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: The biosynthesis of penicillin G (PG) is compartmentalized, which forces penicillin and its intermediates to cross the membrane barriers. Although many aspects around the penicillin intermediates traffic system remain unclosed, the transmembrane transporter protein involvement has been only predicted. In the present work, detection of PG and isopenicillin N (IPN) in Monascus ruber M7 was performed and functions of mfst gene as a transporter were investigated by the combination of gene deletion (Δ mfst ) complementation (Δ mfsT :: mfsT ) and overexpression (M7::PtrpC- mfsT ). While, the feeding of PG pathway precursor side chain and amino acids, i.e., phenylacetic acid, D-valine, and L-cysteine was performed for the interpretation of mfsT gene role as an intermediate transporter. The results showed that, the feeding of phenylacetic acid, D-valine, and L-cysteine possessed a significant effect on morphologies, secondary metabolites (SMs) production of all above-mentioned strains including M. ruber M7. The results of UPLC-MS/MS revealed that, Δ mfsT interrupt the penicillin G (PG) production in M. ruber M7 by blocking the IPN transportation, while PG and IPN produced by the Δ mfsT :: mfsT have been recovered the similar levels to those of M. ruber M7. Conclusively, these findings suggest that the M. ruber M7 is, not only a PG producer, but also, indicate that the mfsT gene is supposed to play a key role in IPN intermediate compound transportation during the PG production in M. ruber M7.

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Genetic Modification of mfsT Gene Stimulating the Putative Penicillin Production in Monascus ruber M7 and Exhibiting the Sensitivity towards Precursor Amino Acids of Penicillin Pathway