The invention relates to cloning, sequencing, analysis and functional study of a biosynthetic gene cluster of a natural product FR901464 which has anti-tumor activity and is generated from pseudomonas as well as the application thereof. The whole gene cluster contains 20 genes: five polyketide synthase genes, one hybrid polyketide/non-ribosomal polypeptide synthase gene, one non-ribosomal polypeptide synthase gene, three independent acyltransferase genes, four genes related to polyketide backbone alkylation, four post-modification genes and two control-related genes. The genetic operation of the biosynthetic gene can block the biosynthesis of FR901464. The provided gene and protein thereof can be used for genetic engineering, protein expression, enzyme catalysis reaction, and the like of the compound and can also be used for searching and discovering compounds that can be used in medicines, industry or agriculture or genes and proteins thereof.