Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

ABSTRACT Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

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Generation of branched-chain amino acids resistant Corynebacterium glutamicum acetohydroxy acid synthase by site-directed mutagenesis

Biotechnology and Bioprocess Engineering ◽

10.1007/s12257-013-0843-x ◽

2014 ◽

Vol 19 (3) ◽

pp. 456-467 ◽

Cited By ~ 6

Author(s):

Yanfeng Guo ◽

Mei Han ◽

Weiliu Yan ◽

Jianzhong Xu ◽

Weiguo Zhang

Keyword(s):

Amino Acids ◽

Corynebacterium Glutamicum ◽

Branched Chain Amino Acids ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Acetohydroxy Acid Synthase ◽

Branched Chain

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Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

Microbial Cell Factories ◽

10.1186/s12934-021-01721-0 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Shengzhu Yu ◽

Bo Zheng ◽

Zhenya Chen ◽

Yi-Xin Huo

Keyword(s):

Amino Acids ◽

Metabolic Engineering ◽

Corynebacterium Glutamicum ◽

Animal Feed ◽

Feedback Inhibition ◽

Value Added ◽

Branched Chain Amino Acids ◽

High Yield ◽

Acetohydroxy Acid Synthase ◽

Branched Chain

Abstract Background Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. Main text Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. Conclusions Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

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Effect of renal tubule-specific knockdown of the Na+/H+exchanger NHE3 in Akita diabetic mice

AJP Renal Physiology ◽

10.1152/ajprenal.00497.2018 ◽

2019 ◽

Vol 317 (2) ◽

pp. F419-F434 ◽

Cited By ~ 13

Author(s):

Akira Onishi ◽

Yiling Fu ◽

Manjula Darshi ◽

Maria Crespo-Masip ◽

Winnie Huang ◽

...

Keyword(s):

Amino Acids ◽

Phosphoenolpyruvate Carboxykinase ◽

Tricarboxylic Acid Cycle ◽

Branched Chain Amino Acids ◽

Tricarboxylic Acid ◽

Proximal Tubules ◽

Wild Type ◽

Kidney Weight ◽

Acid Cycle ◽

Branched Chain

Na+/H+exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.

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Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

Microbiology ◽

10.1099/mic.0.028191-0 ◽

2009 ◽

Vol 155 (8) ◽

pp. 2498-2508 ◽

Cited By ~ 8

Author(s):

Jérôme M. B. Durand ◽

Glenn R. Björk

Keyword(s):

Amino Acids ◽

Type Iii Secretion ◽

Shigella Flexneri ◽

Branched Chain Amino Acids ◽

Haemolytic Activity ◽

Growth Media ◽

Wild Type ◽

Nutritional Environment ◽

Inhibitory Potential ◽

Branched Chain

This paper shows that compounds in defined growth media strongly influence the expression of the effectors of virulence in the human invasive pathogen Shigella flexneri. Ornithine in conjunction with uracil reduces the haemolytic ability of wild-type cultures more than 20-fold and the expression of the type III secretion system more than 8-fold, as monitored by an mxiC : : lacZ transcriptional reporter. mxiC gene expression is further decreased by the presence of methionine or branched-chain amino acids (15-fold or 25-fold at least, respectively). Lysine and a few other aminated metabolites (cadaverine, homoserine and diaminopimelate) counteract the ornithine-mediated inhibition of haemolytic activity and of the expression of a transcriptional activator virF reporter. The complete abolition of invasion of HeLa cells by wild-type bacteria by ornithine, uracil, methionine or branched-chain amino acids establishes that these metabolites are powerful effectors of virulence. These findings provide a direct connection between metabolism and virulence in S. flexneri. The inhibitory potential exhibited by the nutritional environment is stronger than temperature, the classical environmental effector of virulence. The implications and practical application of this finding in prophylaxis and treatment of shigellosis are discussed.

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Analysis of acetohydroxyacid synthase variants from branched-chain amino acids-producing strains and their effects on the synthesis of branched-chain amino acids in Corynebacterium glutamicum

Protein Expression and Purification ◽

10.1016/j.pep.2015.02.006 ◽

2015 ◽

Vol 109 ◽

pp. 106-112 ◽

Cited By ~ 11

Author(s):

Yanfeng Guo ◽

Mei Han ◽

Jianzhong Xu ◽

Weiguo Zhang

Keyword(s):

Amino Acids ◽

Corynebacterium Glutamicum ◽

Branched Chain Amino Acids ◽

Acetohydroxyacid Synthase ◽

Branched Chain

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Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for l-methionine and branched-chain amino acids

Journal of Biotechnology ◽

10.1016/j.jbiotec.2011.06.003 ◽

2012 ◽

Vol 158 (4) ◽

pp. 231-241 ◽

Cited By ~ 50

Author(s):

Christian Lange ◽

Nurije Mustafi ◽

Julia Frunzke ◽

Nicole Kennerknecht ◽

Mirja Wessel ◽

...

Keyword(s):

Amino Acids ◽

Corynebacterium Glutamicum ◽

Branched Chain Amino Acids ◽

Branched Chain ◽

Export System

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Unusual regulation of the uptake system for branched-chain amino acids in Corynebacterium glutamicum

Archives of Microbiology ◽

10.1007/bf00250275 ◽

1993 ◽

Vol 159 (2) ◽

pp. 147-152 ◽

Cited By ~ 7

Author(s):

Eckhard Boles ◽

Holger Ebbighausen ◽

Bernhard Eikmanns ◽

Reinhard Kr�mer

Keyword(s):

Amino Acids ◽

Corynebacterium Glutamicum ◽

Branched Chain Amino Acids ◽

Uptake System ◽

Branched Chain

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Genetic and Biochemical Analysis of the Interaction of Bacillus subtilis CodY with Branched-Chain Amino Acids

Journal of Bacteriology ◽

10.1128/jb.00818-09 ◽

2009 ◽

Vol 191 (22) ◽

pp. 6865-6876 ◽

Cited By ~ 34

Author(s):

Anuradha C. Villapakkam ◽

Luke D. Handke ◽

Boris R. Belitsky ◽

Vladimir M. Levdikov ◽

Anthony J. Wilkinson ◽

...

Keyword(s):

Amino Acids ◽

Bacillus Subtilis ◽

Branched Chain Amino Acids ◽

Site Directed Mutagenesis ◽

Binding Studies ◽

Hydrophobic Pocket ◽

Isoleucine Leucine ◽

Branched Chain

ABSTRACT Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

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