Biomedical Engineering Reference
In-Depth Information
chrysogenum
was later achieved through another environmentally friendly
two-step effi cient approach that was based on the overexpression of the
S. clavuligerus
cefE
gene in a industrial strain of
A. chrysogenum
previously
disrupted in the
cefEF
gene (Velasco et al. 2000). As a consequence, this
strain accumulates DAOC, which is the starting material for 7-ADCA
(Fig. 4).
During the fi rst step, the
cefEF
gene encoding the bifunctional
expandase/hydroxylase was disrupted leading to the accumulation
of PenN by the
A. chrysogenum
mutants. In the following step, the
expandase-encoding
cefE
gene from
S. clavuligerus
(under the control of
the
P. chrysogenum pcbC
gene promoter), which has no relevant DAOC
hydroxylase activity, was introduced in those transformants for DAOC
production. The purifi ed DAOC obtained from the fermentation broth of
A. chrysogenum
is transformed into ketoadipyl-7-ADCA by a D-amino acid
oxidase (DAO) obtained from the basidiomycetous fungus
Rhodotorula
gracilis
. This compound spontaneously reacts with the hydrogen peroxide
produced in this reaction and is decarboxilated yielding glutaryl-7-ADCA.
In the second step, glutaryl-7-ADCA is further hydrolyzed to 7-ADCA
by means of a glutaryl acylase (GLA) obtained from
Acinetobacter
sp.
(Velasco et al. 2000).
Another biological procedure used to modify
A. chrysogenum
for the
biosynthesis of 7-ACA directly by fermentation, was tried before through the
combination of fungal and bacterial genes. This was achieved by introducing
the 7-ACA biosynthetic operon in
A. chrysogenum.
This operon contains
the genes encoding DAO from
Fusarium solani
and GLA from
Pseudomonas
diminuta
and as a consequence,
A. chrysogenum
was endowed with the ability
to convert cephalosporin C into 7-ACA and two side products, 7-ADCA
and 7-aminodecetylcephalosporanic acid (7-DAC). Although the strain
produced detectable levels of 7-ACA, those levels were not signifi cant for
commercial purposes (Isogai et al. 1991).
CONCLUSION
Most of the enzymes involved in cephalosporin biosynthesis are known,
although the molecular mechanisms underlying some of the reactions
still remain obscure (e.g., the multiple reactions performed by the ACV
synthetase to form the LLD-ACV tripeptide or the mechanism of ring
expansion that converts the fi ve-membered thiazolidine ring to the six-
membered dihydrothiazine ring of cephalosporins).
The genes encoding the biosynthetic enzymes have been cloned and
are known to be located in two different chromosomes in the genome of
A.
chrysogenum
. However, further genes involved in transport or regulation
might be still unknown. The involvement of two different gene clusters
suggests that there are two consecutive biosynthetic pathways that need
