Functional expression in Lactobacillus plantarum of xylP encoding the isoprimeverose transporter of Lactobacillus pentosus

Abstract

The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.

FIG. 1

Strategy for the construction of the Lactobacillus-E. coli shuttle plasmid pLPA6(t) and the expression vector pLPA6. The forward primer (5′-TTCTAAGATCTAGGTACCATTAATTGAATTCAGAAAGAAGGC-3′) used in the PCR amplification of xylP generated BglII and KpnI sites (underlined) and a stop codon (indicated in boldface) upstream of the original xylP RBS. The reverse primer (5′-TTAAGGGCCCTCCTTTCTTATCCCATCTTAC-3′) generated an ApaI site (underlined) downstream of the original xylQ RBS. The expression cassette of pLPA6 was derived from plasmid pLP503(t) (a broad-host-range expression vector) (15), which contains the constitutive promoter P_ldh of L. casei ATCC 393, the β-glucuronidase gene (gusA) from E. coli, and the terminator T_cbh of L. plantarum 80 (3). bla, β-lactamase (ampicillin resistance) determinant; ermC, erythromycin resistance determinant; ori−, origin of replication of pGEM; ori+, origin of replication of Lactobacillus plasmid pLP3537 (10); Rep, replication protein gene of pLP3537; 2*T_ldh, two tandemly arranged transcription terminators of the ldh gene of L. casei.

FIG. 2

Time course for isoprimeverose uptake by L. plantarum 80/pLPA6 (○) and L. plantarum 80 wild-type cells (•) at an extracellular pH of 4.5. Cells were energized with l-malate (50 mM), and transport was assayed as described in the text. The amount of isoprimeverose taken up was obtained by calculating the difference of the amount of glucose measured in the presence of l-malate (Table 1, reaction 2) and the amount of glucose measured in the absence of l-malate (Table 1, reaction 1).