Regulation of the PDA1 gene encoding the E1alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae

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 Unique Structural Organization of the Saccharomyces Cerevisiae Pyruvate Dehydrogenase Complex

Microscopy and Microanalysis ◽

10.1017/s1431927600024892 ◽

1998 ◽

Vol 4 (S2) ◽

pp. 954-955

Author(s):

James K. Stoops ◽

Z. Hong Zhou ◽

John P. Schroeter ◽

Steven J. Kolodziej ◽

R. Holland Cheng ◽

...

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Pyruvate Dehydrogenase ◽

Structural Organization ◽

Pyruvate Dehydrogenase Complex ◽

The Other ◽

Oxidative Decarboxylation ◽

Multienzyme Complex ◽

The Core ◽

Dehydrogenase Complex

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.

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Biochemical and Molecular Genetic Aspects of Pyruvate Dehydrogenase Complex from Saccharomyces cerevisiae

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1989.tb14993.x ◽

1989 ◽

Vol 573 (1 Alpha-Keto Ac) ◽

pp. 155-167 ◽

Cited By ~ 5

Author(s):

LESTER J. REED ◽

KAREN S. BROWNING ◽

XIAO-DA NIU ◽

ROBERT H. BEHAL ◽

DAVID J. UHLINGER

Keyword(s):

Saccharomyces Cerevisiae ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Molecular Genetic ◽

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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