Heterologous expression of a novel serine palmitoyltransferase from Sphingobium chungbukense

In the described experiments, we identify and clone a novel serine palmitoyltransferase (SPT) isolated from the gram-negative obligatory aerobic bacteria Sphingobium chungbukense strain KCTC2955. SPT catalyzes the first rate-limiting reaction in the de novo biosynthesis of sphingolipids, in S. chungbukense. The DNA sequence of the cloned SPT gene (1791 bp) has an open reading frame encoding 405 amino acids with a predicted molecular mass of 41 kDa; the sequence contains the consensus PLP-binding motif (GTFSKSXXXXGG) which is conserved among serine palmitoyltransferase. The ribosomal binding site GGGA lays 6 bp upstream of the ORF of 1218 bp, which is initiated by an ATG codon and terminated by TGA. The cloned SPT gene succeeded in overproducing the SPT protein under the T7 promoter of the expression vector pET 21a in Escherichia coli BL21(DE3), in which the product amounted to about 20–30% of the total protein isolated from the cell extract. Sphingobium SPT showed about a 68.4–89.3% homology with other enzymes in the serine palmitoyltransferase family, and amino acid residues proposed to be involved in catalysis were conserved. The activity of purified recombinant-SPT using Ni-NTA metal-affinity chromatography was measured via the radioactivity of the [3H]-3-ketodihydrosphingosone (KDS) formation using l-[3H]-serine. We have identified a novel SPT gene from S. chungbukense KCTC2955 and overexpressed the recombinant SPT in E. coli. This study is a first step in understanding the reaction mechanism of SPT in bacteria as well as the first focused on the genus Sphingobium.