Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum.

BACKGROUND: Clostridium 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. RESULTS: The 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 (MLS (R) ) 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. CONCLUSIONS: The 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.

PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum.

Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is harmful or lethal to these bacteria. Nevertheless, it is known that they can survive limited exposure to air, and often eliminate oxygen or reactive derivatives via NAD(P)H-dependent reduction. This system does apparently contribute to survival after oxidative stress, but is insufficient to establish long-term tolerance of aerobic conditions. Here we show that manipulation of the regulatory mechanism of this defence mechanism can trigger aerotolerance in the obligate anaerobe Clostridium acetobutylicum. Deletion of a peroxide repressor (PerR)-homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment. The mutant strain also revealed higher resistance to H2O2 and activities of NADH-dependent scavenging of H2O2 and organic peroxides in cell-free extracts increased by at least one order of magnitude. Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial PerR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of PerR. This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.

Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation.

In Saccharomyces cerevisiae, acetate is formed by acetaldehyde dehydrogenase (ACDH), a key enzyme of the pyruvate dehydrogenase (PDH) bypass, which fulfils the essential task of generating acetyl-CoA in the cytosol. The role of the five members of the ACDH family (ALD genes) was investigated during anaerobic growth on glucose. Single and multiple ald Delta mutants were generated in the wine-yeast-derived V5 and laboratory CEN.PK strains and analysed under standard (YPD 5 % glucose) and wine (MS 20 % glucose) fermentation conditions. The deletion of ALD6 and ALD5 decreased acetate formation in both strains, demonstrating for the first time that the mitochondrial Ald5p isoform is involved in the biosynthesis of acetate during anaerobic growth on glucose. Acetate production of the ald4 Delta mutant was slightly decreased in the CEN.PK strain during growth on YPD only. In contrast, the deletion of ALD2 or ALD3 had no effect on acetate production. The absence of Ald6p was compensated by the mitochondrial isoforms and this involves the transcriptional activation of ALD4. Consistent with this, growth retardation was observed in ald6 Delta ald4 Delta, and this effect was amplified by the additional deletion of ALD5. A ald Delta null mutant, devoid of ACDH activity, was viable and produced similar levels of acetate to the ald6 Delta ald4 Delta ald5 Delta strain, excluding a role of Ald2p and Ald3p. Thus, acetate is mainly produced by the cytosolic PDH bypass via Ald6p and by a mitochondrial route involving Ald5p. An unknown alternative pathway can compensate for the loss of Ald6p, Ald4p and Ald5p.