Construction and characterization of ack knock‐out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation

Abstract Propionibacterium acidipropionici produces propionic acid from glucose with acetic acid, succinic acid, and CO 2 as byproducts. In this work, inactivation of ack gene, encoding acetate kinase (AK), by gene disruption and integrational mutagenesis was studied as a method to reduce acetate formation in propionic acid fermentation. The partial ack gene of ∼750 bp in P. acidipropionici was cloned using a PCR‐based method with degenerate primers and sequenced. The deduced amino acid sequence had 88% similarity and 76% identity with the amino acid sequence of AK from Bacillus subtilis . The partial ack gene was used to construct a linear DNA fragment with an inserted tetracycline resistance cassette and a nonreplicative integrational plasmid containing a tetracycline resistance gene cassette. These DNA constructs were then introduced into P. acidipropionici by electroporation, resulting in two mutants, ACK‐Tet and TAT‐ACK‐Tet, respectively. Southern hybridization confirmed that the ack gene in the mutant ACK‐Tet was disrupted by the inserted tetracycline resistance gene. As compared to the wild‐type, the activities of AK were reduced by 26% and 43% in ACK‐Tet and TAT‐ACK‐Tet mutants, respectively. The specific growth rate of these mutants was reduced by ∼25% to 0.10/h (0.13/h for the wild‐type), probably because of reduced acetate and ATP production. Both mutants produced ∼14% less acetate from glucose. Although ack disruption alone did not completely eliminate acetate production, the propionate yield was increased by ∼13%. © 2006 Wiley Periodicals, Inc.