The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.

The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaAS͞DhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.

Developing a commercial process for the biological production of n-butanol is challenging as it needs to combine high titer, yield, and productivities. Here we engineer Clostridium acetobutylicum to stably and continuously produce n-butanol on a mineral media with glucose as sole carbon source. We further design a continuous process for fermentation of high concentration glucose syrup using in situ extraction of alcohols by distillation under low pressure and high cell density cultures to increase the titer, yield, and productivity of n-butanol production to the level of 550 g/L, 0.35 g/g, and 14 g/L/hr, respectively. This process provides a mean to produce n-butanol at performance levels comparable to that of corn wet milling ethanol plants using yeast as a biocatalyst. It may hold the potential to be scaled-up at pilot and industrial levels for the commercial production of n-butanol.

BackgroundClostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.ResultsThe method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLSR) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the “marker-less” genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get strains no longer producing acetone or l-lactate.ConclusionsThe restriction-less, marker-less strain described in this study, as well as the maker-less genetic exchange coupled with positive selection, will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

The glycerol oxidative pathway of Clostridium butyricum VPI 1718 plays an important role in glycerol dissimilation. We isolated, sequenced, and characterized the region coding for the glycerol oxidation pathway. Five open reading frames (ORFs) were identified: dhaR, encoding a putative transcriptional regulator; dhaD (1,142 bp), encoding a glycerol dehydrogenase; and dhaK (995 bp), dhaL (629 bp), and dhaM (386 bp), encoding a phosphoenolpyruvate (PEP)-dependent dihydroxyacetone (DHA) kinase enzyme complex. Northern blot analysis demonstrated that the last four genes are transcribed as a 3.2-kb polycistronic operon only in glycerol-metabolizing cultures, indicating that the expression of this operon is regulated at the transcriptional level. The transcriptional start site of the operon was determined by primer extension, and the promoter region was deduced. The glycerol dehydrogenase activity of DhaD and the PEP-dependent DHA kinase activity of DhaKLM were demonstrated by heterologous expression in different Escherichia coli mutants. Based on our complementation experiments, we proposed that the HPr phosphoryl carrier protein and His9 residue of the DhaM subunit are involved in the phosphoryl transfer to dihydroxyacetone-phosphate. DhaR, a potential regulator of this operon, was found to contain conserved transmitter and receiver domains that are characteristic of two-component systems present in the AraC family. To the best of our knowledge, this is the first molecular characterization of a glycerol oxidation pathway in a Gram-positive bacterium.Glycerol can be utilized as a carbon source by bacteria via several metabolic pathways that convert glycerol to dihydroxyacetone-phosphate (DHAP) before DHAP enters the glycolytic pathway. Under aerobic conditions, Escherichia coli phosphorylates glycerol using an ATP-dependent glycerol kinase (19), and glycerol-3-phosphate then is oxidized to DHAP by a membrane-bound FAD-dependent glycerol-3-phosphate dehydrogenase (35). Under anaerobic conditions, Klebsiella pneumoniae and Citrobacter freundii oxidize glycerol using a soluble NAD ϩ -dependent glycerol dehydrogenase (9, 13), and dihydroxyacetone (DHA) then is phosphorylated by a DHA kinase to DHAP (9,13,21). DHA kinases can be grouped into two structurally related families according to the source of the high-energy phosphate: ATP or phosphoenolpyruvate (PEP). ATP-dependent DHA kinases are single-polypeptide two-domain proteins (37), while the PEP-dependent DHA kinases consist of three subunits: DhaK, DhaL, and DhaM (18). DhaK and DhaL are homologous to the amino-terminal K domain and the carboxy-terminal