Genomic analysis of carbon monoxide utilization and butanol production by Clostridium carboxidivorans strain P7

Abstract

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T) possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2) fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T) chromosome. The functionality of these pathways was also confirmed by growth of P7(T) on CO and production of CO(2) as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7(T) was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T) genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.

Figure 1. Growth and native levels of VFA and solvent production of C. carboxidivorans strain P7^T.

A) Strain P7^T growth (OD₆₀₀), CO consumption and CO₂ and H₂ production. B) Strain P7^T native levels of VFA (acetate and butyrate) and solvents (ethanol, butanol and acetone) production.

Figure 2. Phylogenetic tree.

Phylogenetic tree based on complete 16S rRNA sequences from different acetogenic and/or solventogenic Clostridium species, and mesophilic and thermophilic acetogens. Horizontal bar represents sequence divergence. C. carboxidivorans strain P7^T is underlined.

Figure 3. Wood-Ljungdahl pathway in C. carboxidivorans strain P7^T.

Wood-Ljungdahl pathway key enzymes and protein identified in C. carboxidivorans strain P7^T. 1, formate dehydrogenase; 2, formate-tetrahydrofolate ligase; 3 and 4, bifunctional methenyl-tetrahydrofolate cyclohydrolase/methylene-tetrahydrofolate dehydrogenase (NADP+); 5, 5,10-methylene-tetrahydrofolate reductase; 6, 5-methyl-tetrahydrofolate:corrinoid iron-sulfur protein methyltransferase; 7, carbon monoxide dehydrogenase; 8, acetyl-CoA synthase; CFeSP, corrinoid iron-sulfur protein; CODH, additional carbon monoxide dehydrogenase complex. Reactions from the western branch are indicated in blue, those from the eastern branch are indicated in red. The corresponding genes in strain P7^T genome are indicated below the enzyme.

Figure 4. Wood-Ljungdahl pathway gene cluster in C. carboxidivorans strain P7^T.

Comparison between the Wood-Ljungdahl pathway gene cluster from C. carboxidivorans strain P7^T and those from the five groups described by Pierce et al. . cooC, CODH accessory protein; acsA, CODH/ACS complex, CODH subunit; acsB, CODH/ACS complex, ACS subunit; acsC, corrinoid iron-sulfur protein large subunit; fd, ferredoxin; acsF, CODH accessory protein similar to CooC; acsD, corrinoid iron-sulfur protein small subunit; acsE, CODH/ACS complex, methyltransferase subunit; fhs, formyl-tetrahydrofolate synthase; fchA, formimido-tetrahydrofolate cyclodeaminase; folD, bifunctional methylene-tetrahydrofolate dehydrogenase/formyl-tetrahydrofolate cyclohydrolase; hyp, hypothetical protein; metF, methylene-tetrahydrofolate reductase; acoL, CODH/ACS complex, dihydrolipoamide dehydrogenase subunit; gcvH, glycine cleavage system H protein; pulE, ATPase.

Figure 5. Metabolic fermentation pathways of acetyl-CoA in C. carboxidivorans strain P7^T.

1, phosphate acetyltransferase; 2, acetate kinase; 3 and 4, bifunctional acetaldehyde/alcohol dehydrogenase; 5, acetyl-CoA acetyltransferase; 6, coenzyme A transferase; 7, 3-hydroxybutyryl-CoA dehydrogenase; 8, 3-hydroxybutyryl-CoA dehydratase; 9, butyryl-CoA dehydrogenase; 10, phosphate butyryltransferase; 11, butyrate kinase; 12, aldehyde dehydrogenase; 13, NADH-dependent butanol dehydrogenase. The corresponding genes in C. carboxidivorans strain P7^T genome are indicated below or next to each arrow. Dashed arrows indicate that the reactions may not occur in C. carboxidivorans strain P7^T.

Figure 6. Comparison of the bcs gene clusters from C. carboxidivorans strain P7^T and other butanol-producing Clostridium.

Organization and gene composition of the bcs cluster from strain P7^T and from C. saccharobutylicum, C. beijerinckii and C. acetobutylicum as described by Berezina et al. . crt, crotonase; hbd, 3-hydroxybutyryl-CoA dehydrogenase; thl, acetyl-CoA acetyl transferase; bcd, butyryl-CoA dehydrogenase; etfB, electron transport protein, subunit B; etfA, electron transport protein, subunit A.

Figure 7. Confirmation of the presence of a plasmid in C. carboxidivorans strain P7^T.

A) Isolation of plasmid p19 from strain P7^T by standard cesium chloride gradient. Visualization of the two bands of DNA with UV illumination. Arrow indicate the lower band which contains the closed circular plasmid DNA. B) Digestion of the plasmid DNA by EcoRI. 1 kb ladder was used. Sizes are indicated on the left (ladder) and on the right (plasmid DNA).