The Transcriptome and Flux Profiling of Crabtree-Negative Hydroxy Acid-Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism

Jessop‐Fabre, Mathew M.; Dahlin, Jonathan; Biron, Mathias B.; Stovicek, Vratislav; Ebert, Birgitta E.; Blank, Lars M.; Budin, Itay; Keasling, Jay D.; Borodina, Irina

doi:10.1002/biot.201900013

The Transcriptome and Flux Profiling of Crabtree-Negative Hydroxy Acid-Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism

Journal Article · Wed Apr 10 00:00:00 EDT 2019 · Biotechnology Journal

DOI:https://doi.org/10.1002/biot.201900013· OSTI ID:1604701

^[1]; Dahlin, Jonathan ^[1]; ^[1]; Stovicek, Vratislav ^[1]; Ebert, Birgitta E. ^[2]; Blank, Lars M. ^[2]; Budin, Itay ^[3]; Keasling, Jay D. ^[4]; Borodina, Irina ^[1]

Technical Univ. of Denmark, Kongens Lyngby (Denmark)
Aachen Univ. (Germany)
Univ. of California, Berkeley, CA (United States)
Technical Univ. of Denmark, Kongens Lyngby (Denmark); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Shenzhen Inst. for Advanced Technologies (China)

Saccharomyces cerevisiae (S. cerevisiae) is the yeast cell factory of choice for the production of many biobased chemicals. However, it is a Crabtree-positive yeast and so shuttles a large portion of carbon into ethanol. Ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. It is not yet well understood how PDC-negative yeasts are affected when engineered to produce other products than ethanol. In this study, pathways are introduced for the production of three hydroxy acids (lactic, malic, or 3-hydroxypropionic acid [3HP]) into an evolved PDC-negative strain. These strains are characterized via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. Expression of lactic and malic acid biosynthesis pathways improved the maximum specific growth rate (μ_max) of the strain by 64% and 20%, respectively, presumably due to nicotinamide adenine dinucleotide regeneration. All strains show a very high flux (> 90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The study, for the first time, directly compares the flux and transcriptome profiles of several hydroxy acid-producing strains of an evolved PDC-negative S. cerevisiae and suggests directions for future metabolic engineering.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: European Research Council; Novo Nordisk Foundation; USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1604701

Journal Information:: Biotechnology Journal, Journal Name: Biotechnology Journal Journal Issue: 9 Vol. 14; ISSN 1860-6768

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

References (52)

Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose Flikweert, Marcel T.; van der Zanden, Linda; Janssen, Wouter M. Th. M. Yeast, Vol. 12, Issue 3, p. 247-257 https://doi.org/10.1002/(SICI)1097-0061(19960315)12:3<247::AID-YEA911>3.0.CO;2-I	journal	March 1996
Pyruvate Metabolism inSaccharomyces cerevisiae Pronk, Jack T.; Yde Steensma, H.; Van Dijken, Johannes P. Yeast, Vol. 12, Issue 16 https://doi.org/10.1002/(SICI)1097-0061(199612)12:16<1607::AID-YEA70>3.0.CO;2-4	journal	December 1996
Metabolomic and ¹³ C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase: Xylose Metabolic Flux Analysis Wasylenko, Thomas M.; Stephanopoulos, Gregory Biotechnology and Bioengineering, Vol. 112, Issue 3 https://doi.org/10.1002/bit.25447	journal	November 2014
Multi-timescale analysis of a metabolic network in synthetic biology: a kinetic model for 3-hydroxypropionic acid production via beta-alanine Dalwadi, Mohit P.; King, John R.; Minton, Nigel P. Journal of Mathematical Biology, Vol. 77, Issue 1 https://doi.org/10.1007/s00285-017-1189-3	journal	November 2017
Metabolic flux analysis of genetically engineered Saccharomyces cerevisiae that produces lactate under micro-aerobic conditions Nagamori, Eiji; Shimizu, Kazunori; Fujita, Hideaki Bioprocess and Biosystems Engineering, Vol. 36, Issue 9 https://doi.org/10.1007/s00449-012-0870-6	journal	December 2012
Control of lactate production by Saccharomyces cerevisiae expressing a bacterial LDH gene Colombié, S.; Dequin, S.; Sablayrolles, J. M. Enzyme and Microbial Technology, Vol. 33, Issue 1 https://doi.org/10.1016/S0141-0229(03)00082-6	journal	July 2003
Online respiration activity measurement (OTR, CTR, RQ) in shake flasks Anderlei, Tibor; Zang, Werner; Papaspyrou, Manfred Biochemical Engineering Journal, Vol. 17, Issue 3 https://doi.org/10.1016/S1369-703X(03)00181-5	journal	March 2004
Recent advances in biological production of 3-hydroxypropionic acid Kumar, Vinod; Ashok, Somasundar; Park, Sunghoon Biotechnology Advances, Vol. 31, Issue 6 https://doi.org/10.1016/j.biotechadv.2013.02.008	journal	November 2013
Microbial export of lactic and 3-hydroxypropanoic acid: implications for industrial fermentation processes Maris, Antonius J. A. van; Konings, Wil N.; Dijken, Johannes P. van Metabolic Engineering, Vol. 6, Issue 4 https://doi.org/10.1016/j.ymben.2004.05.001	journal	October 2004
Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance Kildegaard, Kanchana R.; Hallström, Björn M.; Blicher, Thomas H. Metabolic Engineering, Vol. 26 https://doi.org/10.1016/j.ymben.2014.09.004	journal	November 2014
Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine Borodina, Irina; Kildegaard, Kanchana R.; Jensen, Niels B. Metabolic Engineering, Vol. 27 https://doi.org/10.1016/j.ymben.2014.10.003	journal	January 2015
Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing Kim, Sujin; Hahn, Ji-Sook Metabolic Engineering, Vol. 31 https://doi.org/10.1016/j.ymben.2015.07.006	journal	September 2015
Cameo: A Python Library for Computer Aided Metabolic Engineering and Optimization of Cell Factories Cardoso, João G. R.; Jensen, Kristian; Lieven, Christian ACS Synthetic Biology, Vol. 7, Issue 4 https://doi.org/10.1021/acssynbio.7b00423	journal	March 2018
Omic data from evolved E. coli are consistent with computed optimal growth from genome‐scale models Lewis, Nathan E.; Hixson, Kim K.; Conrad, Tom M. Molecular Systems Biology, Vol. 6, Issue 1 https://doi.org/10.1038/msb.2010.47	journal	January 2010
HISAT: a fast spliced aligner with low memory requirements Kim, Daehwan; Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 12, Issue 4 https://doi.org/10.1038/nmeth.3317	journal	March 2015
High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method Gietz, R. Daniel; Schiestl, Robert H. Nature Protocols, Vol. 2, Issue 1 https://doi.org/10.1038/nprot.2007.13	journal	January 2007
Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown Pertea, Mihaela; Kim, Daehwan; Pertea, Geo M. Nature Protocols, Vol. 11, Issue 9 https://doi.org/10.1038/nprot.2016.095	journal	August 2016
ARS2/MAGL signaling in glioblastoma stem cells promotes self-renewal and M2-like polarization of tumor-associated macrophages Yin, Jinlong; Kim, Sung Soo; Choi, Eunji Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-16789-2	journal	June 2020
Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator Hanko, Erik K. R.; Minton, Nigel P.; Malys, Naglis Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-01850-w	journal	May 2017
Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional ¹³ C labeling of common amino acids : Central carbon metabolism of Maaheimo, Hannu; Fiaux, Jocelyne; Çakar, Z. Petek European Journal of Biochemistry, Vol. 268, Issue 8 https://doi.org/10.1046/j.1432-1327.2001.02126.x	journal	April 2001
Genetically encoded sensors enable real-time observation of metabolite production Rogers, Jameson K.; Church, George M. Proceedings of the National Academy of Sciences, Vol. 113, Issue 9 https://doi.org/10.1073/pnas.1600375113	journal	February 2016
Expression and Function of a Mislocalized Form of Peroxisomal Malate Dehydrogenase (MDH3) in Yeast McAlister-Henn, Lee; Steffan, Joan S.; Minard, Karyl I. Journal of Biological Chemistry, Vol. 270, Issue 36 https://doi.org/10.1074/jbc.270.36.21220	journal	September 1995
INCA: a computational platform for isotopically non-stationary metabolic flux analysis Young, J. D. Bioinformatics, Vol. 30, Issue 9 https://doi.org/10.1093/bioinformatics/btu015	journal	January 2014
EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae Jensen, Niels B.; Strucko, Tomas; Kildegaard, Kanchana R. FEMS Yeast Research, Vol. 14, Issue 2 https://doi.org/10.1111/1567-1364.12118	journal	March 2014
The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review de Smidt, Olga; du Preez, James C.; Albertyn, Jacobus FEMS Yeast Research, Vol. 8, Issue 7, p. 967-978 https://doi.org/10.1111/j.1567-1364.2008.00387.x	journal	November 2008
Metabolic engineering of Saccharomyces cerevisiae â€ƒfor production of carboxylic acids: current status and challenges Abbott, Derek A.; Zelle, Rintze M.; Pronk, Jack T. FEMS Yeast Research, Vol. 9, Issue 8 https://doi.org/10.1111/j.1567-1364.2009.00537.x	journal	December 2009
Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2 Pacheco, António; Talaia, Gabriel; Sá-Pessoa, Joana FEMS Yeast Research, Vol. 12, Issue 3 https://doi.org/10.1111/j.1567-1364.2012.00790.x	journal	February 2012
Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae Flikweert, Marcel T.; Swaaf, Martin; Dijken, Johannes P. FEMS Microbiology Letters, Vol. 174, Issue 1 https://doi.org/10.1111/j.1574-6968.1999.tb13551.x	journal	May 1999
Transport of carboxylic acids in yeasts Casal, Margarida; Paiva, Sandra; Queirós, Odília FEMS Microbiology Reviews, Vol. 32, Issue 6 https://doi.org/10.1111/j.1574-6976.2008.00128.x	journal	November 2008
Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing Benjamini, Yoav; Hochberg, Yosef Journal of the Royal Statistical Society: Series B (Methodological), Vol. 57, Issue 1 https://doi.org/10.1111/j.2517-6161.1995.tb02031.x	journal	January 1995
Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export Zelle, R. M.; de Hulster, E.; van Winden, W. A. Applied and Environmental Microbiology, Vol. 74, Issue 9 https://doi.org/10.1128/AEM.02591-07	journal	March 2008
Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess Flikweert, M. T.; van Dijken, J. P.; Pronk, J. T. Applied and Environmental Microbiology, Vol. 63, Issue 9 https://doi.org/10.1128/AEM.63.9.3399-3404.1997	journal	September 1997
Characterization of Schizosaccharomyces pombe Malate Permease by Expression in Saccharomyces cerevisiae Camarasa, C.; Bidard, F.; Bony, M. Applied and Environmental Microbiology, Vol. 67, Issue 9 https://doi.org/10.1128/AEM.67.9.4144-4151.2001	journal	September 2001
Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast van Maris, A. J. A.; Geertman, J. -M. A.; Vermeulen, A. Applied and Environmental Microbiology, Vol. 70, Issue 1 https://doi.org/10.1128/AEM.70.1.159-166.2004	journal	January 2004
Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export van Maris, A. J. A.; Winkler, A. A.; Porro, D. Applied and Environmental Microbiology, Vol. 70, Issue 5 https://doi.org/10.1128/AEM.70.5.2898-2905.2004	journal	May 2004
Efficient Production of L-Lactic Acid by Metabolically Engineered Saccharomyces cerevisiae with a Genome-Integrated L-Lactate Dehydrogenase Gene Ishida, N.; Saitoh, S.; Tokuhiro, K. Applied and Environmental Microbiology, Vol. 71, Issue 4 https://doi.org/10.1128/AEM.71.4.1964-1970.2005	journal	April 2005
The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems Kim, K. -S.; Pelton, J. G.; Inwood, W. B. Journal of Bacteriology, Vol. 192, Issue 16 https://doi.org/10.1128/JB.00201-10	journal	April 2010
Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis Schmitt, H. D.; Zimmermann, F. K. Journal of Bacteriology, Vol. 151, Issue 3 https://doi.org/10.1128/JB.151.3.1146-1152.1982	journal	September 1982
Network Identification and Flux Quantification in the Central Metabolism of Saccharomyces cerevisiae under Different Conditions of Glucose Repression Gombert, A. K.; Moreira dos Santos, M.; Christensen, B. Journal of Bacteriology, Vol. 183, Issue 4 https://doi.org/10.1128/JB.183.4.1441-1451.2001	journal	February 2001
Progress in Metabolic Engineering of Saccharomyces cerevisiae Nevoigt, E. Microbiology and Molecular Biology Reviews, Vol. 72, Issue 3 https://doi.org/10.1128/MMBR.00025-07	journal	September 2008
The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems Kim, Kwang-Seo; Pelton, Jeffrey G.; Inwood, William B. Journal of Bacteriology, Vol. 193, Issue 1 https://doi.org/10.1128/jb.01302-10	journal	January 2011
pdc1(0) mutants of Saccharomyces cerevisiae give evidence for an additional structural PDC gene: cloning of PDC5, a gene homologous to PDC1. Seeboth, P. G.; Bohnsack, K.; Hollenberg, C. P. Journal of Bacteriology, Vol. 172, Issue 2 https://doi.org/10.1128/jb.172.2.678-685.1990	journal	January 1990
Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae. Hohmann, S. Journal of Bacteriology, Vol. 173, Issue 24 https://doi.org/10.1128/jb.173.24.7963-7969.1991	journal	January 1991
iMS2Flux – a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis Poskar, C. Hart; Huege, Jan; Krach, Christian BMC Bioinformatics, Vol. 13, Issue 1 https://doi.org/10.1186/1471-2105-13-295	journal	January 2012
An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae Oud, Bart; Flores, Carmen-Lisset; Gancedo, Carlos Microbial Cell Factories, Vol. 11, Issue 1 https://doi.org/10.1186/1475-2859-11-131	journal	January 2012
Connecting extracellular metabolomic measurements to intracellular flux states in yeast Mo, Monica L.; Palsson, Bernhard Ø; Herrgård, Markus J. BMC Systems Biology, Vol. 3, Issue 1 https://doi.org/10.1186/1752-0509-3-37	journal	January 2009
Datura genome reveals duplications of psychoactive alkaloid biosynthetic genes and high mutation rate following tissue culture Rajewski, Alex; Carter-House, Derreck; Stajich, Jason BMC Genomics, Vol. 22, Issue 1 https://doi.org/10.1186/s12864-021-07489-2	journal	March 2021
Aging features of the migratory locust at physiological and transcriptional levels Guo, Siyuan; Yang, Pengcheng; Liang, Bo BMC Genomics, Vol. 22, Issue 1 https://doi.org/10.1186/s12864-021-07585-3	journal	April 2021
Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway Kildegaard, Kanchana R.; Jensen, Niels B.; Schneider, Konstantin Microbial Cell Factories, Vol. 15, Issue 1 https://doi.org/10.1186/s12934-016-0451-5	journal	March 2016
Engineering of Saccharomyces cerevisiae for anthranilate and methyl anthranilate production Kuivanen, Joosu; Kannisto, Matti; Mojzita, Dominik Microbial Cell Factories, Vol. 20, Issue 1 https://doi.org/10.1186/s12934-021-01532-3	journal	February 2021
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Love, Michael I.; Huber, Wolfgang; Anders, Simon Genome Biology, Vol. 15, Issue 12 https://doi.org/10.1186/s13059-014-0550-8	journal	December 2014
Enhanced malic acid production from glycerol with high-cell density Ustilago trichophora TZ1 cultivations Zambanini, Thiemo; Kleineberg, Wiebke; Sarikaya, Eda Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0553-7	journal	July 2016

Similar Records

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae

Journal Article · Sun Sep 26 20:00:00 EDT 2021 · Metabolic Engineering · OSTI ID:1827679

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae

Journal Article · Tue Jan 31 19:00:00 EST 2023 · Biotechnology Journal · OSTI ID:1993836

Related Subjects

13C-based metabolic flux analysis
59 BASIC BIOLOGICAL SCIENCES
Crabtree-negative
central carbon metabolism
hydroxy acid
transcriptomics

The Transcriptome and Flux Profiling of Crabtree-Negative Hydroxy Acid-Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism

Citation Formats

References (52)

Similar Records

Related Subjects