Polyether antibiotics exemplified as lasalocid and monensin contain multiple asymmetric centers. While their biosynthetic mechanism with exquisite stereo-control has attracted great interests, the configurations of the double bonds in a putative linear polyketide intermediate, as well as the modes of oxidative cyclization, have remained unidentified. Due to structual complexity, it is difficult to clalify the biosynthetic mechanism for monensin. We therefore focused lasalocid having more simple structure. Identification of entire lasalocid biosynthetic gene cluster has been accomplished. This cluster contains polyketide synthase gene and genes for epoxidation and hydrolysis presumably responsible for polyether formation. To identify the putative intermediate, we designed deuterium-labeled prelasalocid possessing (E, E) configuration of the trisubstituted olefins. The olefins of right segment 9 installing a deuterium label were constructed utilizing a palladium- and zirconium-mediated conversion of terminal acetylenes. Assembly of 9 with the central segment 10 based on the B-alkyl Suzuki-Miyaura reaction led to 11. After incorporation of the CD_3 group at C12, the resulting 14 was connected with the left segment 16 by an anti-aldol reaction. Based on the highly convergent strategy, the prelasalocid has been synthesized in a stereo-controlled manner.