Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli

Abstract

The butyrogenic genes from Clostridium difficile DSM 1296(T) have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD(+)-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

Fig 1

(A) The butyrate biosynthetic pathway in genetically engineered E. coli. THIA1, thiolase; HBD, 3-hydroxybutyryl-CoA dehydrogenase; CRT2, crotonase; BCD/etfAB2, butyryl-CoA dehydrogenase complex; PBT, phosphate butyryltransferase; BUK, butyrate kinase; FNR, ferredoxin:NADP⁺ oxidoreductase; PntB, NAD(P)⁺ transhydrogenase. (B) The constructed expression plasmid pASG-ButD.wt harboring all eight genes necessary for butyrate production. Ori, ColEl orgin; tetP, Tet promoter; Amp, ampicillin resistance gene.

Fig 2

Purified recombinant enzymes. Coomassie-stained gels with individually purified proteins are shown. C-terminally StrepII-tagged thiolase (45 kDa, lane 1), 3-hydroxybutyryl-CoA dehydrogenase (34 kDa, lane 2), crotonase (30 kDa, line 3), C-terminally StrepII-tagged phosphate butyryltransferase (35 kDa, lane 4), and butyrate kinase (43 kDa, lane 5) (5 to 10 μg each) were separated by 12% SDS-PAGE. Lane 6, 15 μg of the BCD/Etf complex, which is composed of the dehydrogenase (42 kDa) and Etf subunits A (39 kDa) and B (29 kDa), with a C-terminal StrepII tag attached to EtfA, was separated on 15% SDS-PAGE. The sizes of relevant proteins of PageRuler (Fermentas) molecular mass standards (M) are indicated for comparison.

Fig 3

Composition of purified butyryl-CoA dehydrogenases fused to StrepII tags at different sites of individual subunits. Recombinant enzyme variants (20 μg of total protein) with C-terminal StrepII tags fused to EtfA (lane 1), dehydrogenase (lane 3), and EtfB (lane 5) and N-terminally tagged dehydrogenase (lane 2), EtfB (lane 4), and EtfA (lane 6) were separated on 12% SDS-PAGE and subjected to Coomassie staining. Note that only the enzymes represented in lanes 1, 4, and 6 are composed of all three subunits. The positions of relevant proteins in a Precision (Bio-Rad) molecular mass standard (M) are shown for comparison.

Fig 4

Michaelis-Menten kinetics of recombinant butyryl-CoA dehydrogenase. The parameters for the three substrates crotonyl-CoA (A), NADH (B), and ferredoxin (C) were determined independently. The insets show the Lineweaver-Burk plots of the primary data. Kinetics analyses were performed in 50 mM Tris-HCl (pH 7.5) supplemented with 10 mM MgCl₂, 50 mM NaCl, 10 μM FAD, 100 μM NADP⁺, FNR (0.3 U/ml), and 120 ng enzyme per ml. Note that only the level of the substrate in question was varied in the particular experiments, while the substrates not under consideration were kept constant at 50 μM crotonyl-CoA (B and C), 200 μM NADH (A and C), and 10 μM ferredoxin (A and B).

Fig 5

Induced butyrate formation by a recombinant E. coli strain. Escherichia coli Rosetta cells (Novagen) were transformed with the plasmid pASG-ButD.wt. The host cell (A) and the recombinant strain (B) were grown on M₉ medium supplemented with glucose. Glucose consumption (long-dash line), bacterial growth (solid line), acetate levels (dotted line), and butyrate levels (short-dash line) were monitored as indicated. The induction time is indicated by a gray arrow in panel B.