scholarly journals Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

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.

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

2005 ◽  
Vol 71 (1) ◽  
pp. 207-213 ◽  
Author(s):  
Veronika ElišÃ¯Â¿Â½kov� ◽  
Miroslav P�tek ◽  
Jiř� Hol�tko ◽  
Jan Nešvera ◽  
Damien Leyval ◽  
...  
Keyword(s):  
Amino Acids ◽  
Corynebacterium Glutamicum ◽  
Shuttle Vector ◽  
Branched Chain Amino Acids ◽  
Site Directed Mutagenesis ◽  
Enzyme Molecule ◽  
Acetohydroxy Acid Synthase ◽  
Wild Type ◽  
Allosteric Site ◽  
Branched Chain

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.

Rapid Germination of Sulfonylurea-ResistantKochia scopariaL. Accessions is Associated with Elevated Seed Levels of Branched Chain Amino Acids

Weed Science ◽  
1993 ◽  
Vol 41 (1) ◽  
pp. 18-22 ◽  
Author(s):  
William E. Dyer ◽  
Peng W. Chee ◽  
Peter K. Fay
Keyword(s):  
Amino Acids ◽  
Thermal Gradient ◽  
Feedback Inhibition ◽  
Acetolactate Synthase ◽  
Branched Chain Amino Acids ◽  
Resistant Accession ◽  
Early Germination ◽  
Percent Germination ◽  
Soil Temperatures ◽  
Branched Chain

Field observations indicate that sulfonylurea-resistant kochia may germinate at lower soil temperatures and/or germinate more rapidly than susceptible kochia in the absence of herbicide. To investigate this possibility, seeds from three resistant and two susceptible kochia accessions were germinated at temperatures ranging from 4.6 to 13.2 C on thermal gradient plates. At 4.6 and 13.2 C, germination rates of all resistant accessions were higher than susceptible accessions, while germination rates of one resistant accession were higher than susceptible accessions at 7.2 and 10.5 C. Percent germination of all resistant accessions was significantly higher than susceptible accessions after 48 h at 4.6 C. At higher temperatures, percent germination of some resistant accessions was higher after 12 or 24 h, but germination of all accessions was similar at later times. HPLC analysis revealed that seeds from resistant accessions contained about 2-fold higher free levels of branched chain amino acids than seeds from susceptible accessions. The results indicate that mutations conferring resistance to sulfonylurea herbicides in these kochia accessions may concomitantly reduce or abolish acetolactate synthase sensitivity to normal feedback inhibition patterns, resulting in elevated levels of branched chain amino acids available for cell division and growth during early germination.

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

2012 ◽  
Vol 158 (4) ◽  
pp. 231-241 ◽  
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

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

1993 ◽  
Vol 159 (2) ◽  
pp. 147-152 ◽  
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

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Regulation of the Enzymes Involved in Isoleucine, Valine, and Leucine Synthesis

1974 ◽  
Vol 52 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Roderick A. McDonald ◽  
T. Satyanarayana ◽  
J. G. Kaplan
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Feedback Inhibition ◽  
Branched Chain Amino Acids ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Threonine Deaminase ◽  
Branched Chain ◽  
Ph Range

The activities and regulation of the enzymes of the synthetic pathway of branched-chain amino acids were investigated in the fission yeast, Schizosaccharomyces pombe. Previous studies had shown the presence of threonine deaminase (TD) and acetohydroxy acid synthetase (AHAS). The remaining isoleucine–valine enzymes, isomeroreductase (IR), dehydrase, and transaminase B, have now been characterized in cell-free extracts, indicating the presence in this yeast of the complete pathway as demonstrated in other microorganisms. α-Isopropylmalate synthetase (IPMS), the first enzyme of the leucine pathway, has properties of a typical regulatory enzyme; it is most active in the pH range 7.5–8.5, but is most sensitive to feedback inhibition by L-leucine at pH 6.5–7.0. Unlike the situation in baker's yeast, only AHAS and IR appeared to be subject to multivalent repression. TD was relatively resistant to any change in level, and AHAS was repressible by valine included in the growth medium. IPMS was repressed when cells were grown in complex medium; leucine alone did not cause repression, and in contrast with baker's yeast, neither did leucine plus threonine or a combination of all three branched-chain amino acids.

Heterologous Expression and Functional Characterization of Catalytic Subunit of Rice Acetohydroxyacid Synthase

2019 ◽  
Vol 26 (3) ◽  
pp. 176-183
Author(s):  
Ghazaleh Arabzadeh ◽  
Azar Shahpiri
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Functional Characterization ◽  
Branched Chain Amino Acids ◽  
Biochemical Properties ◽  
Catalytic Subunit ◽  
Acetohydroxyacid Synthase ◽  
Branched Chain

Background: Acetohydroxyacid Synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched chain amino acids. AHAS is the common target site of five herbicide chemical groups: sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone. </P><P> Objective: The purification of protein enabled us to study the physical and biochemical properties of the enzyme. In addition in vitro activity of this enzyme was tested in the presence of four different sulfonylureaherbicides and the feedback regulation of enzyme was analyzed in the presence of branched amino acids. Methods: The gene encoding catalytic subunit of rice AHAS (cOsAHAS) without part of the chloroplast transit sequence was cloned into the bacterial expression vector pET41a and heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST).The soluble protein was purified using affinity chromatography. The measurement of GSTOsAHAS activity was performed under optimized conditions at present of branched-chain amino acids and sulfonylurea herbicides independently. Results: The optimum pH and temperature for GST-cOsAHAS activity was 8.0 and 37 °C, respectively. The specific activity and Km value of this enzyme toward pyruvate were 0.08 U/mg and 30 mM, respectively.GST-cOsAHAS was inhibited by herbicides tribenuron, sulfosulfuron, nicosulfuron and bensulfuron while the enzyme was insensitivieto end products. Conclusion: These results suggest that the recombinant form of GST-cOsAHAS is functionally active and carries the binding site for sulfynylurea herbicides. Furthermore, GST-cOsAHAS was insensitive to feedback inhibition by endproducts which indicates the existence of a regulator subunit in rice AHAS as previously has been described in other plant AHASs.

Sign in / Sign up

Export Citation Format

Share Document