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

Abstract

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,more » 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.« less

Authors:
 [1];  [2];  [3];  [4];  [4]; ORCiD logo [1];  [3]; ORCiD logo [4]
  1. Univ. of Toledo, OH (United States)
  2. Kyungsung Univ., Busan (Republic of Korea)
  3. Myongji Univ., Gyeonggi-do (Republic of Korea)
  4. California State Univ., Stanislaus, Turlock, CA (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
Next-Generation BioGreen 21 Program
OSTI Identifier:
1499745
Grant/Contract Number:  
PJ00900705
Resource Type:
Accepted Manuscript
Journal Name:
ACS Omega
Additional Journal Information:
Journal Volume: 3; Journal Issue: 1; Journal ID: ISSN 2470-1343
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Peptides and proteins; Genetics; Monomers; Inhibitors; Inhibition

Citation Formats

Petit, Cecile, Kim, Younghwa, Lee, Sung-Kwon, Brown, Jake, Larsen, Erik, Ronning, Donald R., Suh, Joo-Won, and Kang, Choong-Min. Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis. United States: N. p., 2018. Web. https://doi.org/10.1021/acsomega.7b01597.
Petit, Cecile, Kim, Younghwa, Lee, Sung-Kwon, Brown, Jake, Larsen, Erik, Ronning, Donald R., Suh, Joo-Won, & Kang, Choong-Min. Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis. United States. https://doi.org/10.1021/acsomega.7b01597
Petit, Cecile, Kim, Younghwa, Lee, Sung-Kwon, Brown, Jake, Larsen, Erik, Ronning, Donald R., Suh, Joo-Won, and Kang, Choong-Min. Mon . "Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis". United States. https://doi.org/10.1021/acsomega.7b01597. https://www.osti.gov/servlets/purl/1499745.
@article{osti_1499745,
title = {Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances L-Threonine Biosynthesis},
author = {Petit, Cecile and Kim, Younghwa and Lee, Sung-Kwon and Brown, Jake and Larsen, Erik and Ronning, Donald R. and Suh, Joo-Won and Kang, Choong-Min},
abstractNote = {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.},
doi = {10.1021/acsomega.7b01597},
journal = {ACS Omega},
number = 1,
volume = 3,
place = {United States},
year = {2018},
month = {1}
}

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