DecipheringClostridium tyrobutyricumMetabolism Based on the Whole-Genome Sequence and Proteome Analyses

ABSTRACTClostridium tyrobutyricumis a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence ofC. tyrobutyricumKCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested thatC. tyrobutyricumproduces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently fromClostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels,in vitroCoA transferase assay, and fed-batch fermentation. In addition, the changes in protein expression levels during the course of batch fermentations on glucose were examined by shotgun proteomics. UnlikeC. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways inC. tyrobutyricumdid not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent inC. acetobutylicum, were identified. Such features explain why this organism can produce butyric acid to a much higher titer and better tolerate toxic metabolites. This study presenting the complete genome sequence, global protein expression profiles, and genome-based metabolic characteristics during the batch fermentation ofC. tyrobutyricumwill be valuable in designing strategies for metabolic engineering of this strain.IMPORTANCEBio-based production of chemicals from renewable biomass has become increasingly important due to our concerns on climate change and other environmental problems.C. tyrobutyricumhas been used for efficient butyric acid production. In order to further increase the performance and expand the capabilities of this strain toward production of other chemicals, metabolic engineering needs to be performed. For this, better understanding on the metabolic and physiological characteristics of this bacterium at the genome level is needed. This work reporting the results of complete genomic and proteomic analyses together with new insights on butyric acid biosynthetic pathway and energy conservation will allow development of strategies for metabolic engineering ofC. tyrobutyricumfor the bio-based production of various chemicals in addition to butyric acid.