Acetone production with metabolically engineered strains of Acetobacterium woodii.

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_actthlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters PthlA and Ppta-ack. Batch fermentations using A. woodii [pMTL84151_actthlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased from 1.2mgL(-1)h(-1) in bottle fermentation to 26.4mgL(-1)h(-1) in continuous gas fermentation.

A modified pathway for the production of acetone in Escherichia coli.

A modified synthetic acetone operon was constructed. It consists of two genes from Clostridium acetobutylicum (thlA coding for thiolase and adc coding for acetoacetate decarboxylase) and one from Bacillus subtilis or Haemophilus influenzae (teII(srf) or ybgC, respectively, for thioesterase). Expression of this operon in Escherichia coli resulted in the production of acetone starting from the common metabolite acetyl-CoA via acetoacetyl-CoA and acetoacetate. The thioesterases do not need a CoA acceptor for acetoacetyl-CoA hydrolysis. Thus, in contrast to the classic acetone pathway of Clostridium acetobutylicum and related microorganisms which employ a CoA transferase, the new pathway is acetate independent. The genetic background of the host strains was crucial. Only E. coli strains HB101 and WL3 were able to produce acetone via the modified plasmid based pathway, up to 64mM and 42mM in 5-ml cultures, respectively. Using glucose fed-batch cultures the concentration could be increased up to 122mM acetone with HB101 carrying the recombinant plasmid pUC19ayt (thioesterase from H. influenzae). The formation of acetone led to a decreased acetate production by E. coli.

A rubrerythrin-like oxidative stress protein of Clostridium acetobutylicum is encoded by a duplicated gene and identical to the heat shock protein Hsp21.

Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with proteins predicted to be encoded by the genome of this bacterium revealed that this stress protein is encoded by two almost identical open reading frames CAC3597 and CAC3598. These genes encode a rubrerythrin-like protein with the rubredoxin-like FeS4 domain at the N-terminus and the ferritin-like diiron domain (rubrerythrin domain) at the C-terminus. Thus, the order of the two putative functional domains is reversed compared to "normal" rubrerythrins. This protein is proposed to be involved in the oxidative stress response of strict anaerobic bacteria. Northern blot analysis indicated that hsp21 is induced by heat and oxidative stress (air, H2O2). Hsp21 of C. acetobutylicum can be considered as a "reverse" rubrerythrin and a role of this stress protein, which is conserved among clostridia and other strict anaerobic bacteria, in the heat and oxidative stress response is proposed.

Mutagenesis of conserved charged amino acids in SLH domains of Thermoanaerobacterium thermosulfurigenes EM1 affects attachment to cell wall sacculi.

SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 microg mg(-1)) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.