skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Understanding and eliminating the detrimental effect of thiamine deficiency on the oleaginous yeast Yarrowia lipolytica

Abstract

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de novo, the oleaginous yeast, Yarrowia lipolytica, cannot. In this paper, we used proteomics together with physiological characterization to understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) in the de novo thiamine biosynthesis of Y. lipolytica, and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered the thiamine-prototrophic Y. lipolytica. By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y.more » lipolytica.« less

Authors:
 [1];  [1];  [2];  [1]; ORCiD logo [1]
  1. The Univ. of Tennessee, Knoxville, TN (United States). Dept. of Chemical and Biomolecular Engineering
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Sciences Div.
Publication Date:
Research Org.:
Univ. of Tennessee, Knoxville, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
OSTI Identifier:
1573884
Grant/Contract Number:  
SC0019412
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 86; Journal Issue: 3; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Yarrowia lipolytica; thiamine metabolism; thiamine auxotroph; thiamine prototroph; thiamine-regulated promoters; lipid production; thiamine deficiency

Citation Formats

Walker, Caleb, Ryu, Seunghyun, Giannone, Richard J., Garcia, Sergio, and Trinh, Cong T. Understanding and eliminating the detrimental effect of thiamine deficiency on the oleaginous yeast Yarrowia lipolytica. United States: N. p., 2019. Web. doi:10.1128/AEM.02299-19.
Walker, Caleb, Ryu, Seunghyun, Giannone, Richard J., Garcia, Sergio, & Trinh, Cong T. Understanding and eliminating the detrimental effect of thiamine deficiency on the oleaginous yeast Yarrowia lipolytica. United States. doi:10.1128/AEM.02299-19.
Walker, Caleb, Ryu, Seunghyun, Giannone, Richard J., Garcia, Sergio, and Trinh, Cong T. Fri . "Understanding and eliminating the detrimental effect of thiamine deficiency on the oleaginous yeast Yarrowia lipolytica". United States. doi:10.1128/AEM.02299-19.
@article{osti_1573884,
title = {Understanding and eliminating the detrimental effect of thiamine deficiency on the oleaginous yeast Yarrowia lipolytica},
author = {Walker, Caleb and Ryu, Seunghyun and Giannone, Richard J. and Garcia, Sergio and Trinh, Cong T.},
abstractNote = {Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de novo, the oleaginous yeast, Yarrowia lipolytica, cannot. In this paper, we used proteomics together with physiological characterization to understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) in the de novo thiamine biosynthesis of Y. lipolytica, and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered the thiamine-prototrophic Y. lipolytica. By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica.},
doi = {10.1128/AEM.02299-19},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 3,
volume = 86,
place = {United States},
year = {2019},
month = {11}
}

Journal Article:
Free Publicly Available Full Text
This content will become publicly available on November 8, 2020
Publisher's Version of Record

Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Gene expression changes in thalamus and inferior colliculus associated with inflammation, cellular stress, metabolism and structural damage in thiamine deficiency
journal, March 2006


Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models
journal, October 2013

  • Bunik, Victoria I.; Tylicki, Adam; Lukashev, Nikolay V.
  • FEBS Journal, Vol. 280, Issue 24
  • DOI: 10.1111/febs.12512

 -Keto acid Dehydrogenase Complexes, x. Regulation of the Activity of the Pyruvate Dehydrogenase Complex from beef Kidney Mitochondria by Phosphorylation and Dephosphorylation
journal, January 1969

  • Linn, T. C.; Pettit, F. H.; Reed, L. J.
  • Proceedings of the National Academy of Sciences, Vol. 62, Issue 1
  • DOI: 10.1073/pnas.62.1.234

The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation
journal, May 2014

  • Patel, Mulchand S.; Nemeria, Natalia S.; Furey, William
  • Journal of Biological Chemistry, Vol. 289, Issue 24
  • DOI: 10.1074/jbc.R114.563148

The α-Ketoglutarate–Dehydrogenase Complex: A Mediator Between Mitochondria and Oxidative Stress in Neurodegeneration
journal, January 2005

  • Gibson, Gary E.; Blass, John P.; Beal, M. Flint
  • Molecular Neurobiology, Vol. 31, Issue 1-3
  • DOI: 10.1385/MN:31:1-3:043

Multienzyme complexes
journal, February 1974


Properties and functions of the thiamin diphosphate dependent enzyme transketolase
journal, December 1998

  • Schenk, Gerhard; Duggleby, Ronald G.; Nixon, Peter F.
  • The International Journal of Biochemistry & Cell Biology, Vol. 30, Issue 12
  • DOI: 10.1016/s1357-2725(98)00095-8

Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA)
journal, November 2003


Modulation of thiamine metabolism in Zea mays seedlings under conditions of abiotic stress
journal, October 2008

  • Rapala-Kozik, M.; Kowalska, E.; Ostrowska, K.
  • Journal of Experimental Botany, Vol. 59, Issue 15
  • DOI: 10.1093/jxb/ern253

Purification and characterization of branched chain  -keto acid dehydrogenase complex of bovine kidney
journal, October 1978

  • Pettit, F. H.; Yeaman, S. J.; Reed, L. J.
  • Proceedings of the National Academy of Sciences, Vol. 75, Issue 10
  • DOI: 10.1073/pnas.75.10.4881

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase
journal, March 2003

  • Ciszak, Ewa M.; Korotchkina, Lioubov G.; Dominiak, Paulina M.
  • Journal of Biological Chemistry, Vol. 278, Issue 23
  • DOI: 10.1074/jbc.M300339200

Induced Thiamine (Vitamin b1) Deficiency and the Thiamine Requirement of man: Further Observations
journal, May 1942


Thiamine Deficiency and Neurodegeneration: the Interplay Among Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy
journal, September 2016


Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors
journal, May 1995


Thiamine deficiency disorders: diagnosis, prevalence, and a roadmap for global control programs
journal, August 2018

  • Whitfield, Kyly C.; Bourassa, Megan W.; Adamolekun, Bola
  • Annals of the New York Academy of Sciences, Vol. 1430, Issue 1
  • DOI: 10.1111/nyas.13919

Thiamin (Vitamin B1) Biosynthesis and Regulation: A Rich Source of Antimicrobial Drug Targets?
journal, January 2011

  • Du, Qinglin; Wang, Honghai; Xie, Jianping
  • International Journal of Biological Sciences, Vol. 7, Issue 1
  • DOI: 10.7150/ijbs.7.41

Yarrowia lipolytica : Safety assessment of an oleaginous yeast with a great industrial potential
journal, March 2013

  • Groenewald, Marizeth; Boekhout, Teun; Neuvéglise, Cécile
  • Critical Reviews in Microbiology, Vol. 40, Issue 3
  • DOI: 10.3109/1040841X.2013.770386

Synthetic Biology Expands the Industrial Potential of Yarrowia lipolytica
journal, October 2018


Recent advances in bioengineering of the oleaginous yeast Yarrowia lipolytica
journal, January 2016

  • Shabbir Hussain, Murtaza; M. Rodriguez, Gabriel; Gao, Difeng
  • AIMS Bioengineering, Vol. 3, Issue 4
  • DOI: 10.3934/bioeng.2016.4.493

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica
journal, December 2015

  • Ryu, Seunghyun; Hipp, Julie; Trinh, Cong T.
  • Applied and Environmental Microbiology, Vol. 82, Issue 4
  • DOI: 10.1128/AEM.03582-15

Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism in Yarrowia lipolytica
journal, November 2017

  • Ryu, Seunghyun; Trinh, Cong T.
  • Applied and Environmental Microbiology, Vol. 84, Issue 3
  • DOI: 10.1128/AEM.02146-17

Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose
journal, May 2018

  • Schwartz, Cory; Curtis, Nicholas; Löbs, Ann-Kathrin
  • Biotechnology Journal, Vol. 13, Issue 9
  • DOI: 10.1002/biot.201700584

Exceptional solvent tolerance in Yarrowia lipolytica is enhanced by sterols
journal, July 2019


Simultaneous saccharification and fermentation of cellulose in ionic liquid for efficient production of α-ketoglutaric acid by Yarrowia lipolytica
journal, March 2015

  • Ryu, Seunghyun; Labbé, Nicole; Trinh, Cong T.
  • Applied Microbiology and Biotechnology, Vol. 99, Issue 10
  • DOI: 10.1007/s00253-015-6521-5

Engineering oxidative stress defense pathways to build a robust lipid production platform in Yarrowia lipolytica: Engineering robust microbial oil cell factory
journal, April 2017

  • Xu, Peng; Qiao, Kangjian; Stephanopoulos, Gregory
  • Biotechnology and Bioengineering, Vol. 114, Issue 7
  • DOI: 10.1002/bit.26285

Screening of a thiamine-auxotrophic yeast for α-ketoglutaric acid overproduction: Screening of a α-ketoglutaric acid overproducer
journal, August 2010


Pyruvic acid production by a thiamine auxotroph of Yarrowia lipolytica
journal, July 2004


Thiamine Biosynthesis in Saccharomyces cerevisiae Is Regulated by the NAD+-Dependent Histone Deacetylase Hst1
journal, May 2010

  • Li, Mingguang; Petteys, Brian J.; McClure, Julie M.
  • Molecular and Cellular Biology, Vol. 30, Issue 13
  • DOI: 10.1128/MCB.01590-09

Thiamin metabolism and thiamin diphosphate-dependent enzymes in the yeast Saccharomyces cerevisiae: genetic regulation
journal, June 1998

  • Hohmann, Stefan; Meacock, Peter A.
  • Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol. 1385, Issue 2
  • DOI: 10.1016/S0167-4838(98)00069-7

The vitamin-sensitive promoter P THI11 enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris: The Vitamin-Sensitive Promoter P THI11
journal, July 2016

  • Landes, Nils; Gasser, Brigitte; Vorauer-Uhl, Karola
  • Biotechnology and Bioengineering, Vol. 113, Issue 12
  • DOI: 10.1002/bit.26041

A family of multifunctional thiamine-repressible expression vectors for fission yeast
journal, January 2000


Interdependence of Cell Growth and Gene Expression: Origins and Consequences
journal, November 2010


Growth Rate-Dependent Global Effects on Gene Expression in Bacteria
journal, December 2009


Functional analysis of yeast gene families involved in metabolism of vitamins B 1 and B 6: Biosynthesis of vitamins B 1 and B 6 in yeast
journal, September 2002

  • Rodríguez-Navarro, Susana; Llorente, Bertrand; Rodríguez-Manzaneque, María Teresa
  • Yeast, Vol. 19, Issue 14
  • DOI: 10.1002/yea.916

Thiamine-repressible expression vectors pREP and pRIP for fission yeast
journal, January 1993


A Review of the Biochemistry, Metabolism and Clinical Benefits of Thiamin(e) and Its Derivatives
journal, January 2006

  • Lonsdale, Derrick
  • Evidence-Based Complementary and Alternative Medicine, Vol. 3, Issue 1
  • DOI: 10.1093/ecam/nek009

Molecular Mechanisms of Thiamine Utilization.
journal, May 2001


Genetic Basis of Mitochondrial Function and Morphology in Saccharomyces cerevisiae
journal, March 2002

  • Dimmer, Kai Stefan; Fritz, Stefan; Fuchs, Florian
  • Molecular Biology of the Cell, Vol. 13, Issue 3
  • DOI: 10.1091/mbc.01-12-0588

Thiamine (Vitamin B 1 )
journal, January 2011

  • Fattal-Valevski, Aviva
  • Journal of Evidence-Based Complementary & Alternative Medicine, Vol. 16, Issue 1
  • DOI: 10.1177/1533210110392941

Mitochondrial and Cytosolic Branched-chain Amino Acid Transaminases from Yeast, Homologs of the myc Oncogene-regulated Eca39 Protein
journal, October 1996

  • Kispal, Gyula; Steiner, Harald; Court, Deborah A.
  • Journal of Biological Chemistry, Vol. 271, Issue 40
  • DOI: 10.1074/jbc.271.40.24458

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica
journal, December 2015

  • Shabbir Hussain, Murtaza; Gambill, Lauren; Smith, Spencer
  • ACS Synthetic Biology, Vol. 5, Issue 3
  • DOI: 10.1021/acssynbio.5b00100

New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica
journal, August 2017

  • Trassaert, Marion; Vandermies, Marie; Carly, Fréderic
  • Microbial Cell Factories, Vol. 16, Issue 1
  • DOI: 10.1186/s12934-017-0755-0

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach
journal, September 2011

  • Blazeck, John; Liu, Leqian; Redden, Heidi
  • Applied and Environmental Microbiology, Vol. 77, Issue 22
  • DOI: 10.1128/AEM.05763-11

Protein expression and secretion in the yeast Yarrowia lipolytica
journal, August 2002


Isolation and characterization of ethanol tolerant yeast strains
journal, April 2013

  • Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya
  • Bioinformation, Vol. 9, Issue 8
  • DOI: 10.6026/97320630009421

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission
journal, June 2008

  • Yamane, Takayoshi; Sakai, Hiroaki; Nagahama, Kazuhiro
  • Journal of Bioscience and Bioengineering, Vol. 105, Issue 6
  • DOI: 10.1263/jbb.105.571

High efficiency transformation by electroporation of Yarrowia lipolytica
journal, June 2011


Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds
journal, July 2017

  • Clarkson, Sonya M.; Giannone, Richard J.; Kridelbaugh, Donna M.
  • Applied and Environmental Microbiology, Vol. 83, Issue 18
  • DOI: 10.1128/AEM.01313-17

CharmeRT: Boosting Peptide Identifications by Chimeric Spectra Identification and Retention Time Prediction
journal, June 2018


DanteR: an extensible R-based tool for quantitative analysis of -omics data
journal, July 2012


The Perseus computational platform for comprehensive analysis of (prote)omics data
journal, June 2016

  • Tyanova, Stefka; Temu, Tikira; Sinitcyn, Pavel
  • Nature Methods, Vol. 13, Issue 9
  • DOI: 10.1038/nmeth.3901

Salt Stress in Desulfovibrio vulgaris Hildenborough: an Integrated Genomics Approach
journal, May 2006

  • Mukhopadhyay, A.; He, Z.; Alm, E. J.
  • Journal of Bacteriology, Vol. 188, Issue 11
  • DOI: 10.1128/JB.01921-05

ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks
journal, February 2009


Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
journal, September 1997

  • Altschul, Stephen F.; Madden, Thomas L.; Schäffer, Alejandro A.
  • Nucleic Acids Research, Vol. 25, Issue 17, p. 3389-3402
  • DOI: 10.1093/nar/25.17.3389

Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
journal, January 2013

  • Delic, Marizela; Mattanovich, Diethard; Gasser, Brigitte
  • Microbial Cell Factories, Vol. 12, Issue 1
  • DOI: 10.1186/1475-2859-12-6

nmt2 of fission yeast: A second thiamine-repressible gene co-ordinately regulated withnmt1
journal, August 1994

  • Manetti, Andrea G. O.; Rosetto, Marco; Maundrell, Kinsey G.
  • Yeast, Vol. 10, Issue 8
  • DOI: 10.1002/yea.320100809

Regulation ofTHI4(MOL1), a thiamine-biosynthetic gene ofSaccharomyces cerevisiae
journal, April 1994

  • Praekelt, Uta M.; Byrne, Kerry L.; Meacock, Peter A.
  • Yeast, Vol. 10, Issue 4
  • DOI: 10.1002/yea.320100407

Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches
journal, March 2011

  • Klapper, Maja; Ehmke, Madeleine; Palgunow, Daniela
  • Journal of Lipid Research, Vol. 52, Issue 6
  • DOI: 10.1194/jlr.D011940