Biology Reference
In-Depth Information
fungi, and bacteria, in which they are produced by PKSs which polymerize acyl-Coenzyme
As (CoAs) in a fashion similar to fatty acid synthesis.
Erythromycin A is a clinically important polyketide antibiotic produced by the
Gram-positive bacterium
Saccharopolyspora erythraea
.
154
It is a macrolide antibiotic with an
antimicrobial spectrum similar to that of penicillin, and is often used for people who are
allergic to penicillin to treat respiratory infections, whooping cough, syphilis,
Legionnaires
disease, gastrointestinal infections, and acne. After the discovery and
characterization of the DEBS PKSs,
118,155
157
which synthesize the macrocyclic aglycone of
the antibiotic erythromycin, the biochemical basis for these highly controlled
megasynthases was gradually revealed. Meanwhile, the functional expression of the DEBS
proteins and their mutants in heterologous hosts such as
S. coelicolor
158
and
E. coli
159
allows the rational design of biosynthetic products, and leads to the generation of diverse
polyketide libraries by means of combinatorial cloning of naturally occurring and mutant
PKS modules.
'
One notable example of rational design is the incorporation of unnatural starter units
to change the chain initiation process. Chain initiation on many modular polyketide
synthases is mediated by acyl transfer from the CoA thioester of a dicarboxylic acid,
followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain.
160
Therefore, one approach is to replace the loading domain AT with other AT
domains,
127,160,161
or incorporate synthetic oligoketide precursors into the downstream
DEBS pathway.
131
Kinoshita and coworkers
162
fed several diketide and triketide substrates
to a recombinant
E. coli
strain containing a variant form of the DEBS cluster from which the
first elongation module was deleted. They successfully obtained macrolactone formation
from diketide substrates, but not the triketide substrates. To investigate the structural basis
for selectivity among triketide analogues, Cane and coworkers
163
incubated a series of
N
-
acetyl cysteamine (-SNAC) esters of unsaturated triketides with DEBS module 2
195
TE and
identified some that were good to excellent substrates. Based on this study, Regentin and
coworkers
164
used precursor-directed biosynthesis to produce different triketide lactones
(R-TKLs) in a fermentation process, and found that the R group on the precursor
significantly affected titer (propyl
1
chloromethyl
vinyl). An overproducing strain of
..
.
S. coelicolor
expressing the DEBS module 2
TE protein was found to be best for production
of R-TKLs, and a maximum titer of 500 mg/L 5-chloromethyl-TKL was obtained using 3.5 g/
L precursor. Later, rather than using the KS1
0
system (the DEBS1 protein with an inactivated
ketosynthase domain), Ward and coworkers
165
showed that removing the DEBS loading
domain and first module can lead to a two-fold increase in the utilization of diketide
precursors, and production of 6-dEB analogues (R-6dEB) in
S. coelicolor
and
S. erythraea
exhibited a 10-fold increase.
1
Besides the precursors, the extender unit can also be replaced in the DEBS system. The DEBS
AT domains of each module are the primary gatekeepers for the incorporation
of methylmalonyl-CoA into the polyketide chain. With replacement by heterologous
AT domains that accept malonyl-, ethylmalonyl-, or methoxylmalonyl units, corresponding
building blocks have been verified to be incorporated into the final polyketide
products.
119,123
125,166
168
In addition to swapping domains, 17 new heterologous genes were expressed in a
6-deoxyerythronolide B (6dEB) producer
E. coli
strain, and the resulting strain was capable
of producing the potent antibiotic erythromycin C.
169
Combinatorial polyketide
biosynthesis by de novo design and rearrangement of modular DEBS genes with other
PKS genes allow production of novel derivatives as well.
170
Fourteen modules from eight
PKS clusters were synthesized, and 154 bimodular combinations were created. Nearly half
of the combinations successfully mediated the biosynthesis of a polyketide in
E. coli
, and all
individual modules participated in productive bimodular combinations.
