Genes Encoding Penicillin and Cephalosporin Biosynthesis in Acremonium chrysogenum: Two Separate Clusters are Required for Cephalosporin Production - Biotechnology of Fungal Genes - page 119

affect cephalosporin biosynthesis, but overexpression of this gene results
in a 100% increase in cephalosporin production (Ullán et al. 2002a). On the
other hand, the CefT protein confers resistance to branched chain fatty acids
(e.g., isovaleric acid) and phenylacetic acid as it was concluded from the fact
that these acids were toxic to the cefT disrupted strain (Ullán et al. 2002a).
Heterologous expression of the cefT gene in recombinant P. chrysogenum
cephalosporin-producing strains revealed that the CefT protein is involved
in the secretion of hydrophilic β-lactams, containing the α-aminoadipic acid
side-chain (Ullán et al. 2008) and fl uorescent labeling showed a plasma
membrane location (Nijland et al. 2008).
The cefM gene (Teijeira et al. 2009) encodes an effl ux pump protein
(482 amino acids with a deduced molecular mass of 52.2 kDa) belonging
to the MFS class of membrane proteins, specifi cally to Family 3 (drug
effl ux proteins). This transporter gene is located downstream of the cefD1
gene in the “early” cephalosporin cluster ( Fig. 2) . Targeted inactivation
of the cefM gene by means of the double marker technique resulted in
a drastic reduction in the cephalosporin and PenN secretion and an
important intracellular PenN accumulation. In trans complementation
of the cefM disrupted mutant completely restored the cephalosporin and
penicillin production to wild type levels. In vivo fl uorescence microscopy
analysis using a functional CefM-GFP hybrid protein, showed a probable
microbody membrane location of the CefM protein. In summary, it seems
that the CefM protein is involved in the PenN secretion from the microbody
lumen to the cytosol where it is converted into cephalosporin C (Teijeira
et al. 2009).
Upstream of the cefT gene there is another exporter gene named cefP
(Fig. 2). The cefP gene (Ullán et al. 2010) is 2769 nucleotides long, interrupted
by the presence of three introns and encodes a protein of 866 amino acids
with a deduced molecular mass of 99.2 kDa. Analysis of the amino acid
sequence by bioinformatics tools showed the presence of eleven putative
transmembrane spanners and a Pex19p-binding domain characteristic of
peroxisomal membrane proteins (Rottensteiner et al. 2004). This Pex19p-
binding domain is also present in the CefM protein (Teijeira et al. 2009).
Knock-out strains in cefP accumulate IPN in the culture broths and are
unable to synthesize cephalosporins in an effi cient manner. These results
indicate that the cefP gene is essential for cephalosporin C biosynthesis, since
disrupted mutants are blocked in the conversion of IPN into PenN due to
the lack of the CefP transporter. Confocal microscopy experiments with
the DsRed fl uorescent CefP hybrid protein indicated that the microbodies
reported by Teijeira and co-workers (2009) are authentic peroxisomes since
this hybrid protein co-localizes with the EGFP-SKL peroxisome-targeted
protein (Ullán et al. 2010).