Biomedical Engineering Reference
In-Depth Information
O
O
SNAC
DEBS
(KS1
o
or DKS1
)
HO
OH
R
1
R
1
O
N
OH
OH
R
1
HO
O
HO
R
2
O
OH
R
2
O
O
R
2
O
OH
O
O
O
O
OH
25
(R
1
= Me; R
2
= Et)
26
(R
1
= Me; R
2
= Bn)
27
(R
1
= Me; R
2
= Bu)
28
(R
1
= Et; R
2
= Et)
29
(R
1
= Me; R
2
= CH
2
CH
2
F)
30
(R
1
= Me; R
2
= CH
2
CH
2
N
3
)
SCHEME 14.6
Precursor-directed biosynthesis of selected analogues of erythromycin.
The first reported use of precursor-directed biosynthesis for the production of novel
erythromycin would be more appropriate for the final section of this chapter as it
involved the feeding of fluorinated analogues of 6-dEB to
S. erythraea
for oxidation
and glycosylation [32]. When propionyl-CoAwas identified as the likely primer unit
for DEBS, efforts were undertaken in which acetyl- and butyryl-CoAwere fed to the
native host. The first practically useful breakthrough came when a mutant of the
DEBS pathway was constructed in which the first ketosynthase domain had its
active-site cysteine mutated to an alanine residue. This mutant DEBS was expressed
in
S. coelicolor
CH999, a better understood and more tractable host compared to the
native
S. erythraea
, yielding a bacterial strain that produced no 6-dEB-like
molecules without supplementation. Upon addition of the
N
-acetylcysteamine
(SNAC) thioester of (2
S
,3
R
)-2-methyl-3-hydroxypentanoic acid (NDK), however,
the production of 6-dEB was observed [33]. This diketide-SNAC ester is analogous
to the biosynthetic intermediate that is passed from module 1 to module 2 in DEBS.
Its successful incorporation into the DEBS pathway demonstrated that module 2 was
capable of accepting substrates directly in the absence of a functional module 1. In
the initial study, it was demonstrated that this KS1 null mutant was capable of not
only accepting NDK, the diketide analogue of the natural substrate derived from
propionate, but also accepting bulkier substrates in the form of a chain lengthened
diketide derived from butyrate as well as a precursor with a terminal phenyl group,
leading to compounds
, respectively (Scheme 14.6). After the successful
incorporation of diketides in which the terminal ethyl group was varied, the ability
to alter the
a
-methyl group was examined, leading to the production of compound
26
and
27
28
(Scheme 14.6) [34]. A diketide in which the
a
-methyl group was replaced by a
methoxy moiety was also successfully used to produce the corresponding 6-dEB
analogue [35].
The first two of these analogues were isolated and fed to amutant of
S. erythraea
incapable of producing 6-dEB for post-PKS processing and both were oxidized and
glycosylated as expected to give bioactive compounds. This work began a decade of
extensive observation on the biosynthesis of unnatural erythromycin analogues that
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