Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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On the Specificity of the Herbicide Chlorsulfuron in Intact Spinach Chloroplasts

Zeitschrift für Naturforschung C ◽

10.1515/znc-1985-11-1229 ◽

1985 ◽

Vol 40 (11-12) ◽

pp. 917-918 ◽

Cited By ~ 3

Author(s):

Uwe Homeyer ◽

D. Schulze-Siebert ◽

G. Schultz

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Acid Synthesis ◽

Spinach Chloroplasts ◽

Transamination Reaction ◽

Dehydrogenase Complex

Abstract In vitro incubation of intact spinach chloroplasts with 1 mᴍ Pyruvate was used to study the specificity of action of the herbicide Chlorsulfuron on the synthesis of valine, alanine and fatty acids. As a result, increasing concentrations of the herbicide strongly inhibited valine synthesis while fatty acid synthesis via pyruvate dehydrogenase complex (PDC) and alanine formation by transamination reaction was promoted.

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In vitro synthesis and processing of components of the Ascaris suum pyruvate dehydrogenase complex

Molecular and Biochemical Parasitology ◽

10.1016/0166-6851(88)90113-2 ◽

1988 ◽

Vol 29 (1) ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

V RUFF ◽

S DESAI ◽

E DUBRUL ◽

R KOMUNIECKI

Keyword(s):

Pyruvate Dehydrogenase ◽

Ascaris Suum ◽

Pyruvate Dehydrogenase Complex ◽

In Vitro Synthesis ◽

Synthesis And Processing ◽

Dehydrogenase Complex

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Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

Biochemical Journal ◽

10.1042/bj2890081 ◽

1993 ◽

Vol 289 (1) ◽

pp. 81-85 ◽

Cited By ~ 35

Author(s):

J Quinn ◽

A G Diamond ◽

A K Masters ◽

D E Brookfield ◽

N G Wallis ◽

...

Keyword(s):

Escherichia Coli ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Structural Studies ◽

Gene Encoding ◽

E Coli ◽

Inner Lipoyl Domain ◽

Dehydrogenase Complex ◽

Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

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Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase

Biochemical Journal ◽

10.1042/bj2480351 ◽

1987 ◽

Vol 248 (2) ◽

pp. 351-358 ◽

Cited By ~ 22

Author(s):

B Laber ◽

N Amrhein

Keyword(s):

Pyruvate Dehydrogenase ◽

Potent Inhibitor ◽

Pyruvate Dehydrogenase Complex ◽

Anthocyanin Synthesis ◽

Anaerobic Conditions ◽

First Order ◽

Saturation Kinetics ◽

Thiamin Pyrophosphate ◽

Dehydrogenase Complex

The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate.

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