Microbial solvent formation revisited by comparative genome analysis

Abstract

Background: Microbial formation of acetone, isopropanol, and butanol is largely restricted to bacteria belonging to the genus Clostridium. This ability has been industrially exploited over the last 100 years. The solvents are important feedstocks for the chemical and biofuel industry. However, biological synthesis suffers from high substrate costs and competition from chemical synthesis supported by the low price of crude oil. To render the biotechnological production economically viable again, improvements in microbial and fermentation performance are necessary. However, no comprehensive comparisons of respective species and strains used and their specific abilities exist today.

Results: The genomes of a total 30 saccharolytic Clostridium strains, representative of the species Clostridium acetobutylicum, C. aurantibutyricum, C. beijerinckii, C. diolis, C. felsineum, C. pasteurianum, C. puniceum, C. roseum, C. saccharobutylicum, and C. saccharoperbutylacetonicum, have been determined; 10 of them completely, and compared to 14 published genomes of other solvent-forming clostridia. Two major groups could be differentiated and several misclassified species were detected.

Conclusions: Our findings represent a comprehensive study of phylogeny and taxonomy of clostridial solvent producers that highlights differences in energy conservation mechanisms and substrate utilization between strains, and allow for the first time a direct comparison of sequentially selected industrial strains at the genetic level. Detailed data mining is now possible, supporting the identification of new engineering targets for improved solvent production.

Keywords: Acetone; Butanol; C. beijerinckii; C. saccharobutylicum; C. saccharoperbutylacetonicum; Clostridium acetobutylicum; Phylogeny; Solvents.

Fig. 1

Historical development of industrial ABE strains: only sequenced strains are indicated. Data stem from Jones [7]

Fig. 2

Core/Pan genome analysis of 44 clostridial genomes: a simplified Venn diagram showing the core and the pan genome of all 44 solventogenic clostridia. The number of genome-specific OGs is depicted in the respective ellipse. Ortholog detection was done with blastp and the Proteinortho software [8] with a similarity cutoff of 50% and an E value of 1e−10

Fig. 3

MLSA tree of 44 sequenced solventogenic clostridia: a maximum likelihood tree of 44 solventogenic clostridial genomes was inferred with 500 bootstraps with RAxML [9] and visualized with Dendroscope [10]. Genomes sequenced within this study were marked with a red asterisk and type strains marked with a T

Fig. 4

Average nucleotide identity analysis of the 44 sequenced strains: ANI analysis based on MUMmer alignment of the genome sequences was performed and visualized using PYANI [13]

Fig. 5

Central metabolism of solventogenic clostridia: Color codes indicate the presence or absence of specific enzymes in the various species of solventogenic clostridia. Position and colors are always conserved from left to right: First row C. acetobutylicum, C. beijerinckii/C. diolis, C. puniceum; second row C. saccharobutylicum, C. saccharoperbutylacetonicum, Clostridium sp.; third row C. roseum/C. aurantibutyricum, C. pasteurianum, C. felsineum. Blanks (white) indicate absence of respective enzymes

Fig. 6

Structure of the sol operon: structure of the sol operon based on Tblastx comparison of representative members of the different subclades. An E value cutoff of 1e−10 was used and visualization were done with the program Easyfig [21]

Fig. 7

Localization of the sol operon: the localization of the sol operon in the megaplasmid pSOL1 of C. acetobutylicum is compared with the localization in the chromosome of C. aurantibutyricum, C. roseum, and C. felsineum. Visualization was done with Easysfig [21] (tblastx, E value cutoff of 1e−10). The GC-content of the C. acetobutylicum sol operon is depicted in comparison to the flanking regions