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Strain improvement through random mutagenesis, screening and selection has provided us with spontaneous mutants which could produce more ML-236B than the original isolate, Penicillium citrinum SANK18767. The objective of the present study is to clarify how a high-producing mutant No. 41520 acquired the ability to produce 500 times more ML-236B than the original isolate on a molecular basis. Southern blot analysis and sequence comparison revealed that amplification of the ML-236B biosynthetic gene cluster and alteration of nucleotides within the loci had not occurred in the genome of No. 41520. On the other hand, a differential hybridization and Northern blot analysis showed that expression levels of the nine biosynthetic genes mlcA to mlcH and mlcR in No. 41520 increased greatly as compared to those in the original isolate. These data suggested that the increase in ML-236B production was partly due to increased expression of genes involved in ML-236B biosynthesis. Morphological differences and higher consumption of carbon source would also affect ML-236B production in No. 41520. Functional analysis revealed that a gene, orf1 next to mlcR, was not involved in the ML-236B biosynthesis, but it was involved in the transcriptional activation of genes along with the ML-236B gene cluster. Titer enhanced mutations might have occurred in the regulation system for transcription activation of the ML-236B biosynthetic genes in the mutants of P. citrinum.

Molecular basis of ML-236B production in the high-producing mutant No. 41520 of Penicillium citrinum