MOESM1 of Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida

Additional file 1: Figure S1. Growth of Pseudomonas citronellolis LA18T on levulinic acid and pyruvic acid. OD600 values were measured. Symbols: black circle, levulinic acid; white circle, pyruvic acid. The vertical bar indicates the variation between two independent experiments. Figure S2. SDS-PAGE analysis of the soluble and insoluble fractions from recombinant E. coli cells carrying pCo7-387CoA-L and pCold™ IV. Lanes: M, molecular mass marker; 1 and 2, insoluble and soluble fractions from JM109 cells carrying pCo7-387CoA-L; 3, soluble fraction from JM109 cells carrying pCold™ IV; 4 and 5, insoluble and soluble fractions from DH5α cells carrying pCo7-387CoA-L. The crude extracts were applied to SDS-PAGE at a concentration of 0.5 μg/mL. Figure S3. Unrooted phylogenetic tree showing the positions of LvaE2 from Pseudomonas citronellolis (closed circle), LvaE from Pseudomonas putida (open circle), FadD from E. coli (closed triangle), and some other fatty acyl-CoA synthetases (FadK, open triangle; PrpE, closed square) based on multiple alignment of related proteins. The amino acid sequences used for phylogenetic analysis were collected through BLAST searches ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) using LvaE2, LvaE, E. coli FadD (long-chain fatty acid-CoA ligase; YP_006120162.1), E. coli FadK (short chain acyl-CoA synthetase; NP_416216.5), and E. coli PrpE (propionyl-CoA synthetase; NP_414869.1) as queries against the RefSeq database. As a result, 55, 79, 99, 55, and 62 sequences with similarity to LvaE2, LvaE, FadD, FadK, and PrpE, respectively, were obtained. The major constituents are: Group I (including FadD), long-chain fatty acid-CoA ligases; Group II, AMP-binding proteins; Group III (including LvaE2), long-chain fatty acid-CoA ligases; Group IV (including LvaE), acyl-CoA synthetases; Group V (FadK), short-chain acyl-CoA synthetases and cyclohexane carboxylate CoA ligases; and Group VI (PrpE), propionyl-CoA synthetases and acetate CoA ligases.