Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis

Petit, Cecile; Kim, Younghwa; Lee, Sung-Kwon; Brown, Jake; Larsen, Erik; Ronning, Donald R.; Suh, Joo-Won; Kang, Choong-Min

doi:10.1021/acsomega.7b01597

Title: Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis

Journal Article · Mon Jan 29 00:00:00 EST 2018 · ACS Omega

DOI:https://doi.org/10.1021/acsomega.7b01597· OSTI ID:1499745

Petit, Cecile ^[1]; Kim, Younghwa ^[2]; Lee, Sung-Kwon ^[3]; Brown, Jake ^[4]; Larsen, Erik ^[4];

^[1]; Suh, Joo-Won ^[3];

^[4]

Univ. of Toledo, OH (United States)
Kyungsung Univ., Busan (Republic of Korea)
Myongji Univ., Gyeonggi-do (Republic of Korea)
California State Univ., Stanislaus, Turlock, CA (United States)

L-Threonine is an important supplement in the food industry. It is currently produced through fermentation of Escherichia coli but requires additional purification steps to remove E. coli endotoxin. To avoid these steps, it is desirable to use Corynebacterium glutamicum, a microorganism generally regarded as safe. Engineering of C. glutamicum to increase production of l-threonine has mainly focused on gene regulation as well as l-threonine export or carbon flux depletion. In this study, we focus on the negative feedback inhibition produced by L-threonine on the enzyme homoserine kinase (ThrB). Although L-threonine binds to allosteric sites of aspartate kinase (LysC) and homoserine dehydrogenase (Hom), serving as a noncompetitive inhibitor, it acts as a competitive inhibitor on ThrB. This is problematic when attempting to engineer enzymes that are nonresponsive to increasing cellular concentrations of L-threonine. Using primary structure alignment as well as analysis of the Methanocaldococcus jannaschii ThrB (MjaThrB) active site in complex with L-threonine (inhibitor of ThrB) and L-homoserine (substrate of ThrB), a conserved active-site alanine residue (A20) in C. glutamicum ThrB (CglThrB) was predicted to be important for differential interactions with L-threonine and L-homoserine. Through site-directed mutagenesis, we show that one variant of C. glutamicum ThrB, CglThrB-A20G, retains wild-type enzymatic activity, with dramatically decreased feedback inhibition by L-threonine. Additionally, by solving the first Corynebacterium X-ray crystal structure of homoserine kinase, we can confirm that the changes in L-threonine affinity to the CglThrB-A20G active site derive from loss of van der Waals interactions.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: Next-Generation BioGreen 21 Program

Grant/Contract Number:: PJ00900705

OSTI ID:: 1499745

Journal Information:: ACS Omega, Vol. 3, Issue 1; ISSN 2470-1343

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: ENGLISH

Citation Metrics:

Cited by: 9 works

Citation information provided by
Web of Science

References (26)

Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids Reinscheid, Dieter J.; Kronemeyer, Wolfgang; Eggeling, Lothar Applied and Environmental Microbiology, Vol. 60, Issue 1 https://doi.org/10.1128/aem.60.1.126-132.1994	journal	January 1994
Activity of Exporters of Escherichia coli in Corynebacterium glutamicum, and Their Use to Increase L-Threonine Production Diesveld, Ramon; Tietze, Nadine; Fürst, Oliver Microbial Physiology, Vol. 16, Issue 3-4 https://doi.org/10.1159/000142530	journal	July 2008
PHENIX: a comprehensive Python-based system for macromolecular structure solution. Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Apollo - University of Cambridge Repository https://doi.org/10.17863/cam.45787	text	January 2010
Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum Simic, Petra; Willuhn, Juliane; Sahm, Hermann Applied and Environmental Microbiology, Vol. 68, Issue 7 https://doi.org/10.1128/aem.68.7.3321-3327.2002	journal	July 2002
Genome-wide analysis of the l-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation Rückert, C.; Pühler, A.; Kalinowski, J. Journal of Biotechnology, Vol. 104, Issue 1-3 https://doi.org/10.1016/s0168-1656(03)00158-5	journal	September 2003
The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins Kalinowski, Jörn; Bathe, Brigitte; Bartels, Daniela Journal of Biotechnology, Vol. 104, Issue 1-3 https://doi.org/10.1016/s0168-1656(03)00154-8	journal	September 2003
Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum Möckel, B.; Eggeling, L.; Sahm, H. Journal of Bacteriology, Vol. 174, Issue 24 https://doi.org/10.1128/jb.174.24.8065-8072.1992	journal	December 1992
Molecular Functions of Conserved Aspects of the GHMP Kinase Family ^† Andreassi, John L.; Leyh, Thomas S. Biochemistry, Vol. 43, Issue 46 https://doi.org/10.1021/bi048963o	journal	November 2004
A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by l-threonine Archer, J. A. C.; Solow-Cordero, D. E.; Sinskey, A. J. Gene, Vol. 107, Issue 1 https://doi.org/10.1016/0378-1119(91)90296-n	journal	October 1991
Regulation of Aspartate Family Amino Acid Biosynthesis in Brevibacterium flavum Miyajima, Ryuichi; Otsuka, Shin-Ichiro; Shiio, Isamu The Journal of Biochemistry, Vol. 63, Issue 2 https://doi.org/10.1093/oxfordjournals.jbchem.a128754	journal	February 1968
The thrH Gene Product of Pseudomonas aeruginosa Is a Dual Activity Enzyme with a Novel Phosphoserine:Homoserine Phosphotransferase Activity Singh, S. Kumar; Yang, Kun; Karthikeyan, Subramanian Journal of Biological Chemistry, Vol. 279, Issue 13 https://doi.org/10.1074/jbc.m311393200	journal	December 2003
A functionally split pathway for lysine synthesis in Corynebacterium glutamicium Schrumpf, B.; Schwarzer, A.; Kalinowski, J. Journal of Bacteriology, Vol. 173, Issue 14 https://doi.org/10.1128/jb.173.14.4510-4516.1991	journal	July 1991
Structural Basis for the Catalysis and Substrate Specificity of Homoserine Kinase ^† Krishna, S. Sri; Zhou, Tianjun; Daugherty, Matthew Biochemistry, Vol. 40, Issue 36 https://doi.org/10.1021/bi010851z	journal	September 2001
Influence of threonine exporters on threonine production in Escherichia coli D., Kruse; R., Krämer; L., Eggeling Applied Microbiology and Biotechnology, Vol. 59, Issue 2-3 https://doi.org/10.1007/s00253-002-0987-7	journal	July 2002
Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum Mateos, L. M.; Pisabarro, A.; Pátek, M. Journal of Bacteriology, Vol. 176, Issue 23 https://doi.org/10.1128/jb.176.23.7362-7371.1994	journal	December 1994
Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799 Colón, G. E.; Jetten, M. S.; Nguyen, T. T. Applied and Environmental Microbiology, Vol. 61, Issue 1 https://doi.org/10.1128/aem.61.1.74-78.1995	journal	January 1995
Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions Henkin, Tina M.; Yanofsky, Charles BioEssays, Vol. 24, Issue 8 https://doi.org/10.1002/bies.10125	journal	July 2002
Systems and synthetic metabolic engineering for amino acid production – the heartbeat of industrial strain development Becker, Judith; Wittmann, Christoph Current Opinion in Biotechnology, Vol. 23, Issue 5 https://doi.org/10.1016/j.copbio.2011.12.025	journal	October 2012
Mechanism of Concerted Inhibition of α2β2-type Hetero-oligomeric Aspartate Kinase from Corynebacterium glutamicum Yoshida, Ayako; Tomita, Takeo; Kuzuyama, Tomohisa Journal of Biological Chemistry, Vol. 285, Issue 35 https://doi.org/10.1074/jbc.M110.111153	journal	August 2010
Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli Livshits, Vitaliy A.; Zakataeva, Natalia P.; Aleshin, Vladimir V. Research in Microbiology, Vol. 154, Issue 2 https://doi.org/10.1016/S0923-2508(03)00036-6	journal	March 2003
Structure and Mechanism of Homoserine Kinase Zhou, Tianjun; Daugherty, Matthew; Grishin, Nick V. Structure, Vol. 8, Issue 12 https://doi.org/10.1016/S0969-2126(00)00533-5	journal	December 2000
Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega Sievers, Fabian; Wilm, Andreas; Dineen, David Molecular Systems Biology, Vol. 7, Issue 1 https://doi.org/10.1038/msb.2011.75	journal	January 2011
A new bioinformatics analysis tools framework at EMBL-EBI Goujon, M.; McWilliam, H.; Li, W. Nucleic Acids Research, Vol. 38, Issue Web Server https://doi.org/10.1093/nar/gkq313	journal	May 2010
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010

Cited By (1)

Improvement of l-Valine Production by Atmospheric and Room Temperature Plasma Mutagenesis and High-Throughput Screening in Corynebacterium glutamicum Han, Guoqiang; Xu, Ning; Sun, Xieping ACS Omega, Vol. 5, Issue 10 https://doi.org/10.1021/acsomega.9b02747	journal	March 2020

Similar Records

Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

Journal Article · Sun Jan 01 00:00:00 EST 1989 · Sov. Genet. (Engl. Transl.); (United States) · OSTI ID:1499745

Beskrovnaya, O Yu; Fonshtein, M Yu; Kolibaba, L G; +2 more

Structural and Functional Characterization of YdjI, an Aldolase of Unknown Specificity in Escherichia coli K12

Journal Article · Fri Jul 19 00:00:00 EDT 2019 · Biochemistry · OSTI ID:1499745

Huddleston, Jamison P.; Thoden, James B.; Dopkins, Brandon J.; +4 more

Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

Journal Article · Fri Nov 21 00:00:00 EST 2008 · Arch. Biochem. Biophys. · OSTI ID:1499745

Rice, E A; Bannon, G A; Glenn, K C; +3 more

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Peptides and proteins
Genetics
Monomers
Inhibitors
Inhibition

Title: Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis

Citation Formats

References (26)

Cited By (1)

Similar Records

Related Subjects