Thioesterase-Catalyzed Aminoacylation and Thiolation of Polyketides in Fungi

Article abstract of DOI:10.1021/jacs.9b01083

Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Product release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize aminoacylated polyketides in an ATPindependent fashion. The KU42 TE domain selects cysteine and homocysteine and catalyzes transthioesterification using the side-chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these HRPKS-TE enzymes showcases the functional diversity of HRPKS enzymes and provides potential TE domains as biocatalytic tools to diversify HRPKS structures.

Full text of DOI:10.1021/jacs.9b01083

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Article
Thioesterase-catalyzed aminoacylation and thiolation of polyketides in fungi
Man-Cheng Tang, Curt R. Fischer, Jason V Chari, Dan Tan, Sundari Suresh, Angela Chu,
Molly Miranda, Justin Smith, Zhuan Zhang, Neil Garg, Robert P St. Onge, and Yi Tang
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 03 May 2019
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Thioesterase-catalyzed aminoacylation and thiolation of polyketides
in fungi
Man-Cheng Tang,1† Curt R. Fischer,3† Jason V. Chari,2 Dan Tan,1 Sundari Suresh,3,4 Angela Chu,3,4 Molly Mi-
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randa,3 Justin Smith,3 Zhuan Zhang,1 Neil K. Garg,2 Robert P. St. Onge,3,4* Yi Tang1,2*
1 Departments of Chemical and Biomolecular Engineering, 2 Chemistry and Biochemistry, University of California, Los Angeles, Cali-
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fornia 90095, United States; Stanford Genome Technology Center, Stanford University School of Medicine, and Stanford ChEM-H
Institute, Palo Alto, CA 94304, United States. 4 Department of Biochemistry, Stanford University School of Medicine, Palo Alto, CA
94304, United States.
KEYWORDS. Natural product, cysteine, aminoacylation, templated biosynthesis
ABSTRACT: Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Prod-
uct release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to
date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs
that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize amino-
acylated polyketides in an ATP-independent fashion. The KU42 TE domain selects cysteine and homocysteine, and catalyzes transthioesteri-
fication using the side chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct
amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these
HRPKS-TE enzymes showcase the functional diversity of HRPKS enzymes, and provides potential TE domains as biocatalytic tools to diversify
HRPKS structures.
cific amino acid followed by acyl transfer to the thiolation (T) do-
INTRODUCTION
main. The condensation domain (C) then serves as a HRPKS re-
leasing domain in principle, by transferring the completed polyke-
tide acyl chain from the ACP domain of HRPKS module to the T
domain in the NRPS module as an aminoacylated polyketide, and
forming an amide bond (Figure 1B).10
In thiol-templated biosynthesis catalyzed by polyketide synthases
(PKSs) and nonribosomal peptide synthetases (NRPSs), product
release is an essential step to ensure enzyme turnover. In bacterial
PKSs where multidomain modules are arranged in an assembly line
like fashion, C-terminal thioesterase (TE) domains are used to re-
lease the polyketide product via hydrolysis or macrocyclization.1
Fungal highly reducing PKSs (HRPKSs)2, on the other hand, consist
of a single set of catalytic domains that is used iteratively. The do-
main architecture of fungal HRPKSs resembles a single module of
bacterial type I modular PKSs. In contrast to bacterial product re-
lease strategies, many fungal HRPKSs studied to date use a separate,
dissociated TE enzyme or acyltransferase to release the polyketide
chain through hydrolysis or transesterification (Figure 1A).3 Both
examples are represented in the lovastatin biosynthetic pathway:4
the TE LovG hydrolyzes dihydromonacolin L acid from LovB; while
the acyltransferase LovD transfers -methylbutyrate from LovF to
monacolin L acid and afford lovastatin acid(Figure 1A). In tandem
PKS systems, product of the HRPKS can be offloaded by the N-ter-
minal starter unit–ACP transacylase (SAT) domain of a nonreduc-
ing PKS (NRPKS).5 Other modes of product release from HRPKS
not catalyzed by TE have been reported in several recent studies.6
A
LovB
LovG
thioesterase
HO
OH
KS MAT DH MT ER0 KR ACP
C
OH
O
hydrolysis
LovC
O
S
PKS
elogation
cycles
H
H
ER
H2O
HO
R
dihydromonacolin L acid
HO
O
OH
LovD
acyltransferase
LovF
KS MAT DH MT ER KR ACP
OH
O
O
transesterification
H
O
S
PKS
elogation
cycles
Monacolin J
Lovastatin acid
B
HRPKS module
NRPS module
KS MAT DH ER KR ACP
C
A
T
S
R/DKC
O
AMP
O
O
S
O
HO
O
A
A
PKS
elogation
cycles
O
H2N
R2
H2
N
R2
H2N
R2
AMP
ATP
OH
PPi
R1
Many fungal polyketides are aminoacylated and further modified
to have significant biological activities, exemplified by cytochalasin
E (antiangiogensis),7 pseurotin A (immunosuppressant)8 and
UCS1025A (anticancer).9 These natural products are synthesized
by HRPKS-NRPS hybrid enzymes.10 The adenylation domain in the
C-terminal NRPS module uses one ATP molecule to activate a spe-
aminoacylation
C
T
S
HO
releasing
and
tailoring
O
O
O
O
O
R1
N
H
N
R2
H
aspyridone A
aminoacylated polyketide
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Figure 1. Known product releasing mechanisms of fungal HRPKSs. (A)
Punctularia strigosozonata and Hydnomerulius pinastri, respec-
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5
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Product release is catalyzed by a separate TE or acyltransferase through
hydrolysis or transesterification; (B) C domain serves as the releasing
domain in the biosynthesis of fungal aminoacylated polyketide prod-
ucts. Domain abbreviations: KS, ketosynthase; MAT, malonyl-
CoA:ACP transacylase; DH, dehydratase; MT, methyltransferase; ER,
enoyl reductase; KR, ketoreductase; ACP, acyl carrier protein; C, con-
densation; A, adenylation; T, thiolation; R, reductase.
tively, contain genes encoding HRPKS-TE belonging to this family
(Figure 2).14 Bioinformatic analysis indicated that within the KU42
cluster, genes encoding an HRPKS-TE, three P450s, an oxidoreduc-
tase, a PAP2-like protein, a hypothetical protein, and a major facili-
tator superfamily transporter (Figure 2A) are found. When KU42
HRPKS-TE and the three P450s were introduced together into en-
gineered yeast strain RC01, two aminoacylated products (1 and 2, as
shown in Figure 2A) were identified.14 In parallel, genes from KU43
clusters encode an HRPKS-TE, four P450s, two carboxyl methyl-
transferases (C-MTs), and others (Figure 2B). The expression of
the KU43 HRPKS-TE together with the four P450s and two C-MTs
in engineered yeast strains resulted in the production of an aminoac-
ylated polyketide 3 (Figure 2B).14 The structures of aminoacylated
polyketides were surprising based on known fungal HRPKS pro-
gramming rules, since enzymes responsible for amino acid activation
and incorporation, such as NRPS or CoA-ligase, are absent in the
two gene clusters.
The iterative nature of fungal HRPKSs dictates that a single set of
domains is used repeatedly during elongation and tailoring of the
polyketide chain. Hence, the releasing domain/enzyme is necessary
to ensure that the correct polyketide product, once formed, is re-
leased promptly. Previous studies showed that in the absence of re-
leasing domains, the HRPKS comes to a halt due to occupancy of
the ACP domain,11 or functions aberrantly to yield products of in-
correct sizes.12 Therefore, understanding the different mechanisms
of product release is a key aspect of decoding HRPKS programming
rules. Genome mining of fungal HRPKSs with an emphasis on find-
ing unusual product release domains can lead to discovery of new
products. For example, we previously identified a clade of HRPKSs
that are terminated with a C-terminal domain with sequence homol-
ogy to carnitine O-acyltransferase (cAT).13 The reversible nature of
the cAT was a key feature that enabled complete execution of Tv6-
931 programming steps.
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Here we studied the biosynthetic mechanisms of these HRPKS-
TE enzymes and showed the amino acids are directly incorporated
by the fused TE domains. In the case of KU42, we demonstrate the
TE domain can catalyze a transthioesterification with thiol contain-
ing amino acids followed by a S-N shift to form the amide bond.
RESULTS AND DISCUSSIONS
Sequence analysis of the KU42 TE domain. 1 and 2 are closely
related compounds containing a S, N-diacylated homocysteine. The
acyl groups appear to derive from the same dienoyl triketide precur-
sor. In the absence of any apparent amino acid activation domain,
we hypothesized that 1 and 2 were formed by the KU42 HRPKS-TE
domain, and homocysteine is used to release the polyketide chains.
The amino and the thiol groups of homocysteine serve as nucleo-
philes to perform aminoacylation and thiolation, respectively, to
form the diacylated adduct. We propose the amino acid specificity is
likely controlled by the TE domain. Sequence analysis revealed that
the TE domain, like many other PKS TE domains, is a member of
the serine / hydrolase domain superfamily.15 The TE domain
contains a cysteine residue (C2040) instead of serine as the con-
served active site nucleophile, while the other two residues of the
conserved catalytic triad, D2056 and H2225, have the usual identity
(Figure S1).1b, 16
Homology searches against predicted fungal protein sequences
revealed that similar TE domains are encoded on a diverse set of fun-
gal genomes. Of 500 BLAST hits retrieved (with E values between
2e-41 and 0.127), thirty-seven coded for cysteine as the active site
nucleophile, while 452 had serine (seven coded for alanine at this
position; three other sequences had other residues or alignment gaps
in the conserved position). The occurrence of cysteine or serine in
this key active site position was independent of phylogeny (Figure
S2), suggesting that mutation to cysteine has occurred multiple
times in the evolution of this domain. Cysteine active site nucleo-
philes have been observed in some starter-unit:ACP transacylase do-
mains in fungal nonreducing PKSs, although the biochemical reason
for this substitution over serine is unclear.17 A recent report from the
Wencewicz group reported a cysteine nucleophile in a TE domain is
required for cyclization of the -lactone product obafluorin.18 It was
proposed that the thioester intermediate is at a higher ground state
energy compared to the oxyester, and thus is favorable in driving -
lactone formation.18
Figure 2. Genome mining of fungal secondary metabolite biosynthetic
gene clusters containing a gene encoding HRPKS-TE megasynthase. (A)
KU42 cluster mined from P. strigosozonata and the production of 1 and
2 in yeast. (B) KU43 cluster mined from H. pinastri and the production
of 3 in yeast. HP, hypothetical protein; PAP2, type 2 phosphatidic acid
phosphatase; MFS, major facilitator superfamily; C-MT, carboxyl me-
thyltransferase. Shown are the traces of extracted-ion chromatogram. In
control traces from strains transformed with vector only, these com-
pounds were not found.
Recently, our genome mining efforts identified a group of fungal
HRPKSs with fused terminal TE domains that differ from all charac-
terized HRPKSs to date. These architecturally distinct HRPKSs had
no known associated products.14 Two such gene clusters, KU42 and
KU43, mined from the genomes of basidiomycete fungal species,
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Functional characterization of KU42-HRPKS-TE. To identify
with 4 as the predominant product (Figure S4). ATP was not neces-
sary in these assays to activate the amino acids, as expected since
there is no adenylation domain in the enzyme. The collective in vivo
and in vitro assays confirm the HRPKS-TE alone is able to add two
molecules of the polyketide product onto either cysteine (preferred
substrate) or homocysteine, forming the S, N-diacylated products
without the use of a condensation or adenylation domain.
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the product synthesized by KU42-HRPKS-TE alone, the gene was
cloned under ADH2 promoter on a 2μ plasmid and transformed into
the engineered S. cerevisiae strain BJ5464-NpgA.11 After four days of
culturing followed by extraction with ethyl acetate (containing 1%
acetic acid), we observed accumulation of several new metabolites
(Figure 3A). The major product is 4 with molecular weight (MW)
of 309, and the minor product is 5 with MW of 323. These com-
pounds were isolated and characterized by NMR (Table S3 and S4,
Figure S12-S21). Compound 4 was elucidated to be disorbyl cyste-
ine, in which both of the thiol group and the amine group of cysteine
were acylated with sorbic acid (Figure 3A). Based on the value of
proton coupling constants obtained from 1H NMR spectrum
(J=11.2 Hz for H2-H3 and H2’-H3’, 14.0 Hz for H4-H5 and H4’-
H5’) and published literature values for sorbate with differently ole-
fin stereochemistry,19 the double bond configurations in 4 were as-
signed to be 2-cis-4-trans-sorbyl. Similar to 4, the structure of 5 was
KU42 HRPKS-TE substrate scope and proposed reaction mech-
anism. We then investigated the function of TE domain in KU42
HRPKS-TE. We first constructed the TE active site mutant C2040A,
in which the putative cysteine nucleophile was mutated into alanine.
When transformed into yeast, the mutant lost the ability to biosyn-
thesize 4 and 5 (Figure 3A), indicating that both polyketide chain
release and amino acid incorporation require the cysteine nucleo-
phile. Mutation of the putative general base H2225 to either Ala or
Asn similarly abolished the function of the enzyme. When the active
site nucleophile was changed from cysteine to serine (C2040S) and
expressed in yeast, we also did not observe formation of either 4 or
5. We further subjected these culture supernatants to untargeted
metabolite analysis using XCMS (Figure S5).20 This analysis re-
vealed that the C2040S mutant, but not C2040A or H2225A, pro-
duced an unique compound not produced by wild type HRPKS-TE,
and has (ion) mass of 113.0597 Da, corresponding to the molecular
formula of sorbic acid (C6H8O2) plus a proton. This peak indicates
the C2040S mutant retained the ability to synthesize the polyketide
product, but is unable to perform aminoacylation or thiolation. In-
stead, the product appears to be prone to hydrolysis, analogous to
the cysteine to serine mutation observed in obafluorin TE domain.18
Although it is not clear whether such hydrolysis occurs as a S2040-
sorbyl oxyster, or direct hydrolysis from the ACP, this result con-
firms the necessity of the cysteine as an active site nucleophile.
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also elucidated in which the
L-cysteine unit in compound 4 was re-
placed by -homocysteine (Figure 3A). 5 is therefore the likely oxi-
L
dized to 1 and 2 when the P450 enzymes were coexpressed in yeast
(Figure 2A).
We attempted to express and purify the standalone TE domain
from E. coli BL21(DE3), but failed to obtain soluble protein. We
were successful in expression of the TE domain together with up-
stream ACP domain, ACP-TE, as N-terminal His6 tagged fusion pro-
tein (Figure S3). To test the function of ACP-TE, acyl-S-N-acetyl
cysteamine (SNAC) compounds were added to the enzyme and an-
alyzed by LC-MS to determine whether one or two acyl groups are
transferred to the amino acid nucleophile. Due to instability of sor-
byl-SNAC, we used an available acyl-SNAC, (S)-3-hydroxyoc-
tanoyl-SNAC (12)21, as a substrate analog (Figure 4A). Compared
to the negative control, we were able to detect formation of three dif-
ferent products (Figure S6): one has the MW of 419, which is ex-
pected for the diacylated product 13; another product has the MW
of 277, which corresponds to the MW of the monoacylated product
14; the third compound has the MW of 552, which corresponds to
that of 15, a dimer of 14 formed though oxidative coupling of free
thiol groups to form a disulfide (Figure 4A). When tris-(2-carboxy-
ethyl)phosphine (TCEP) was included in the assay, formation of 15
was no longer detected and only 13 and 14 were formed. This sup-
ports the proposed relationship of 14 and 15. Together with the mu-
tagenesis results, these results confirmed that the TE domain is in-
deed responsible for the acylation reactions.
Figure 3. Characterization of KU42 HRPKS-TE in vivo and in vitro.
Shown are the extracted-ion chromatogram. (A) Expression of HRPKS-
TE and C2040A mutant in yeast and the structures of the compounds
produced; (B) LC-MS analysis of the in vitro assays of purified HRPKS-
TE in the presence of malonyl-CoA, NADPH and
cysteine.
L-cysteine or L-homo-
In order to confirm that KU42 HRPKS-TE alone is sufficient for
the biosynthesis of 4 and 5, recombinant enzyme (246.2 kDa) was
purified from yeast to homogeneity by anti-FLAG resin column
chromatography (Figure S3). Assay of the enzyme in the presence
The substrate scope of the TE domain was further assayed using
different acyl-SNAC and amino acid substrates (Figure 4B). When
of malonyl-CoA, NADPH, and
(Figure 3B). When -cysteine was replaced by
production of 5 was confirmed (Figure 3B). When the enzyme was
assayed in the presence of equimolar amount of -cysteine and -ho-
mocysteine (1 mM), the production of both 4 and 5 was detected
L
-cysteine yielded 4 as expected
L
-cysteine was used as the nucleophile, several acyl-SNAC substrates
L
L
-homocysteine, the
with different acyl chain length (from C4 to C8) could be used as the
acyl donors by TE domain. Among the C4 substrates, only butyryl-
SNAC was accepted by the TE domain (Figure S6). Using butyryl-
L
L
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SNAC as the acyl donor, different amino acid nucleophiles were and trans-double bonds. Several HRPKSs have been proposed to bi-
1
2
3
4
5
6
7
8
tested for formation of mono- and dibutyryl adducts. One key trend
that emerged, which is consistent with amino acid incorporated into
4 and 5, is that amino acids without a free thiol group cannot be used
osynthesize polyketides with both cis and trans olefins, such as in the
sordarin pathway.23 Cox and coworkers recently reported StPKS1
from Strobilurus tenacellus can produce a polyketide containing
both cis- and trans-olefins.24 However, the programming rules that
govern the formation of mixed olefins from a single set of KR and
DH domains are unknown. A working hypothesis is that the KR do-
main reduces the -keto functionality with opposite stereochemistry
after the two chain elongation steps. The two oppositely configured
alcohol substrates binds in the DH active site differently for syn elim-
ination, leading to either cis or trans products. We previously
as nucleophiles, such as
cysteine can also be accepted by the TE domain as nucleophile to
form the dibutyryl- -cysteine product. Importantly, when N-acetyl-
-cysteine was used as the nucleophile, we observed formation of a
L-serine and L-methionine. Interestingly, D-
D
L
monobutyryl adduct by LC-MS. This product, unlike 14, does not
undergo air oxidation to form a dimer, suggesting that the free thiol
group is acylated by the butyryl unit.
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showed in the hypothemycin HRPKS, the stereochemistry (L or D)
of KR-catalyzed -ketoreduction is chain-length dependent.25 It is
also well documented that stereochemistry of the reduced alcohol
can affect the stereoselectivity of the DH-catalyzed dehydration re-
action.26
To test this hypothesis, we first verified the HRPKS product is in-
deed the 2-cis-4-trans sorbyl chain, and the TE domain is not in-
volved in trans-cis isomerization of the second olefin. We con-
structed a truncated version of KU42 HRPKS-TE lacking the TE do-
main (HRPKS ΔTE). Assay of the truncated enzyme in the presence
of only malonyl-CoA yielded triacetic acid lactone (Figure S7), con-
firming the elongation domains are active. Addition of NADPH led
to no detectable polyketide products, since there is no releasing do-
main (Figure S7). To enable release, we added a heterologous TE
domain from an unrelated PKS13, which was previously demon-
strated to have promiscuous substrate specificity in ACP-thioester
hydrolysis,27 into the reaction mixture. Under this condition, we ob-
served turnover of a new product that had the similar UV spectrum
with 2-trans-4-trans-sorbic acid, but different retention time (Figure
S7). We synthesized an authentic standard of 2-cis-4-trans-sorbic
acid (Figure S28), which matched that released from the enzymatic
assay. This confirmed the polyketide produced by KU42 HRPKS is
indeed the 2-cis-4-trans-sorbate, and no post-PKS isomerization of
a trans-trans product is required.
Figure 4. Assaying KU42 TE domain with different acyl and amino acid
substrates reveal biochemical mechanism. (A) Product analysis of the in
vitro assays of KU42 ACP-TE in the presence of L-homocysteine and 12.
In the negative control, boiled enzyme was used. (B) Summary of prod-
ucts formed by using different amino acids and acyl-SNAC substrates.
We next examined the stereoselectivity of the KR domain towards
-keto substrates of different sizes. We assayed the HRPKS-TE with
three different β-ketoacyl-SNAC compounds in the presence of
NADPH, and analyzed the -keto reduced products using chiral
HPLC and synthetic standards (Figure 5). The diketide acetoace-
tyl-SNAC (6)25 and triketide 3-oxohexanoyl-SNAC (7)25 were both
reduced to the corresponding β-hydroxyacyl-SNAC, and the reten-
From these in vitro assay results of TE domain with different sub-
strates, a possible catalytic mechanism for the formation of diacyl-
ated products 4 and 5 can be proposed. We propose the TE domain
is a bona fide acyltransferase that can catalyze the transthioesterifi-
cation between the side chain thiol nucleophile and the sorbyl-S-
tion time matched to that of the 6D and 7D (the D configuration of
ACP thioester to release the product. When L-homocysteine is used
the hydroxyl products),25 respectively (Figure 5). Hence the KR
specificity does not vary with chain length. We then assayed the
product stereochemistry using 3-oxo-4-trans-hexenoyl-SNAC (8)28,
of which the acyl chain is the natural triketide of the KR domain.
as the releasing nucleophile, TE domain would catalyze the trans-
thioesterification reaction to yield S-sorbyl homocysteine. This is
followed by a spontaneous intramolecular S-to-N acyl shift to form
the N-sorbyl homocysteine to regenerate a free thiol group. This
type of uncatalyzed rearrangement reaction is widely used in native
chemical ligation to synthesize peptides and small proteins.22 With
the thiol group now free to react, the TE domain catalyzes the sec-
ond transthioesterification reaction of N-sorbyl homocysteine to
form the disorbyl homocysteine 5 (Figure 6). Therefore, the TE do-
main does not catalyze direct aminoacylation, but instead catalyzes
the unprecedented release of polyketide products using the side
chain thiol of cysteine or homocysteine.
Surprisingly, the product elution time matched that of 8
verify the stereochemistry of this product, we scaled up the reaction
and purified sufficient amounts of 8 by HPLC. The structure was
L. To further
L
verified by NMR analysis (Table S5, Figure S22 and S23) and the
27
optical rotation ([α]D +13, c =0.2, CHCl3) matched to the re-
25
ported value of 8
L
([α]D +17, c =0.42, CH2Cl2).28 Our studies
therefore revealed an unexpected layer of fungal HRPKS program-
ming complexity, in that the stereoselectivity of the KR domain de-
pends on the degree of unsaturation of the substrate. In this case,
changing from a single bond in the substrate (in 7) to the double
bond (in 8) completely reversed the stereochemical outcome. The
Delineating the stereoselectivity of KR and DH domains of KU42
HRPKS-TE in the synthesis of 2-cis-4-trans-sorbyl polyketide chain.
A surprise in the structures of 4 and 5 is that they contain both cis-
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stereochemical switch from diketide 6 (in
D
configuration) to trike-
acetylcysteamine through a Michael addition and a retro-Michael
addition (see Figure S9).31 N-acetylcysteamine itself could form
through the decomposition of acyl-SNAC substrates. Although we
were not able to prove directly the dehydration specificity of the DH
domain in these experiments, our demonstration of unexpected KR
programming rules and comparison to knowledge accumulated for
the bacterial DH domains, can rationalize the formation of cis and
trans olefin in the same polyketide product by a single fungal
HRPKS (Figure 6).
1
2
3
4
5
6
7
8
tide 8 (in configuration) is most likely the cause for the subsequent
L
different dehydration stereochemical outcomes.
Characterization of the roles of the three P450s from KU42 clus-
ter. Our results showed that the major product of KU42 HRPKS-TE
when expressed in yeast or assayed in vitro is 4, the disorbyl-L-cyste-
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ine. However, we also showed that when KU42 HRPKS-TE was co-
expressed with three P450s enzymes from KU42 cluster in yeast, the
products were 1 and 2, which are derived from the homocysteine
containing 5 (Figure 2A).13 The two hydroxyl groups in 1 and 2 are
presumably formed from water-mediated opening of an epoxide pre-
cursor. To explore the roles of these P450s in the biosynthesis of 1
and 2, co-expression experiments of the HRPKS-TE and individual
P450 in S. cerevisiae RC0132 were performed (Figure S10). Coex-
pression with either P450-2 or P450-3 did not lead to the formation
of 1 and 2. When KU42 HRPKS was co-expressed with P450-1,
both 1 and 2 could be detected (Figure S10). Therefore, we assign
P450-1 to be an epoxidase that catalyzes the selective epoxidation of
one of the cis olefin in 5 to form an unstable product 16, which can
be readily hydrolyzed to form 1 and 2 under culturing condition
(Figure 6). P450-1 appears to be selective for the homocysteine ad-
duct 5, as no cysteine derived analogs of 1 and 2 can be detected in
the culture.
The overall biosynthetic pathway to 1 and 2 from KU42 HRPKS-
TE is shown in Figure 6. Formation of the 2-cis-4-trans-sorbyl
polyketide chain is proposed to involve the programmed stereose-
lectivities of the KR domain, which in turn influences the dehydra-
tion stereochemistry. The TE domain then catalyzes two trans-
thioesterification steps using either cysteine or homocysteine. The
second transthioesterification follows an S-N rearrangement, to af-
ford 4 or 5, respectively. While 4 does not appear to be further pro-
cessed, 5 is selectively epoxidized by P450-1 to 16, which can be at-
tacked by water to afford both 1 and 2. The exact natural product of
the KU42 pathway is unknown and is subject to future studies.
Figure 5. Biochemical characterization of the function of KU42-
HRPKS-TE KR domain in vitro. Shown are the HPLC traces at 240 nm.
PKS DH domains generally catalyze the syn dehydration of their
substrates.29 Cane and coworkers showed that in the biosynthesis of
fostriecin, the double bond configurations produced by DH do-
mains were determined by the stereo-configuration of the β-hydroxy
group that controlled by KR domains.26b We therefore propose that
because of the different stereochemistry of the -hydroxyl group in
6
D
and 8
DH domain, leading to trans and cis geometry of the dehydrated ole-
fin, respectively. When diketide substrate 6 was incubated with
L, the substrates will bind differently in the active site of the
KR
ACP
S
DH
KR
KS
NADPH
S
NADPH
S
S
S
1 acetyl-CoA
1 malonyl-CoA
O
O
O
O
O
O
D
O
trans
HO
(D)
HO
KU42 HRPKS-TE, we detected the formation of a new product 9
(MW 187) (Figure S8). The compound was purified from scaled-up
in vitro assays and shown to be trans-crotonoyl-SNAC (Table S6,
Figure S24 and S25). No dehydration product was detected when
(L)
DH
HS
NH2
O
TE
SH
H
N
S
NH2
O
HO
S-to-N
acyl shift
TE
S
HS
6L
was used as the substrate (Figure S8), indicating strict substrate
L-homocysteine
ACP
S
O
O
HO
HO
O
spont.
O
specificity of the DH domain at the diketide stage. When 8 was
L
transthioesterification
O
tested as the substrate, we also detected the formation of a dehydra-
tion product 10 (MW 213) (Figure S8). However after purification
from scaled-up in vitro assays, the product was shown to be 2-trans-
4-trans-sorbyl-SNAC (Table S7, Figure S26 and S27), not the ex-
pected 2-cis-4-trans-sorbyl-SNAC. Assaying DH domains that pro-
duce cis-configured olefin has documented difficulties due to isom-
erization of the double bond that is  to the thioester.26d, 30 Simi-
larly, we proposed that the formation of 2-trans-4-trans-sorbyl-
SNAC is due to the rapid cis-to-trans isomerization of 2-cis-4-trans
sorbyl product, which could be catalyzed by nucleophiles such as N-
TE
S
cis
transthioesterification
O
epoxide
opening
HO
O
HO
O
P450-1
O2
O
O
O
O
1
2
and
S
N
H
S
N
H
O
5
16
Figure 6. The proposed biosynthetic pathway of 5.
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Factors that influence cysteine to homocysteine incorporation detectable production of 3 when the mutant HRPKS-TE was co-ex-
1
levels. Our in vitro experiments clearly indicated HRPKS-TE in fact
prefers cysteine over homocysteine, yet based on in vivo reconstitu-
tion results with P450-1, homocysteine is the preferred substrate to
yield the natural product of the pathway. Therefore, the in vivo ratio
of cysteine to homocysteine should impact product distributions,
and the roles of other enzymes in the cluster. To test this idea, we
utilized CRISPRi-mediated gene repression, coupled to a targeted
metabolomics readout. We integrated the KU42 HRPKS-TE gene
into a yeast haploid strain, and mated this strain to 41 CRISPRi
strains33 expressing different guide RNAs directed against one of 17
genes involved in homocysteine and cysteine biosynthetic pathways
(Figure S11). The resulting diploid strains were then cultured in 96-
well plates in the presence or absence of anhydrotetracycline (ATc;
which induces guide RNA expression). The relative titers of cyste-
ine-derived 4 and homocysteine-derived 5 in each well were meas-
ured via LC/MS. Among the genes tested, repression of CYS4,
which encodes for cystathionine -synthase that converts homocys-
teine to cystathionine (a cysteine precursor), was found to result in
the greatest shift in relative abundance of each product. We observed
a ~ 5x increase in the titer of 5 and a ~ 2x decrease in titer of 4 (Figure
S11), for an overall ~10 fold change in the relative amounts of 5 and
4.
pressed with C-MT-1 in yeast (Figure S29).
2
3
4
5
6
7
8
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KU43-HRPKS-TE is responsible for the biosynthesis of 3. Our
studies above showed how the KU42 HRPKS-TE can synthesize dis-
orbyl homocysteine products, using TE to catalyze transthioesterifi-
cation and release the HRPKS product. In contrast, the product of
KU43 HRPKS-TE, 3, is formed by a direct amidation between oc-
tanoate and leucine methyl ester (Figure 7). Analysis of KU43-
HRPKS-TE indicated that it contains the domain organization of
KS-MAT-DH-ER-KR-ACP-TE. We reasoned the TE domain could
be responsible for the amidation reaction since no enzyme responsi-
ble for amino acid activation and incorporation is present in the
KU43 gene cluster. Different from the KU42-HRPKS TE, sequence
alignment results indicated that the active site of KU43-HRPKS TE
is serine, the same as the canonical TEs (Figure S1).
Figure 7. Biosynthesis of 3. (A) Product profiles of S. cerevisiae BJ5464-
NpgA transformed with KU43 cluster genes. Shown are the extracted
MS spectra. (B) The proposed biosynthetic pathway of 3.
Summary. In this study, we characterized two fungal HRPKSs
with fused C-terminal TE domains, a new domain architecture
among fungal PKSs. Both KU42 and KU43 HRPKS-TEs produced
natural products that have not been reported previously, showing
the potential of studying novel HRPKS enzymes for natural product
mining. We demonstrated that both TE domains are involved in
product release to synthesize aminoacylated polyketides in an ATP-
independent fashion. The reactions catalyzed by the two TE do-
mains, however, are very different. The KU42 TE recognizes thiol
containing amino acids, and uses the side chain thiol as nucleophile
to perform chain release via transthioesterfication. The product can
then undergo S-N acyl shift, followed by a second transthioesterifi-
cation to produce the diacylated amino acid. Although the trans-
thioesterification reactions are known for TE enzymes, the use of
cysteine or homocysteine side chain thiol for polyketide product re-
lease is reported for the first time. In contrast, the KU43 TE recog-
nizes leucine methyl ester and uses its amino group as nucleophile to
release the polyketide chain produced by the HRPKS.
Expression of HRPKS-TE alone in BJ5464-NpgA did not yield
any new products (Figure 7A). When either C-MT-1 or C-MT-2
(Figure 2B), which are highly identical to each other (96% protein
sequence identity), from the cluster was coexpressed with KU43
HRPKS-TE, we detected the production of 3 (Figure 7A). Based on
these results, we proposed that C-MT-1 or C-MT-2 is responsible
for the biosynthesis of L-leucine methyl ester, which could be used
as the nucleophile by KU43 HRPKS-TE to release the octanoate
chain biosynthesized by KU43-HRPKS (Figure 7B). To support
this hypothesis, we repeated the heterologous expression experi-
ment of KU43 HRPKS-TE with the addition of L-leucine methyl es-
ter as the releasing substrate. As expected, the production of 3 was
detected (Figure 7A). Based on these results, we proposed the TE
domain of KU43 HRPKS-TE catalyzes polyketide chain release by
In conclusion, our results show that novel programming rules are
present in HRPKS-TEs to biosynthesize different natural products.
These TE domains, along with others that can be mined from un-
characterized biosynthetic pathways, can serve as potential biocata-
lytic tools for diversifying polyketide structures.
using L-leucine methyl ester to yield 3 (Figure 7B). Different from
the KU42 HRPKS TE, which uses cysteine as the active site nucleo-
phile to catalyze the transthioesterification reaction to yield thio-
lated polyketide, the KU43 HRPKS-TE uses serine as the active site
nucleophile to capture the polyketide product as an oxyester, fol-
lowed by amide bond formation to yield the aminoacylated polyke-
tide. This is in sharp contrast to the ATP-dependent aminoacylation
catalyzed by NRPS domains. Requirement of the serine nucleophile
in catalysis was verified by site-directed mutagenesis. Mutation of
the active site serine of KU43-HRPKS TE to alanine resulted in no
EXPERIMENTAL METHODS
Strains and General DNA Manipulation Techniques. E. coli TOP
10 and E. coli DH10b were used for cloning, following standard re-
combinant DNA techniques. E. coli BL21(DE3) was used for pro-
tein expression. Saccharomyces cerevisiae strain BJ5464-NpgA11
(MATα ura3−52 his3-Δ200 leu2- Δ1 trp1 pep4::HIS3prb1 Δ1.6R
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chromatography to near homogeneity. The purified protein was
can1 GAL) and RC0132 were used as the yeast expression hosts. Pol-
1
2
3
4
5
6
7
8
ymerase chain reactions were performed using Platinum Pfx DNA
polymerase (Invitrogen). DNA restriction enzymes were used as
recommended by the manufacturer (New England Biolabs). DNA
primers used in this study were synthesized by Integrated DNA
Technologies and are listed in Supporting Information, Table S1.
PCR products were confirmed by DNA sequencing by Laragen, Cal-
ifornia, USA.
buffer-exchanged and concentrated to final buffer (50 mM HEPES,
50 mM NaCl, 10% glycerol, pH 7.0). Aliquots of the purified protein
were stored at -80 °C. Protein concentration was determined by the
Bradford method34 using a BSA calibration curve. The yield of puri-
fied KU42 ACP-TE is ~37 mg/mL.
Activity Assay for KU42-HRPKS-TE and Its Mutants. For in vitro
synthesis of 4, 10 μM KU42-HRPKS-TE was incubated with 1 mM
L-cysteine, 2 mM malonyl-CoA and 4 mM NADPH in 100 mM
HEPES (pH 7.0) buffer. The reaction mixture was incubated at
30 °C overnight. The reaction was quenched with equal volume of
acetonitrile. After centrifugation, the supernatant was subjected for
LC-MS analysis. For in vitro synthesis of 5, the assay was performed
using the same condition except the L-cysteine was replaced by L-
homocysteine. All the experiments were done in triplicate. In the
manuscript, data collected from one set of experiments are shown.
For in vitro synthesis of 2-cis-4-trans-sorbic acid, 10 μM KU42
HRPKS ΔTE was incubated with 2 mM malonyl-CoA, 4 mM
NADPH, and 5 μM PKS13-TE in 100 mM HEPES (pH 7.0) buffer.
The reaction mixture was incubated at 30 °C overnight. The reaction
was quenched with equal volume of acetonitrile. After centrifugation,
the supernatant was subjected for LC-MS analysis.
Assay for KU42 ACP-TE Activity Using Different Amino Acid
Substrates and Acyl-SNAC Substrates. For in vitro activity test of
KU42 ACP-TE, the following condition was used: the reaction mix-
ture contains 50 μM KU42 ACP-TE, 1 mM amino acid substrate,
and 2 mM acyl-SNAC substrate, and incubated at 30 °C for 6 hours.
The reaction was quenched by adding equal volume of acetonitrile.
After centrifugation, the supernatant was subjected for LC-MS anal-
ysis. When tested with different amino acid substrates, butyryl-
SNAC was used as the acyl-SNAC substrate. When tested with dif-
ferent acyl-SNAC substrate, L-cysteine was used as the amino acid
substrate. All the experiments were done in triplicate. In the manu-
script, data collected from one set of experiments are shown.
Assay for the Functions of KU42 HRPKS KR Domain and DH Do-
main. To assay the activity of KU42 HRPKS KR domain, 10 μM
KU42-HRPKS-TE was incubated with 1 mM β-ketoacyl-SNAC and
2 mM NADPH at 30 °C for 4 hours. The reaction was quenched with
equal volume of acetonitrile. After centrifugation, the supernatant
was subjected for LC-MS analysis.
Compound Isolation and Purification. To isolate compound 4
and 5, a seed culture of the BJ5464-NpgA strain harboring the ex-
pression plasmid of KU42-HRPKS-TE was grown in 40 mL SDCt
(uracil dropout) media at 28 °C. After 2 days, this seed culture was
then used to inoculate 4 L of YPD medium and cultured for 3 days
at 28 °C. The cell pellets was removed by centrifugation and the su-
pernatant was collected and combined. The pH value of the super-
natant was adjusted to 3 by adding concentrated HCl. Then the su-
pernatant was extracted with EtOAc twice and the organic layer was
combined and concentrated. The crude residue was injected to
ISCO-CombiFlash® Rf 200 (Teledyne Isco, Inc) with silico 40g col-
umn chromatography for separation. Fractions containing the target
compounds were combined, and the target compounds were further
purified by semi-preparative HPLC using reverse-phase column
(Phenomenex Kinetics, C18, 5 μm, 100 Å, 10 × 250 mm). The titers
of compound 4 and 5 are ~1 mg/mL and ~0.3 mg/mL, respectively.
Expression and Purification of KU42-HRPKS-TE and Its Mu-
tants. For protein expression of KU42-HRPKS-TE and its mutants
in yeast strain BJ5464-NpgA, the corresponding genes were cloned
into pXW55 vector by yeast in vivo homologous recombination us-
ing Frozen-EZ Yeast Transformation II Kit™ (Zymo Research). Sin-
gle colonies of S. cerevisiae BJ5464-NpgA cells that had been trans-
formed with the expression plasmids were inoculated into 20 mL
SDCt (uracil dropout) media and incubated at 28 °C for 2 days.
Then, the starter culture was inoculated into 2 L YPD media and in-
cubated at 28 °C. After 2 days, the cell pellets were harvested and
suspended in lysis buffer (50 mM NaH2PO4, 150 mM NaCl, 10 mM
imidazole, pH 8.0). The cells were lysed by sonication and the cellu-
lar debris was removed by high-speed centrifugation (16000 rpm, 1
hour). The enzyme was purified from the supernatant by using anti-
FLAG resin column. The fractions containing the target protein
were pooled, buffer-exchanged, and concentrated to final buffer (50
mM HEPES, 50 mM NaCl, 10% glycerol, pH 7.0) using an Amicon
Ultra-15 (100 kDa) centrifugal filter. Aliquots of the purified pro-
teins were stored at -80 °C. Protein concentration was determined
by the Bradford method34 using a BSA calibration curve. The yield
of purified KU42-HRPKS-TE is ~2 mg/mL.
Expression and purification of KU42 ACP-TE. The gene frag-
ment encoding for KU42 ACP-TE was cloned into pET28a between
the NdeI and XhoI restriction sites. Single colonies of E. coli
BL21(DE3) cells that had been transformed with the expression
plasmid was inoculated into 3 mL LB media containing 50 mg/L
kanamycin and incubated overnight at 37 °C. Then, the starter cul-
ture was inoculated into 500 mL LB media containing 50 mg/L kan-
amycin, and grew at 37 °C. When the OD600 reached 0.6, the culture
was cooled to 16 °C and induced with 0.1 mM IPTG. Cells were har-
vested after 24 hours by centrifugation. The purification was then
carried out at 4 °C. After re-suspended in lysis buffer (50 mM
NaH2PO4, 150 mM NaCl, 10 mM imidazole, pH 8.0), the cells were
lysed by sonication. And the cellular debris was removed by high-
speed centrifugation (16000 rpm, 30 min). Then, the enzyme was
purified from the supernatant by using Ni-NTA agarose affinity
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To assay the activity of KU42 HRPKS DH domain, 10 μM KU42-
HRPKS-TE was incubated with 1 mM β-hydroxyacyl-SNAC at
30 °C for overnight. The reaction was quenched with equal volume
of acetonitrile. After centrifugation, the supernatant was subjected
for LC-MS analysis.
ASSOCIATED CONTENT
Experimental details, spectroscopic and computational data. This mate-
rial is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
yitang@ucla.edu
bstonge@stanford.edu
Author Contributions
†These authors contributed equally.
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
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2005, 186-188. (c) Eley, K. L.; Halo, L. M.; Song, Z.; Powles, H.; Cox, R. J.;
ACKNOWLEDGMENT
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Tenellin in the Insect Pathogenic Fungus Beauveria bassiana. Chembio-
chem. 2007, 8, 289-297. (d) Boettger, D.; Hertweck, C. Molecular Diversity
Sculpted by Fungal PKS-NRPS Hybrids. Chembiochem. 2013, 14, 28-42.
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Xie, X.; Kealey, J. T.; Da Silva, N. A.; Vederas, J. C.; Tang, Y. Complete Re-
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Polyketide Synthase Product Chain-Length Control by Partnering Thiohy-
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Liu, N.; Hillenmeyer, M. H.; Tang, Y. Reversible Product Release and Re-
capture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase
Domain. Angew. Chem. Int. Ed. 2017, 56, 9556-9560.
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H.-C.; Li, J.; Naughton, B.; Cherry, J.; Miranda, M.; Li, Y. F.; Chu, A. M.;
Hennessy, J. R.; Vandova, G. A.; Inglis, D.; Aiyar, R.; Steinmetz, L. M.; Davis,
R. W.; Medema, M. H.; Sattely, E.; Khosla, C.; St.Onge, R. P.; Tang, Y.;
Hillenmeyer, M. H. HEx: A heterologous expression platform for the discov-
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15. Cantu, D. C.; Chen, Y.; Reilly, P. J. Thioesterases: A new perspective
based on their primary and tertiary structures. Protein Sci. 2010, 19, 1281-
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Functions and Mechanisms. Curr. Protein Pept. Sci. 2000, 1, 209–235. (b)
Rauwerdink, A.; Kazlauskas, R. J. How the Same Core Catalytic Machinery
Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic
Triad of α/β-Hydrolase Fold Enzymes. ACS Catal. 2015, 5, 6153–6176.
17. Winter, J. M.; Cascio, D.; Dietrich, D.; Sato, M.; Watanabe, K.; Sawaya,
M. R.; Vederas, J. C.; Tang Y. Biochemical and Structural Basis for Control-
ling Chemical Modularity in Fungal Polyketide Biosynthesis. J. Am. Chem.
Soc. 2015, 137, 9885-9893.
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This work was supported by the NIH 1 U01 GM110706-01 Genome to
Natural Products Initiative to Stanford Genome Technology Center, of
which YT is a subward recipient. The work in the lab of YT is also sup-
ported by 1R35GM118056. The work at Stanford University was also
supported by R01GM121932-01A1 to RPS. Chemical characteriza-
tion studies were supported by shared instrumentation grants from the
NSF (CHE-1048804) and the NIH NCRR (S10RR025631). The au-
thors acknowledge Maureen Hillenmeyer, Colin Harvey, Marnix
Medema, Mohammad Alanjary, and Elizabeth Sattely for valuable tech-
nical discussions.
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