In vivo substrate specificity of HMO transporters was examined by heterologously expressing the gene clusters in lnbX- and gltA-deficient B. longum 105-A strain with pMSK65 carrying the genes encoding intracellular exo-glycosidases for HMO depolymerization (Fig. 1, A and B) (16). An In-Fusion HD Cloning kit (Clontech Laboratories, Mountain View, CA, USA) was used for ligation henceforth. pMSK65 was constructed as follows: First, the constitutive xfp promoter region (29) and 1,2-α-l-fucosidase gene of B. infantis (Blon_2335) were amplified by polymerase chain reaction (PCR), and the fragments were inserted into Pst I– and Sal I–digested pBFS38 (29), generating pMSK36. Then, the PCR-amplified xfp promoter region, N-acetylglucosaminidase gene (Blon_0459), and LNT β-1,3-galactosidase gene (Blon_2016) were inserted into the Bgl II site of pMSK36 in this order, which yielded pMSK59. Last, the xfp promoter region, 1,3/1,4-α-l-fucosidase gene (Blon_2336), and sialidase gene (Blon_2348) were amplified, and the three fragments were inserted into the Not I site of pMSK59 in that order, resulting in pMSK65.

The gene clusters of FL transporter-1 (Blon_0341–0343) and FL transporter-2 (Blon_2202–2204) from B. infantis JCM 1222T and FL transporter-2 (BBKW_1838–1840) from B. kashiwanohense JCM 15439T that were amplified by PCR were inserted into the Nde I site of pJW241 (30) (a compatible plasmid with pMSK65) under the control of the xfp promoter. The resulting plasmids were introduced into pMSK65-harboring B. longum 105-A ΔlnbX ΔgltA strain. The primers used for strain construction are listed in table S3 (nos. 1 to 12). The PCR-amplified fragments were sequenced to ensure that no base changes other than those planned had occurred.

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