Abstract
ABSTRACTClostridium tyrobutyricum is 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 of C. tyrobutyricum KCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested that C. tyrobutyricum produces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently from Clostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels, in vitro CoA 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. Unlike C. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways in C. tyrobutyricum did not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent in C. 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 of C. tyrobutyricum will be valuable in designing strategies for metabolic engineering of this strain.
Highlights
Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals
One interesting feature of the C. tyrobutyricum genome is its compactness; its genome size is about 78% and 65% of that of its closest relatives C. kluyveri and C. ljungdahlii, respectively (Table 1)
C. acetobutylicum was chosen as the positive control, as it has PTB and butyrate kinase (BK) activities. (C) Specific activities of butyrate:acetate coenzyme A (CoA) transferase in the C. tyrobutyricum Korean Collection for Type Cultures (KCTC) 5387 crude extract
Summary
Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. C. tyrobutyricum can be employed as a great microbial cell factory for the production of various chemicals due to several advantages including its ability to grow on a minimal medium, high metabolic flux toward butyric acid, and tolerance to the product at high concentrations [6] Despite such potential, C. tyrobutyricum has received less attention as an industrial host strain compared to other clostridial species, mainly due to our limited understanding on its genotypic and metabolic characteristics at the genome level or scale. C. tyrobutyricum has received less attention as an industrial host strain compared to other clostridial species, mainly due to our limited understanding on its genotypic and metabolic characteristics at the genome level or scale Most studies of this bacterium so far have been focused on the optimization of fermentation processes [7,8,9,10]. The lack of complete genome sequence and transcriptomic and/or proteomic studies during the course of fermentation has limited our understanding of the physiological characteristics of C. tyrobutyricum for subsequent metabolic engineering at the systems level
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