Solvent synthesis pathways
The key enzymes involved in the synthesis of butanol, ethanol and acetone were searched in the genome of GSU5 and other sequencedThermoanaerobacterium strains using the RAST annotation Server, BLAST and the Biocyc database collection in order to analyze the corresponding pathways.
As shown in Table 2, all genes coding for key enzymes of butanol synthesis were detected in GSU5 and all sequenced T. thermosaccharolyticum strains (locus tags in Table S3). Genes coding for the crotonase (crt ), butyryl-CoA dehydrogenase (bcd ), electron transfer flavoprotein (etfAB ), 3-hydroxybutyryl-CoA dehydrogenase (hbd ), and acetyl-CoA C-acetyltransferase (thl ), are located in a cluster, followed by but , that codes for a butyryl-CoA: acetate-CoA transferase, and rex , corresponding to a redox dependent transcriptional regulator (Figure 2). In the well-known butanol producer Clostridium acetobutylicumseven of the genes are also in a genomic cluster, the bcs operon, albeit with a different organization: in this organism thl is located in a different region, rex is situated upstream fromcrt , and but is absent.
Putative binding sites for the transcriptional regulator Rex (ROP: Rex operator site) were observed upstream from thl (Figure 2) and adhE in the genome of GSU5, suggesting that Rex could regulate the expression of these genes in Thermoanaerobacterium . Butanol synthesis genes were also found in Thermoanaerobacterium sp. RBIITD but with a genetic organization different from the one present in T. thermosaccharolyticum, since thl is located in the bcsoperon but rex is located upstream from crt (Figure 2). In contrast, these genes were absent in T. xylanolyticum LX-11, T. saccharolyticum JW/SL-YS485 , T. saccharolyticum NTOU1, T. aotearoense SCUT27 and Thermoanaerobacterium sp. PSU-2 , which suggests that they are not capable of producing butanol (Table 2).
There are 5 genes encoding alcohol dehydrogenases distributed throughout the genome of GSU5 and one encoding an aldehyde dehydrogenase (ald ). This last gene was present in about half of the analyzed genomes, including three out of five sequenced T. thermosaccharolyticum (Table 2). All Thermoanaerobacteriumstrains carry adhE , coding for the bifunctional alcohol/aldehyde dehydrogenase, in a monocistronic operon with a similar location in all genomes analyzed, as well as bhd and two other alcohol dehydrogenases. Interestingly, one of the dehydrogenases, adh3 , was located upstream from genes coding for a NADH-dependent reduced ferredoxin: NADP oxidoreductase (nfnAB ) in all genomes.
The well characterized C. acetobutylicum produces butanol and acetone through the ABE (Acetone-Butanol-Ethanol) pathway, so the possibility that T. thermosaccharolyticum produced this compound was studied. The genes that code for acetone synthesis enzymes were searched in the genomes of T. thermosaccharolyticum GSU5 and in all available Thermoanaerobacterium genomes using the genes from C. acetobutylicum as queries. None of the genomes analyzed carried ctfAB or adc , the genes that code for the butyrate-acetoacetate CoA-transferase (subunits A and B), and the acetoacetate decarboxylase respectively (Table 2).
Genes corresponding to enzymes involved in the synthesis of acetate and butyrate (Table 2) were also identified. T. thermosaccharolyticumGSU5 presents the gene coding for butyryl-CoA: acetate CoA- transferase (But) which catalyzes the conversion of butyryl-CoA to butyrate, located downstream from the butanol synthesis genes. This gene was also found in all other strains of this species, and in Thermoanaerobacteriumsp. RBIITD, albeit in this last case but is located in a different part of the genome (Figure 2). On the other hand T. xylanolyticum LX-11, T. saccharolyticum JW/SL-YS485, T. saccharolyticum NTOU1, T. aotearoense SCUT27 and T. sp.PSU-2 carry the genes coding for Ptb (that catalyzes the synthesis of butyryl-CoA to butyryl-P), and Buk (that catalyzes the conversion of butyryl-P to butyrate).