Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Article abstract of DOI:10.1002/ange.202004991

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non-native polyketide substrates. Reactions pairing wildtype PKS modules with non-native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non-cognate TE domains was severely reduced. In contrast, non-native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.

Full text of DOI:10.1002/ange.202004991

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Titel: Probing Selectivity and Creating Structural Diversity Through
Hybrid Polyketide Synthases
Autoren: David H. Sherman, Aaron A Koch, Jennifer Schmidt, Andrew
Lowell, Douglas Hansen, Katherine Coburn, and Joseph
Chemler
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Probing Selectivity and Creating Structural Diversity Through
Hybrid Polyketide Synthases
Aaron A. Koch, Jennifer J. Schmidt, Andrew N. Lowell, Douglas A. Hansen, Katherine M. Coburn,
Joseph A. Chemler, and David H. Sherman*
Abstract: Engineering polyketide synthases (PKS) to produce new
metabolites requires an understanding of catalytic points of failure
during substrate processing. Growing evidence indicates the
processing of non-native substrates. We demonstrate that
engineered modules containing heterologous substrate-
a
matched TE domain generally restore polyketide production.
Initial experiments attempted to convert full-length synthetic
polyketide intermediates (1-3, Figure 1A)[4] into the corresponding
macrolactones (4-8) using the terminal two modules from the
pikromycin (Pik) and erythromycin (DEBS) systems, and the
penultimate module from the juvenimicin (Juv) pathway. These
modules share similar domain architecture and ability to process
advanced polyketide substrates into their respective
macrolactone natural products (4, 6, 9-10, Figure 1B). Notably,
the presence or absence of a ketoreductase (KR) domain within
a PKS module influences if the final macrolactone contains a 3-
hydroxy (4, 7) or 3-keto (5, 6) functionality. The use of penultimate
modules for cyclization required the incorporation of a TE in place
of C-terminal docking domains, modifications that have been
designed previously for Pik Module 5 (Mod5) and DEBS Mod5.[5]
thioesterase (TE) domain as
a significant bottleneck within
engineered PKS systems. We created a series of hybrid PKS modules
bearing exchanged TE domains from heterologous pathways and
challenged them with both native and non-native polyketide
substrates. Reactions pairing wildtype PKS modules with non-native
substrates primarily resulted in poor conversions to anticipated
macrolactones. Likewise, product formation with native substrates
and hybrid PKS modules bearing non-cognate TE domains was
severely reduced. In contrast, non-native substrates were converted
by most hybrid modules containing a substrate compatible TE, directly
implicating this domain as the major catalytic gatekeeper and
highlighting its value as a target for protein engineering to improve
analog production in PKS pathways.
Engineered polyketide synthases (PKSs) are often found to be
highly inefficient, displaying significant decreases in product yield
or failing to generate the anticipated metabolite entirely compared
to a wild type module against its native substrate.[1] Determining
the point(s)-of-failure in these engineered pathways remains
challenging due to the dependence on downstream domains to
successfully process intermediates into mature, detectable small
molecules.[2] In contrast, targeted in vitro reactions in which
isolated PKS modules process full-length synthetic substrates
provides information about decreased catalytic function within a
PKS module. Rigorous characterization of the products generated
in engineered PKS reactions is crucial to understanding and
optimizing catalysis and metabolite production in these systems.
Previous in vitro experiments have demonstrated that PKS
modules can process non-native substrates in some cases and
have delineated obstacles that limit engineering of PKS
systems.[ The present study focuses on how substitution of the
thioesterase (TE) domain within a PKS module effects its ability
to process native and non-native substrates. Herein, TE domains
are shown to be a primary point of catalytic inefficiency in the
Wild-type PKS modules (Figure 1A) efficiently processed
native substrates in vitro into corresponding macrolactones
(Figure 1C, underlined), while modules presented with non-native
substrates typically showed a severe decrease in product
formation. The Pik Mod5-Pik TE hybrid protein efficiently
processed native substrate (1) to 10-dml (4) with 75% conversion;
however, when DEBS Mod5-DEBS TE and DEBS Mod6-DEBS
TE were reacted with 1 as a non-native substrate, poor
conversions of 1.2 and 6.6% were observed, respectively.
Likewise, reactions with 2 followed a similar trend. Consistent with
our previous report[4b], Pik Mod6-Pik TE efficiently processes 2 to
narbonolide (6). Although DEBS Mod6-DEBS TE produced
substantial levels of 3-hydroxy-narbonolide
7 (due to the
presence of a KR in the DEBS Mod6 domain architecture), all
other non-native reaction pairings failed to produce appreciable
levels of either macrolactone 6 or 7 from 2. Furthermore, all
reactions containing 3 failed to generate the predicted product (8).
3]
This analysis of PKS modules with native and non-native
substrates reinforces the challenge of designing and engineering
[6]
PKS modules for synthetic biology applications. Recently, we
have shown[ that TE modifications have a significant effect on
the catalytic processing of non-native substrates by PKS modules.
We concluded that the decreased yields from reactions containing
non-native substrates (Figure 1C) are at least partially due to
ineffective processing within the TE domain.
7]
[*]
Prof. D. H. Sherman
Life Sciences Institute, Departments of Medicinal Chemistry,
Chemistry, Microbiology & Immunology
The University of Michigan
2
10 Washtenaw Avenue, Ann Arbor, MI 48109-2216n (USA)
To determine if the decreased yields originate from
compatibility issues between the polyketide intermediates and the
TE domain in each terminal module, we generated a series of
PKS fusion proteins where the native TE domain was exchanged
for a corresponding domain from a heterologous system (see
Supporting Information). Modules were chosen from the
functionally related Pik, DEBS, and Juv biosynthetic pathways
which generate macrolactones that vary in size from 12- to 16-
membered rings (Figure 1B). All modules tested possess the core
ketosynthase (KS), acyl transferase (AT), and acyl carrier protein
E-mail: davidhs@umich.edu
Dr. A. A. Koch, Dr. J. J. Schmidt, Dr. D. A. Hansen, K. M. Coburn,
Dr. J. A. Chemler
Life Sciences Institute, The University of Michigan (USA)
Dr. A. N. Lowell
Current Address: Department of Chemistry, Virginia Tech
Blacksburg VA 24061 (USA)
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie
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4
a
4b
4c
Figure 1. (a) Reaction scheme of PKS modules processing their native full-length synthetic substrates 1 , 2 , 3 to
the corresponding macrolactones. Substrate 2 is generated by in situ photolysis of the 2-nitrobenzyloxymethyl ether
NBOM) protected native hexaketide.4 (b) Domain architecture of the PKS modules used in this study and the native
b
(
macrolactone products from each respective biosynthetic pathway. (c) Table of percent conversion for reactions with
substrates 1-3. The product yield for each PKS module processing its native substrate is underlined. Conversion to
each macrolactone was monitored by HPLC with data represented as the mean ± standard deviation where n = 3.
ND = not detected.
(
ACP) domains critical for extension of a polyketide with an
and second, how does a match between the TE domain and the
incoming linear (acyl-ACP) intermediate affect the efficiency of
product formation? In all reactions where 1 was a non-native
substrate (e.g. DEBS Mod5, DEBS Mod6, and Juv Mod6 with 1),
we achieved significant conversions to 10-dml (4, Table 1) when
the substrate (1) matched the TE domain (Pik TE, underlined
yields). Our results demonstrate that the KS-AT-ACP-KR
domains of these PKS modules maintain a suitable level of
catalytic function when the downstream TE domain (Pik TE) is
correctly matched to the incoming unnatural intermediate (1). This
conclusion is supported by the 21, 17, 41, and 75% conversions
from the reactions containing, DEBS Mod5-Pik TE, DEBS Mod6-
Pik TE, Juv Mod6-Pik TE, Pik Mod5-Pik TE, respectively (Table
additional acyl unit, and all except Pik Mod6 contain
a
ketoreductase (KR) domain, which reduces the post-extension -
keto group to a hydroxyl (i.e., Figure 1A, 4 versus 5). This
provided hybrid PKS modules with either a cognate TE from its
own biosynthetic pathway or non-cognate TE domain from the
other two pathways (Figure 1, Table 1). We then characterized
the effect of the non-cognate TE on the ability of the hybrid PKS
modules to process native and non-native substrates.
Our initial expansion tested the flexibility of the hybrid TE
modules using 1 as the substrate (Table 1). While all the hybrid
modules share the same domain configuration (KS-AT-KR-ACP-
TE) and are expected to produce 10-dml (4) from 1, 3-keto-10-
dml (5) has also been reported as a result of domain-skipping.[3]
HPLC quantification of the reaction products compared to
authentic standards of 4 and 5[7b] provided new insights into two
key questions: First, can PKS module-TE domain hybrids from
different type I PKS pathways function in an engineered context,
1). These results also indicate that the DEBS and Juv TE domains
are unable to efficiently generate 12-membered macrolactones as
the percent conversions of hybrid modules containing these TE
domains were low, even when the substrate (1) was native to the
PKS module (Pik Mod5). The observation that pairing of the TE
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domain to the native polyketide substrate considerably enhances
product yield reinforces the value of assessing the innate
substrate specificity of TE domains in the context of engineered
PKSs. Although attenuated yields may also be attributed to one
or more of the other PKS catalytic domains (e.g. KS, AT, KR,
ACP), the TE has been identified as a central feature for effective
off-loading and cyclization of polyketides.[7] Selection or
modification of a TE within a module can therefore increase
Table 2. Evaluation of PKS TE hybrid modules with 2. [a]
Table 1. Evaluation of PKS TE hybrid modules with 1,
[
a]
the native substrate of Pik Mod5.
[
a] Substrate 2 is generated by in situ photolysis of the 2-
nitrobenzyloxymethyl ether (NBOM) protected native
4
b
hexaketide. Conversion to 6 or 7 was monitored by
HPLC with data represented as the mean ± standard
deviation where n = 3. Trace = detected by LC-MS but
below the detection limit of HPLC. ND = not detected.
functionality present in the linear polyketide chain (Figure 2). The
native linear seco-acid product of Pik Mod6 is unreduced (i.e. keto
group) at the C-3 position and its cognate TE displays a
preference for this ACP-bound substrate. Surprisingly, when
substituted onto the DEBS Mod6 as the DEBS Mod6-Pik TE
hybrid, the Pik TE retains its preference for the 3-keto ACP-
tethered intermediate and selectively catalyzes cyclization to 6.
This result suggests that the binding affinity of the unreduced
ACP-tethered intermediate is higher toward the non-cognate Pik
TE rather than to its own natural KR domain, thus leading to
release of 6 instead of KR-mediated reduction of the β-keto group
at C-3 and release of 7. This ability of the Pik TE to strongly
interact with C-3 keto intermediates is also observed in the
reaction of 1 with DEBS Mod6-Pik TE leading to 5 (Table 1).
[
a]Reactions where the TE domain is paired with the
substrate are underlined. Conversion to 4 or 5 was
monitored by HPLC with data represented as the mean
±
MS but below the detection limit of HPLC. ND= not
detected.
standard deviation where n = 3. Trace=detected by LC-
product yields and processing of non-native substrates.
We next expanded our studies to assess substrate flexibility
by probing hybrid TE modules for the ability to accept and process
Pik hexaketide 2[4b] into 14-membered macrolactones using
HPLC quantification against known standards (Table 2). Similar
to the results using 1, substitution of the cognate Pik TE domain
in Pik Mod6 with either the DEBS or Juv TE domains led to a
severe decrease in the ability of Pik Mod6 to convert 2 into
macrolactone 6. However, when reacted with DEBS Mod6, 2
could be converted into 6 and 7. Due to the domain architecture
of DEBS Mod6, the expected product is C-3-OH macrolactone 7.
Compound 7 is produced with the DEBS and Juv TE fusion
proteins, but with the Pik TE, C-3-keto macrolactone 6 is favored.
This result suggests a preference of the various TE domains for
functionality at the C-3 position that matches their native chain
elongation intermediate when offloading 14-membered rings.
Thus, the hybrid DEBS Mod6-Pik TE produces predominantly
Next, we tested the ability of hybrid TEs to retain activity when
coupled with an upstream module, an interaction critical to late-
stage PKS engineering as it ensures that a hybrid protein will
function correctly in the context of other modules. Reactions were
performed using 3[4c] in its native context with Juv Mod6, which
must then pass an elongated intermediate to Juv Mod7-TE
hybrids for an additional extension and cyclization (Table 3). LC-
MS analysis of these reactions revealed that DEBS, Juv, and Pik
TE domains all possessed the ability to generate a 16-membered
macrolactone when fused to Juv Mod7. HPLC quantification of
tylactone (9) production demonstrated Juv Mod6 + Juv Mod7-Juv
TE to be the most efficient (34%), followed closely by Juv Mod7-
DEBS TE (30%). This result not only shows that PKS hybrid TE
modules are catalytically competent when used with upstream
modules, but also further supports selectivity in the TE for 3-
hydroxy or 3-keto functionality.
narbonolide (6), suggesting that this hybrid mediates skipping of
the KR domain [
keto group at C-3, as dictated by the match between the TE and
substrate oxidation state (Table 2).
5a, 8]
, in preference for macrolactonization with the
Having identified the crucial role of the TE domain in
engineered PKS systems, we sought to harness hybrid TE
modules to produce unnatural macrolactones. Accordingly, we
These data show that the ability of a TE domain to effectively
cyclize an advanced polyketide intermediate depends on the
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Figure 2. Biocatalytic mechanism of the processing of 2 by DEBS Mod6. Individual PKS-TE fusion proteins produce
divergent products apparently due to innate affinities between the -keto thioester and the TE versus KR.
linear acid with subsequent spontaneous hemiketalization and
Table 3. Reaction of 3 with Juv Mod6 + Juv Mod7 TE
hybrids.
[
a]
dehydration. Addressing this issue was approached using two
methods: first a serine to alanine TE inactivating mutant was
generated in Juv Mod6-Pik TE (Juv Mod6-Pik TES148A) to
decouple the activity of the TE domain from that of the module.
Second, a rapid time course analysis was conducted using LC-
MS analysis to monitor the release of seco-acid and/or hemiketal
products as well as formation of the dehydrated pyran moiety.
Initial analytical reactions with Juv Mod6-PikTES148A failed to
produce recognizable seco-acid/hemiketal products.
Next, we evaluated the time course experiments with the goal
of understanding the origin of the dehydrated pyran (see
Supporting Information). These efforts yielded emergence of a
substance corresponding to either the seco-acid and/or hemiketal
upon incubation for 1-2 hours. Semi-preparative scale up and
characterization of this compound to determine its identity as a
linear acid, hemiketal or a mixture of the two proved challenging,
however, a fraction consisting of a mixture of four isomers was
obtained. Efforts to separate these molecules by subsequent
rounds of purification proved futile indicating the mixture is likely
interconverting. NMR characterization yielded nearly complete
assignment of the four compounds, which include three linear
chain stereoisomers (sites of epimerization are unclear) as well
as a single hemiketal constituent 12. In order to confirm that this
mixture could spontaneously dehydrate, the initial mixture was
subjected to buffered conditions with and without Juv Mod6-Pik
TE. Monitoring the product profile over time demonstrated partial
conversion to the dehydrated pyran 13 irrespective of Juv Mod6-
Pik TE enzyme. These results indicate that the extended
hexaketide is likely offloaded from the PKS module as the seco-
acid, which rapidly interconverts between the hemiketal and the
seco-acid, and subsequently dehydrates to generate the final
isolated product 13 (Scheme 1).
[
a] Conversion to 9 was monitored by HPLC with data
represented as the mean ± standard deviation where n =
. Trace=detected by LC-MS but below the detection limit
3
of HPLC.
utilized the Juv Mod6 TE hybrids and screened with substrate 3
(
the natural substrate for this module), to probe for the ability to
generate the predicted 14-membered diene macrolactone 8
Scheme 1). Analytical experiments assessed by LC-MS initially
indicated that a product corresponding to the correct mass was
formed only when Juv Mod6 was paired with the Pik TE domain.
To confirm this initial result, we performed a 0.1 mmol scale
reaction and purified the product by preparatory HPLC.
Surprisingly, the isolated product was a pyran containing seco-
acid 13 (Scheme 1) as opposed to the expected macrolactone 8.
The historical reliance on in vivo reactions and assessing
(
fermentation
extracts
by
liquid
chromatography-mass
spectrometry (LC-MS) analysis of the culture medium has limited
the ability to characterize new structures.[2a, 2b] LC-MS analysis
can provide ambiguous data with isomeric molecules generating
identical mass spectra, precluding definitive structural assignment.
Importantly, pyran 13 and the anticipated macrolactone 8 share
the same exact mass and were ultimately distinguished by a full
complement of NMR studies (see Supporting Information).
The work described herein provides further insight into the
substrate flexibility of type I modular PKS TE domains and their
application in PKS engineering. The TE domain is a critical
catalytic gatekeeper for the processing of unnatural substrates
into macrolactones and other new molecules, and partially
Initial efforts aimed at evaluating the origin of this new
dehydrated pyran functionality centered on discerning whether it
resulted directly from TE mediated catalysis, or by offloading the
explains previous studies where non-native substrates failed to
be processed by engineered PKS modules.[5a] To explore the
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Scheme 1. Reaction of Juv Mod6-Pik TE with Tyl hexaketide 3 results in pyran 13, which is identical in mass to
macrolactone 8. This outcome was only achieved via extension of Tyl hexaketide through Juv Mod6-Pik TE and
hydrolytic offloading to the linear seco acid 11, which can rapidly interconvert to the hemiketal containing product 12.
Finally, the hemiketal is spontaneously dehydrated to produce the observed pyran 13. Stereochemistry displayed is
that of the native product 11 and subsequent hemiketalization (12). *represent likely points of inverted stereochemistry
seen in the isolated product.
extent of this limitation and overcome it, we generated a series of
type I PKS modules fused with TE domains from three related
biosynthetic pathways and assessed each for catalytic activity
Acknowledgements
with select polyketide substrates to emulate the final catalytic
steps in engineered pathways. Although the TE interactions are
not the only factor involved in polyketide assembly and offloading
We gratefully acknowledge NIH grant R35 GM118101, and the
Hans W. Vahlteich Professorship (D.H.S.). This research was
partially supported by Rackham Merit Predoctoral Fellowships
(
e.g. Juv Mod6 hybrids fail to process 2, Table 2), we achieved
robust catalysis of non-native substrates through PKS modules
when the TE domain was matched with the substrate from its
native PKS. In addition to explaining the attenuated yields of
polyketides obtained from engineered PKSs described in
previous studies,[7] the current work generates further
experimental support for the critical role of the TE domain in the
processing of unnatural intermediates. These results are in
accord with allied efforts performed in fungal iterative PKSs, which
(
D.A.H. and A.A.K.), T32-CA009676 (A.A.K. training fellowship),
and an American Foundation for Pharmaceutical Education
Predoctoral Fellowship (D.A.H.). We thank Dr. Zachary Litman
for critical reading of this manuscript.
Keywords: Polyketides • Biosynthesis • Biocatalysis • Natural
products • PKS Engineering
identified the TE domain as a key gatekeeper in the production of
fungal polyketides.[9]
[1]
K. J. Weissman, Nat. Prod. Rep. 2016, 33, 203-230.
a) R. McDaniel, A. Thamchaipenet, C. Gustafsson, H. Fu, M.
[
2]
Betlach, M. Betlach, G. Ashley, Proc. Natl. Acad. Sci. U. S. A.
Furthermore, our results indicate that identity of the TE
domain may also alter the sequence of catalytic events that
occurs in engineered PKS modules. This possibility is highlighted
by the product divergence when DEBS Mod6 TE hybrids process
the unnatural substrate 2, giving either 6 (3-keto) or 7 (3-hydroxy)
macrolactones predominantly (Figure 1). Recent work from
Kalkreuter et al. also noted the gatekeeper role of the KR by
observing that assembly of unnatural polyketide intermediates
resulted in a propensity to skip the PKS module reductive step,
setting keto functionality at the respective C-atom.[10] Future
structural and protein engineering studies will focus on delineating
the role of the TE domain in the processing of unnatural
substrates. By exploring how non-native TE domains can
contribute to diminished product formation observed with
combinatorial biosynthesis and directed pathway engineering
strategies, we will be able to more effectively design bacterial type
I PKS pathways for enhanced production of polyketide natural
product analogs, a long-standing goal in secondary metabolite
biosynthesis and synthetic biology.[1, 3, 11]
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V. Santi, Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 11740-11745; c)
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C. Khosla, D. E. Cane, J. Am. Chem. Soc. 2001, 123, 2495-2502;
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Kittendorf, J. D. Mortison, Y. J. Yoon, D. H. Sherman, J. Am.
Chem. Soc. 2013, 135, 11232-11238; b) D. A. Hansen, A. A. Koch,
D. H. Sherman, J. Am. Chem. Soc. 2015, 137, 3735-3738; c) A. N.
Lowell, M. D. DeMars, S. T. Slocum, F. Yu, K. Anand, J. A.
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Schultz, D. H. Sherman, J. Am. Chem. Soc. 2017, 139, 7913-
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Angewandte Chemie
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
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Improved catalysis with
engineered polyketide synthases:
Pairing wild-type polyketide
synthases with non-native substrates
largely failed to produce the
Aaron A. Koch, Jennifer J. Schmidt,
Andrew N. Lowell, Douglas A. Hansen,
Katherine M. Coburn, Joseph A.
Chemler, and David H. Sherman
Page No. – Page No.
anticipated products. A series of
hybrid modules bearing heterologous
thioesterase domains were
generated and employed to alleviate
the observed catalytic bottleneck,
resulting in the efficient processing of
non-native substrates and an
Probing Selectivity and Creating
Structural Diversity Through Hybrid
Polyketide Synthases
unexpected path to product diversity.
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