Construction and characterization of pta gene‐deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation

Abstract

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.

Clostridium ljungdahlii represents a microbial production platform based on syngas

“…Transformation of E. coli was performed according to Inoue et al (38) or Dower et al (39). C. ljungdahlii was electroporated using a protocol for C. tyrobutyricum (30). Modifications: For preparation of competent cells a 50-mL culture of C. ljungdahlii in early exponential growth phase was used to inoculate 50 mL fresh medium containing 40 mM DL-threonine.…”

Butyric acid production from red algae by a newly isolated Clostridium sp. S1

“…S1 fermentation. Error bars represent 1 standard deviation of triplicate experiments (Zhu et al 2005), a small production of acetic acid by Clostridium sp. S1 is an advantage in enhancing selectivity and yield of butyric acid.…”

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

“…For the purpose of completely eliminating acetate production, it would be better to knock out pta gene, coded for phosphotransacetylase. It has been reported for several microorganisms that pta gene is located upstream of ack in the same operon and pta inactivation also leads to ack inactivation and results in significant reduction in acetate production (Green and Bennett, 1998;Green et al, 1996;Zhu et al, 2005). It is The fermentation was carried out with the synthetic medium without tetracycline for the wild-type.…”

“…The partial gene fragment in the nonreplicative plasmid can recombine with the internal region of the target gene on the parental chromosome, resulting in the insertional inactivation of the target gene. Integrational mutagenesis using nonreplicative plasmids containing host partial pta gene has been successfully used to create pta-disruption mutants of C. acetobutylicum and C. tyrobutyricum with significantly reduced PTA and AK activities and acetate production (Green and Bennett, 1998;Green et al, 1996;Zhu et al, 2005). In this work, both methods were used to create ack-disruption mutants of P. acidipropionici that showed similar transformation efficiency and fermentation kinetics.…”