Construction and Characterization of ack Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid and Hydrogen Production

Abstract

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.

Biochemical Production and Separation of Carboxylic Acids for Biorefinery Applications

“…Several reviews have been published on different strains used for butyric acid production and seven different genera have been investigated for their potential industrial applications [56,61]. Some of the most commonly studied and industrially used strains includes C. tyrobutyricum [62,63], C. acetobutylicum [64,65], and C. thermobutyricum [66,67]. A major drawback of using clostridial strains for butyric acid production is the formation of other side products such as acetic acid, ethanol, butanol, acetaldehyde, and lactate, as well as gases such as CO 2 and H 2 .…”

Blocking the butyrate-formation pathway impairs hydrogen production in <italic>Clostridium perfringens</italic>

“…Inactivation of ack (encoding acetate kinase), thus inactivating the acetate formation pathway in Clostridium tyrobutyricium, enhanced hydrogen and butyrate production from glucose. However, no significant effect on acetate formation was identified [11]. In a previous study with the same strain, inactivation of pta, encoding phosphotransacetylase which is another key enzyme in the acetate formation pathway, had little effect on hydrogen production.…”

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from maltose and soluble starch by overexpressing α-glucosidase

“…All recombinant plasmids constructed in this work were transformed into the mutant strain of C. tyrobutyricum ATCC 25755 with ack knockout developed in a previous study (Liu et al 2006). Clostridial growth medium (CGM) was used to culture C. tyrobutyricum at 37°C under anaerobic conditions.…”