[Objective] Completely reconstruction of the biosynthetic pathway of whiE in Escherichia coli, isolation and purification of new compounds synthesized in the recombinant strain for elucidation of the biosynthetic pathway of whiE.[Methods] Construction of single-gene recombinant plasmids of whiE-ORFII, whiE-ORFVII, whiE-ORFI, and SDS-PAGE analysis of the fusion protein expression; isoschizomers Xba I/ Spe Ⅰ were adopted to assemble multiple genes in a single plasmid construct. The recombinant plasmid was then introduced into Escherichia coli BAP1 for heterologous expression, and the fermentation products was detected by high performance liquid chromatography (HPLC); the normal phase silica gel column and the reverse semi-preparative column were used sequentially to separate the fermentation products, the quadrupole high resolution mass spectrometry (Q-TOF MS) was used to identify the molecular weight of the fermentation products.[Results] whiE-ORFII, whiE-ORFVII and whiE-ORFI were solubly expressed; no new product was detected when these three genes were added to the strain BTw95 individually; two compounds ZYC-1 and ZYC-2 can be detected in the strains coupled with whiE-ORFII & whiE-ORFVII, whiE-ORFI & whiE-ORFVII as well as all the three genes. Q-TOF MS analysis in negative ion mode showed that the[M-H]^- of ZYC-1 is 419.0748 with a proposed molecular formula, C₂₃H₁₆O₈ and[M-H]^- of ZYC-2 is 465.0743 with a proposed molecular formula, C₂₄H₁₈O₁₀.[Conclusion]] This study achieved the heterologous reconstruction of the spore pigment whiE biosynthetic pathway in E. coli. Two dodecaketide type II polyketides were identified and isolated. The biosynthetic pathway of the spore pigment whiE was also proposed.