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_act_thlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4 mM). 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 P_thlA and P_pta-ack. Batch fermentations using A. woodii [pMTL84151_act_thlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high K_m 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_act_thlA] was increased from 1.2 mg L⁻¹ h⁻¹ in bottle fermentation to 26.4 mg L⁻¹ h⁻¹ in continuous gas fermentation.