Biomedical Engineering Reference
In-Depth Information
genes responsible for cephalosporin C biosynthesis (available as a single
copy in the genome), are arranged in two groups or clusters (
Fig. 2)
located
in two different chromosomes (Skatrud and Queener 1989, Gutiérrez et al.
1999, Ullán et al. 2002b). The
pcbAB
,
pcb
C,
cefD1
and
cefD2
genes are linked
(Gutiérrez et al. 1999, Ullán et al. 2002b) in the so-called “early” cluster
(Gutiérrez et al. 1991), while the “late” cluster is formed only by the
cefEF
and
cefG
genes (Gutiérrez et al. 1992). In an industrial
A. chrysogenum
strain
(394-4) the “early” cluster was mapped to chromosome VI and the “late”
cluster to chromosome II (Skatrud and Queener 1989). Nevertheless, in
A.
chrysogenum
C10 (ATCC 48272) a different localization has been reported
for both the “early” and “late” cephalosporin clusters; chromosomes VII
(4.6 Mb) and I (2.2 Mb), respectively (Gutiérrez et al. 1991, Gutiérrez et
al. 1999). This different chromosomal location of cephalosporin clusters
indicates that signifi cant chromosome rearrangements have occurred
during strain improvement (Smith et al. 1991, Gutiérrez et al. 1999) giving
rise to changes in size and electrophoretic mobility of the chromosomes. It
seems that a smaller chromosome is resolved in the
A. chrysogenum
394-4
strain (but not in C10 strain) and chromosome I of
A. chrysogenum
C10
corresponds to chromosome II of the 394-4 strain.
Early Cluster: Genes for Isopenicillin N and
Penicillin N Biosynthesis
The two early enzymatic steps of the β-lactam biosynthetic pathway that
result in the formation of IPN, are common to all the classical β-lactam
producers (Martín et al. 2010). As indicated above,
A. chrysogenum
ACV
synthetase is a non-ribosomal peptide synthetase, which is a very large
multifunctional protein of 3712 amino acids (Gutiérrez et al. 1991). ACV
synthetase is encoded by an intron-free gene of 11136 bp named
pcbAB
(Gutiérrez et al. 1991). Similar genes occur in the fungal and bacterial
penicillin, cephalosporin and cephamycin clusters (Liras and Martín 2006).
The
pcbAB
gene is clustered with the
pcbC
gene (also known in
A.
nidulas
as
ipnA
) that encodes the IPN synthase (cyclase). In
A. chrysogenum
,
the
pcbAB
and
pcbC
genes (Fig. 2), in contrast to bacteria, are in divergent
orientation having a bidirectional promoter region (Liras and Martín 2006).
IPN is the fi rst bioactive compound of the β-lactam antibiotic pathway. Later,
in all the cephalosporin and cephamycin producers the L-α-aminoadipic
side chain of IPN is isomerized to the D enantiomer to form PenN.
As indicated in the previous section, the conversion of IPN into PenN
in
A. chrysogenum
involves the concerted action of an IPN-CoA synthetase
and an IPN-CoA epimerase encoded by two linked genes,
cefD1-cefD2
respectively, located downstream of the
pcbC
gene. The fi rst gene,
cefD1
, has
