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

Abstract

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.

Importance: 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.

FIG 1

Genome atlas view of the C. tyrobutyricum KCTC 5387 (ATCC 25755) chromosome and the pCTK01 plasmid. The outermost ring shows the coordinates of the chromosome or plasmid. The coding sequences in the forward and reverse strands are colored blue and orange, respectively. tRNAs and rRNAs are indicated by red and green, respectively. The fourth and fifth rings represent the deviations of GC content and GC skew, i.e., (G − C)/(G + C), from their average values, respectively; purple, negative deviation (below average); green, positive deviation (above average).

FIG 2

Summary of the C. tyrobutyricum shotgun proteomics. (A) Batch fermentation profiles used for the sample preparation. Symbols: closed circles, glucose; closed squares, OD₆₀₀; closed diamonds, acetic acid; closed triangles, butyric acid; open squares, ethanol. (B) A Venn diagram of proteins identified at each sampling point for label-free shotgun proteomic data. The number of proteins is shown in each area. (C) Histograms that present relative protein fold changes at C4 and C6 compared to C2 based on TMT-tagged proteome samples.

FIG 3

Protein changes of the central metabolic pathway in C. tyrobutyricum. The protein changes are from TMT-labeled identification results. Genes which products were identified are indicated in blue. Genes indicated in black are those genes whose products were not identified in TMT-labeled samples but were identified in label-free samples. Genes indicated in red are those genes whose products were not identified both in TMT-labeled and label-free peptide samples. Glucose-6-P, glucose-6-phosphate; Fd_ox, oxidized ferredoxin.

FIG 4

Butyric acid production in C. tyrobutyricum KCTC 5387 (ATCC 25755) depends on butyrate:acetate CoA transferase, not phosphotransbutyrylase (PTB) and butyrate kinase (BK). (A) Hypothesized pathway of butyric acid production in C. tyrobutyricum KCTC 5387. (B) Specific activities of PTB and BK in the crude extracts of C. tyrobutyricum (Cty) and C. acetobutylicum (Cac) ATCC 824. C. acetobutylicum was chosen as the positive control, as it has PTB and BK activities. (C) Specific activities of butyrate:acetate CoA transferase in the C. tyrobutyricum KCTC 5387 crude extract. The assay was performed in the presence (+) or absence (−) of sodium acetate (SA). (D) RT-PCR of four candidate CoA transferase genes in the wild-type (WT) and cat1::int strains of C. tyrobutyricum. The thiolase gene was chosen as the internal control.