xie, Zhi, Lee, Patrick K. H., Shi, Leming, Ruiz-Perez, Carlos A., Zambrano, Maria Mercede, Siam, Rania, Ouf, Amged, Richard, Hugue, Lafontaine, Ingrid, Wieler, Lothar H., Semmler, Torsten, Prithiviraj, Bharath, Nedunuri, Narasimha, Mehr, Shaadi, Banihashemi, Kambiz, Lista, Florigio, Anselmo, Anna, Suzuki, Haruo, Kuroda, Makoto, Yamashita, Riu, Sato, Yukoto, Kaminuma, Eli, Aranda, Celia M. Alpuche, Martinez, Jesu, Dada, Christopher, Dybwad, Mariu, Oliveira, Manuela, Schuster, Stephan, Siwo, Geoffrey H., Jang, Soojin, Seo, Sung Chul, Hwang, Sung Ho, Ossowski, Stephan, Bezdan, Daniela, Chaker, Salama, Chatziefthimiou, Aspassia D., Udekwu, Kla, Liungdahl, Per, Sezerman, Ugur, Meydan, Cem, Elhaik, Eran, Gonnet, Gaston, Schriml, Lynn M., Mongodin, Emmanuel, Huttenhower, Curti, Gilbert, Jack, Eisen, Jonathan, Hirschberg, David, Hernandez, Mark, Mcgrath, Ken, Mcgrath, Leanne, Gray, Andrew, Osuolale, Olayinka, Segata, Nicola, Fillo, Silvia, Iraola, Gregorio, Zhou, Yiming, Chang, Yujun, Li, Yang, Zhend, Yuanting, Hou, Wanwan, Ramirez, Adan, Cepeda, Martha, Desnues, Christelle, Rascovan, Nicola, Baron, Colin, Nagarajan, Niranjan, Ercolini, Danilo, Menary, Wayne, Tighe, Scott, Donia, Mohamed, Levy, Shawn, Benito, Joseph, Jones, Angela, Kasarskis, Andrew, Maritz, Julia, Jorgensen, Ellen, Neches, Russell, Livelli, Tom, Barnetche, Jesus Martinez, Pasolli, Edoardo, Greenfield, Nick, Hasan, Nur, Brownstein, John, Nozick, Linda, Michels, Harold, Schriml, Lynn, Brownstein, Catherine, Garbarino, Jeanne, Lyons, Abby, and Zhu, Jeff

Subjects
0301 basic medicine, Microbiology (medical), Built environment, Antimicrobial resistance markers, [SDV]Life Sciences [q-bio], 030106 microbiology, BIOINFORMÁTICA, Biosynthetic gene cluster, Genomics, Biology, Meeting Report, Microbiology, Biosynthetic gene clusters, Microbiome, Next-generation sequencing, 12. Responsible consumption, Metagenomic, 03 medical and health sciences, Data visualization, Urban planning, Databases, Genetic, 11. Sustainability, City Planning, Architecture, Environmental planning, Ecosystem, Metadesign, business.industry, Antimicrobial resistance marker, Biotechnology, 030104 developmental biology, Research Design, 13. Climate action, Metagenomics, Public Health, Sample collection, business, Human
Abstract

International audience; The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium is a novel, interdisciplinary initiative comprised of experts across many fields, including genomics, data analysis, engineering, public health, and architecture. The ultimate goal of the MetaSUB Consortium is to improve city utilization and planning through the detection, measurement, and design of metagenomics within urban environments. Although continual measures occur for temperature, air pressure, weather, and human activity, including longitudinal, cross-kingdom ecosystem dynamics can alter and improve the design of cities. The MetaSUB Consortium is aiding these efforts by developing and testing metagenomic methods and standards, including optimized methods for sample collection, DNA/RNA isolation, taxa characterization, and data visualization. The data produced by the consortium can aid city planners, public health officials, and architectural designers. In addition, the study will continue to lead to the discovery of new species, global maps of antimicrobial resistance (AMR) markers, and novel biosynthetic gene clusters (BGCs). Finally, we note that engineered metagenomic ecosystems can help enable more responsive, safer, and quantified cities.

Published
2016

26. In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response

Author

Rebnegger, Corinna, Graf, Alexandra B, Valli, Minoska, Steiger, Matthias G, Gasser, Brigitte, Maurer, Michael, and Mattanovich, Diethard

Subjects
Filamentous growth, Unfolded protein response, Fungal Proteins, Recombinant protein secretion, Gene Expression Regulation, Fungal, Specific growth rate, Models, Biological, Research Articles, Yeast, Pichia, Recombinant Proteins
Abstract

Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h(-1) . At very slow growth (μ = 0.015 h(-1) ) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes.

Published
2013

28. Pichia Pastoris exhibits high viability and a low maintenance energy requirement at near-zero specific growth rates

Author

Rebnegger, Corinna, Vos, Tim, Graf, Alexandra B., Valli, Minoska, Pronk, Jack T., Daran-Lapujade, Pascale, and Mattanovich, Diethard

Subjects
Fungal Proteins, Glycerol, Kinetics, Glucose, Methanol, Trehalose, Biomass, Energy Metabolism, Pichia, Biotechnology, Transcription Factors
Abstract

The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1 to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than the mS estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes. IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grown P. pastoris in a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found that P. pastoris decreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.

Published
2016

33. BMC Biology / Systems-level organization of yeast methylotrophic lifestyle

Author

Rußmayer, Hannes, Buchetics, Markus, Gruber, Clemens, Valli, Minoska, Grillitsch, Katlheinz, Modarres, Gerda, Guerrasio, Raffaele, Klavins, Kristaps, Neubauer, Stefan, Drexler, Hedda, Steiger, Matthias, Troyer, Christina, Al Chalabi, Ali, Krebiehl, Guido, Sonntag, Denise, Zellnig, Günther, Daum, Günther, Graf, Alexandra B., Altmann, Friedrich, Koellensperger, Gunda, Hann, Stephan, Sauer, Michael, Mattanovich, Diethard, and Gasser, Brigitte

Published
2015

34. The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium inaugural meeting report

Author

Chernomoretz, Ariel, Stolovitzky, Gustavo, Labaj, Pawel P., Graf, Alexandra B., Darling, Aaron, Burke, Catherine, Noushmehr, Houtan, Moraes, Milton Ozorio, Dias-Neto, Emmanuel, Guo, Yongli, Xie, Zhi, Lee, Patrick, Shi, Leming, Ruiz-Perez, Carlos A., Mercedes Zambrano, Maria, Siam, Rania, Ouf, Amged, Richard, Hugues, Lafontaine, Ingrid, Wieler, Lothar H., Semmler, Torsten, Ahmed, Niyaz, Prithi-viraj, Bharath, Nedunuri, Narasimha, Mehr, Shaadi, Banihashemi, Kambiz, Lista, Florigio, Anselmo, Anna, Suzuki, Haruo, Kuroda, Makoto, Yamashita, Riu, Sato, Yukoto, Kaminuma, Eli, Alpuche Aranda, Celia M., Martinez, Jesus, Dada, Christopher, Dybwad, Marius, Oliveira, Manuela, Schuster, Stephan, Siwo, Geoffrey H., Jang, Soojin, Seo, Sung Chul, Hwang, Sung Ho, Ossowski, Stephan, Bezdan, Daniela, Chaker, Salama, Chatziefthimiou, Aspassia D., Udekwu, Klas, Ljungdahl, Per, Sezerman, Ugur, Meydan, Cem, Elhaik, Eran, Gonnet, Gaston, Schriml, Lynn M., Mongodin, Emmanuel, Huttenhower, Curtis, Gilbert, Jack, Mason, Christopher E., Eisen, Jonathan, Hirschberg, David, Hernandez, Mark, Chernomoretz, Ariel, Stolovitzky, Gustavo, Labaj, Pawel P., Graf, Alexandra B., Darling, Aaron, Burke, Catherine, Noushmehr, Houtan, Moraes, Milton Ozorio, Dias-Neto, Emmanuel, Guo, Yongli, Xie, Zhi, Lee, Patrick, Shi, Leming, Ruiz-Perez, Carlos A., Mercedes Zambrano, Maria, Siam, Rania, Ouf, Amged, Richard, Hugues, Lafontaine, Ingrid, Wieler, Lothar H., Semmler, Torsten, Ahmed, Niyaz, Prithi-viraj, Bharath, Nedunuri, Narasimha, Mehr, Shaadi, Banihashemi, Kambiz, Lista, Florigio, Anselmo, Anna, Suzuki, Haruo, Kuroda, Makoto, Yamashita, Riu, Sato, Yukoto, Kaminuma, Eli, Alpuche Aranda, Celia M., Martinez, Jesus, Dada, Christopher, Dybwad, Marius, Oliveira, Manuela, Schuster, Stephan, Siwo, Geoffrey H., Jang, Soojin, Seo, Sung Chul, Hwang, Sung Ho, Ossowski, Stephan, Bezdan, Daniela, Chaker, Salama, Chatziefthimiou, Aspassia D., Udekwu, Klas, Ljungdahl, Per, Sezerman, Ugur, Meydan, Cem, Elhaik, Eran, Gonnet, Gaston, Schriml, Lynn M., Mongodin, Emmanuel, Huttenhower, Curtis, Gilbert, Jack, Mason, Christopher E., Eisen, Jonathan, Hirschberg, David, and Hernandez, Mark

Abstract

The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium is a novel, interdisciplinary initiative comprised of experts across many fields, including genomics, data analysis, engineering, public health, and architecture. The ultimate goal of the MetaSUB Consortium is to improve city utilization and planning through the detection, measurement, and design of metagenomics within urban environments. Although continual measures occur for temperature, air pressure, weather, and human activity, including longitudinal, cross-kingdom ecosystem dynamics can alter and improve the design of cities. The MetaSUB Consortium is aiding these efforts by developing and testing metagenomic methods and standards, including optimized methods for sample collection, DNA/RNA isolation, taxa characterization, and data visualization. The data produced by the consortium can aid city planners, public health officials, and architectural designers. In addition, the study will continue to lead to the discovery of new species, global maps of antimicrobial resistance (AMR) markers, and novel biosynthetic gene clusters (BGCs). Finally, we note that engineered metagenomic ecosystems can help enable more responsive, safer, and quantified cities.

Published
2016
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35. Pichia Pastoris exhibits high viability and a low maintenance energy requirement at near-zero specific growth rates

Author

Rebnegger, C. (author), Vos, T. (author), Graf, Alexandra B. (author), Valli, Minoska (author), Pronk, J.T. (author), Daran-Lapujade, P.A.S. (author), Mattanovicha, Diethard (author), Rebnegger, C. (author), Vos, T. (author), Graf, Alexandra B. (author), Valli, Minoska (author), Pronk, J.T. (author), Daran-Lapujade, P.A.S. (author), and Mattanovicha, Diethard (author)

Abstract

The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/S max) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (mu;) of 0.025 h-1 to near-zero specific growth rates (mu; -1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.10± 1 mg glucose per g biomass and h, which was 3-fold lower than themS estimated from fastergrowing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low , a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes., BT/Industrial Microbiology

Published
2016
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41. Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Author

Prielhofer, Roland, Cartwright, Stephanie, Graf, Alexandra B., Valli, Minoska, Bill, Roslyn, Mattanovich, Diethard, Gasser, Brigitte, Prielhofer, Roland, Cartwright, Stephanie, Graf, Alexandra B., Valli, Minoska, Bill, Roslyn, Mattanovich, Diethard, and Gasser, Brigitte

Abstract

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very diffe

Published
2015

46. Systems-level organization of yeast methylotrophic lifestyle

Author

Rußmayer, Hannes, primary, Buchetics, Markus, additional, Gruber, Clemens, additional, Valli, Minoska, additional, Grillitsch, Karlheinz, additional, Modarres, Gerda, additional, Guerrasio, Raffaele, additional, Klavins, Kristaps, additional, Neubauer, Stefan, additional, Drexler, Hedda, additional, Steiger, Matthias, additional, Troyer, Christina, additional, Al Chalabi, Ali, additional, Krebiehl, Guido, additional, Sonntag, Denise, additional, Zellnig, Günther, additional, Daum, Günther, additional, Graf, Alexandra B., additional, Altmann, Friedrich, additional, Koellensperger, Gunda, additional, Hann, Stephan, additional, Sauer, Michael, additional, Mattanovich, Diethard, additional, and Gasser, Brigitte, additional

Published
2015
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