New insights into the isopenicillin N transport in Penicillium chrysogenum

• Published in: Metabolic engineering 2014-03, Vol.22, p.89-103
• Main Authors: Fernández-Aguado, M., Martín, J.F., Rodríguez-Castro, R., García-Estrada, C., Albillos, S.M., Teijeira, F., Ullán, R.V.
• Format: Article
• Language: English
• Subjects: Biological Transport, Active - physiology; Carrier Proteins - genetics; Carrier Proteins - metabolism; Fungal Proteins - genetics; Fungal Proteins - metabolism; Isopenicillin N; Major facilitator transporter; Penicillin biosynthetic pathway; Penicillins - metabolism; Penicillium chrysogenum; Penicillium chrysogenum - genetics; Penicillium chrysogenum - metabolism; Peroxisomal membrane protein
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2014.01.004

In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRed protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane. •The penM gene encodes a membrane protein in Penicillium chrysogenum.•penM gene silencing and oveerexpresion affects the benzylpenicillin production.•6-APA feeding increases the benzylpenicillin production in SilM35 (silenced strain).•Localization studies showed that PenM protein is located in the peroxisomal membrane.•PenM could be implicated in the import of IPN to the peroxisome from the cytosol.