J. Nat. Prod. 2009, 72, 469–472
469
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In Vitro Biosynthesis of Unnatural Enterocin and Wailupemycin Polyketides
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John A. Kalaitzis, Qian Cheng, Paul M. Thomas, Neil L. Kelleher, and Bradley S. Moore*
Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, UniVersity of California at San Diego,
La Jolla, California 92093-0204, and Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801
ReceiVed September 23, 2008
Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family
of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme
syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have
generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated
further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis
pathways.
Enzymes in organic synthesis are typically employed in stere-
ochemical resolution and functional group alteration reactions due
to their regiochemical and stereoselective prowess. In the synthesis
of polyketide natural products, for instance, biosynthetic enzymes
have been utilized as powerful biocatalysts in numerous transforma-
tions that include oxidation, glycosylation, and macrocyclization
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reactions. In some cases, however, de novo syntheses of complex
products from simplified building blocks have been reported that
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nicely illustrate the power of multienzymatic synthesis.
We recently reconstituted the biosynthesis of the antibiotic
polyketide enterocin from benzoic and malonic acids, which
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involved upward of a dozen recombinant and commercial enzymes.
By controlling the composition of the biosynthetic enzymes, we
were able to produce a series of natural wailupemycin and enterocin
polyketides derived from the marine bacterium Streptomyces
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maritimus. In this study we set out to evaluate a collection of
benzoic acid substrate analogues in the enzymatic assembly of a
library of enterocin and wailupemycin derivatives in which we could
control the input of substrates and enzyme biocatalysts.
Figure 1. In vitro biosynthesis and structures of wailupemycins F
and G and desmethyl-5-deoxyenterocin (R ) benzyl) by recom-
binant enterocin enzymes. Replacement of the benzoic acid primer
with other mono- and disubstituted benzoates and heteroaromatic
carboxylates yielded new structural analogues (see Figure 3).
The enterocin and wailupemycin polyketides are assembled from
benzoic acid and seven molecules of malonyl-CoA by the enc type
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II polyketide synthase (PKS) complex (Figure 1). Initiation of the
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biosynthetic reaction is carried out by EncN, which catalyzes the
ATP-dependent activation and transfer of benzoate to the acyl
carrier protein (ACP), EncC. The migration of the benzoyl unit
from EncC to the ketosynthase heterodimer EncA-EncB allows for
the subsequent malonation of holo-EncC by malonyl-CoA:ACP
transacylase (FabD), which sets up the first Claisen condensation
reaction between the benzoyl and malonyl units. This process is
repeated six additional times to yield an octaketide that is further
processed by the ketoreductase EncD to yield a pathway intermedi-
ate common to the wailupemycin and enterocin natural products.
Without further enzyme processing, this reactive intermediate
undergoes nonenzymatic cyclization reactions to give wailupemy-
cins D-G. Alternatively, the key rearrangement catalyst EncM
oxidatively converts the linear polyketide intermediate in a Favor-
skii-like reaction into the enterocin tricyclic scaffold. Further
tailoring by the O-methyltransferase EncK and the cytochrome P450
hydroxylase EncR completes the pathway to enterocin.
the biosynthetic pathway in a test tube. This provided us the
opportunity to compare the synthesis of unnatural enc-based
analogues produced in vitro against a prior collection prepared in
vivo. We previously exploited the exogenous S. maritimus pathway
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to benzoyl-CoA to create a small library of enterocin and
wailupemycin analogues through mutasynthesis in which the
disruption of the phenylalanine ammonia lyase encP gene allowed
for pathway rescue by chemical complementation with a series of
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aryl acids. However, although the benzoate:CoA ligase EncN
displayed a broad in vitro substrate tolerance, this agility was not
mirrored in vivo for the whole pathway reconstitution with unnatural
primers. Thus in a very controlled manner by limiting substrates
and enzyme biocatalysts, we set out to explore whether we could
extend the in vivo library.
The ultimate success of this enzymatic total synthesis approach
with unnatural substrates initially hinges upon the ability of EncN
to transfer unnatural substrates to the ACP EncC. We monitored
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All of the enterocin pathway enzymes have been prepared as
recombinant proteins and subsequently reconstituted to recapitulate
this transfer reaction by FTMS analysis of the holo-EncC protein
(12 145.11 Da; 12 144.97 Da theor, ∆ 0.14 amu). Upon incubation
with EncN, benzoate was transferred in typical nonribosomal
peptide synthetase fashion, resulting in a +104 Da mass shift to
12 249.55 Da (12 249.00 Da theor, ∆ 0.55 amu) (Figure 2A/B).
While benzoic acid is the preferred physiological substrate of
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Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute
and State University for his pioneering work on bioactive natural products.
*
Corresponding author. Tel: (858) 822-6650. Fax: (858) 558-3702.
E-mail: bsmoore@ucsd.edu.
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EncN, as demonstrated with competition binding experiments with
Scripps Institution of Oceanography.
University of Illinois.
Skaggs School of Pharmacy and Pharmaceutical Sciences.
‡
salicylic and 2,3-dihydroxybenzoic acids (Figure 2C), in its absence,
EncN preferentially activates and loads salicylate (12 265.56 Da;
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0.1021/np800598t CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/12/2009
Kalaitzis, John A.
