Biomedical Engineering Reference
In-Depth Information
The rapamycin PKS is initially charged with 4,5-dihydroxycyclohex-1-ene
carboxylic acid (DHCHC). This precursor molecule is then passed through a series of
elongation steps with the final linear molecule having L-pipecolate appended to the
terminus immediately prior to cyclization to form prerapamycin. This L-pipecolate
coupling and the subsequent cyclization are carried out by the NRPS gene product of
rapP
. The macrocycle is then acted upon by a series of tailoring enzymes including
P450 monooxygenases and SAM methyltransferases to yield the final rapamycin
molecule [12].
Prior to the identification of DHCHC as the likely priming molecule for the
rapamycin PKS, initial precursor-directed approaches toward the production of
rapamycin analogues were undertaken by feeding L-pipecolate analogues, such as
L-proline, to cultures of
S. hygroscopius
, leading to the production of compounds
such as
(Scheme 14.2) [13]. This work increased titers of the unnatural products
through the chemical inhibition of
L
-pipecolate biosynthesis with (
7
)-nipectoic acid,
a novel approach that presents a potentially simpler alternative to the often seen
strategy of genetic modification. This inhibitor was fed along with the sulfur-
containing
L
-pipecolate analogues, which successfully led to the production of
compounds similar to
[14].
After DHCHC had been identified as the primer for the rapamycin PKS, a more
traditional study was undertaken in which a series of commercially available
carboxylic acids were fed to
S. hygroscopius
, several of which were successfully
incorporatedwith
6
8
being a representative example (Scheme 14.2). This approachwas
extended when a mutant of
S. hygroscopius
was produced in which the shikimic acid
pathway, the means by which cells access DHCHC, was disrupted. This system led to
the incorporation of several precursors that had shown little or no acceptance in the
wild-type organism and the production of the subsequent prerapamycin analo-
gues [15-17].
This increased substrate tolerance by the mutant system allowed a more in-
depth study of the requirements on a substrate for incorporation into the rapamycin
biosynthetic pathway. It was ultimately concluded that a hydrogen bond acceptor is a
requirement for recognition by the PKS system.
OMe
HO
O
X
n
H
3
CO
OH
O
R
O
H
5 (
X = C,
n
= 1)
O
O
OH
6 (
X = S,
n
= 1)
7 (
X = C,
n
= 0)
O
X
N
O
MeO
R
OH
HO
n
OH
H
O
O
8 (
X = C,
n
= 1)
SCHEME 14.2
Various precursor-directed approaches toward the production of selected
rapamycin analogues.
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