Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses.
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. 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.



Bio-based production of chemicals from renewable biomass has become increasingly important due to our concerns on climate change and other environmental problems. C. tyrobutyricum has 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 of C. tyrobutyricum for the bio-based production of various chemicals in addition to butyric acid.
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, Yuseong-gu, Daejeon, South Korea.
Joungmin
J
Lee
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, Yuseong-gu, Daejeon, South Korea.
Yu-Sin
YS
Jang
Department of Biomolecular and Chemical Engineering, Dongyang University, Yeongju, Gyeongbuk, South Korea.
Mee-Jung
MJ
Han
Mass Spectrometry Research Center, Korea Basic Science Institute, Cheongju, Chungbuk, South Korea.
Jin Young
JY
Kim
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, Yuseong-gu, Daejeon, South Korea Bioinformatics Research Center, KAIST, Daejeon, South Korea BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, KAIST, Daejeon, South Korea Institute for the BioCentury, KAIST, Daejeon, South Korea leesy@kaist.ac.kr.
Sang Yup
SY
Lee
D000693:Anaerobiosis; D020148:Butyric Acid / Q000378:metabolism*; D048014:Clostridium tyrobutyricum / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D020869:Gene Expression Profiling; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D010957:Plasmids; D020543:Proteome / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D020543:Proteome; D020148:Butyric Acid; D005947:Glucose