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

Abstract

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 (Δmfst) complementation (ΔmfsT::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, ΔmfsT interrupt the penicillin G (PG) production in M. ruber M7 by blocking the IPN transportation, while PG and IPN produced by the ΔmfsT::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.

Taurine-mediated gene transcription and cell membrane permeability reinforced co-production of bioethanol and Monascus azaphilone pigments for a newly isolated Monascus purpureus

“…De novo RNA-Seq assays uncovered a series of key genes for M. purpureus treated with taurine. (1) For DEGs, the enhanced effect of product accumulation for MFS, transcription factor and FSH1 had been proofed for Monascus strains [16,43,44]. The enriched kinase activity was supported by cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway in GO analysis for MonAzPs production of M. purpureus [45].…”

The ABCT31 Transporter Regulates the Export System of Phenylacetic Acid as a Side-Chain Precursor of Penicillin G in Monascus ruber M7

“…The PG was extracted from the respective M. ruber M7 (wild-type) and Δ abct 31 by the previously defined method (Ramzan et al, 2019 ).…”

“…The previously reported method (Ullán et al, 2008 ; Ramzan et al, 2019 ) was used for the analysis of intra- and extracellular PG contents.…”

“…The mass profile of the extract was produced using electrospray ionization (ESI) coupled Acquity TQD tandem quadruple mass spectrometer (Waters, Manchester, UK). The conditions for the ESI-MS to detect MS were set according to Ramzan et al ( 2019 ). The presence of PG metabolites in the extract was confirmed using a PG standard with a purity of more than 98.0 percent (Sigma-Aldrich) (Lopes et al, 2013 ).…”

“…can create both useful and harmful secondary metabolites (SMs), such as Monascus pigments, aminobutyric acid, dimurumic acid, and monacolin K, and a typical type of mycotoxin, which is known as citrinin (Ozcengiz and Demain, 2013 ; Yuliana et al, 2017 ; Chen and Pan, 2019 ). Recently, Chen ( 2015 ) found a probable gene cluster having liability for β-lactam synthesis in the genome of M. ruber M7, and one β-lactam biosynthesis gene was identified in the gene cluster (Ramzan et al, 2019 ). M. purpureus was also proved to produce γ-lactam according to Wei et al ( 2017 ).…”