Phoslactomycins (PLMs) are inhibitors of protein serine/threonine phosphatase 2A showing diverse and important antifungal, antibacterial and antitumor activity. PLMs are polyketide natural products and produced by several Streptomyces species. The PLMs biosynthetic gene clusters were identified from Streptomyces platensis SAM-0654 and localized in two separate genomic regions, consisting of 27 open reading frames that encode polyketide synthases (PKSs), enzymes for cyclohexanecarboxyl-CoA (CHC-CoA) and ethylmalonyl-CoA (Em-CoA) synthesis, enzymes for post-PKS modifications, proposed regulators, and putative resistance transporters. Bioinformatic analysis and inactivation experiment of regulatory genes suggest that PnR1 and PnR2 are two positive regulators of PLMs biosynthesis. Gene transcription analysis by reverse transcriptase PCR (RT-PCR) of the PLMs gene cluster demonstrated that PnR1 and PnR2 activate the transcription of the structural biosynthetic genes while PnR2 specially governs the transcription of pnR1 in a higher level.