Engineered Biosynthesis of Fungal 4-Quinolone Natural Products

Article abstract of DOI:10.1021/acs.orglett.0c02426

Quinolone-containing natural products are widely found in bacteria, fungi, and plants. The fungal quinolactacins, which are N-methyl-4-quinolones, display a wide spectrum of biological activities. Here we uncovered a concise nonribosomal peptide synthetase pathway involved in quinolactacin A biosynthesis from Penicillium by using heterologous reconstitution and in vitro enzymatic synthesis. The N-desmethyl analog of quinolactacin A was accessed through the construction of a hybrid bacterial and fungi pathway in the heterologous host.

Full text of DOI:10.1021/acs.orglett.0c02426

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Engineered Biosynthesis of Fungal 4‑Quinolone Natural Products
Mengting Liu, Masao Ohashi,* and Yi Tang*
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ABSTRACT: Quinolone-containing natural products are widely
found in bacteria, fungi, and plants. The fungal quinolactacins,
which are N-methyl-4-quinolones, display a wide spectrum of
biological activities. Here we uncovered a concise nonribosomal
peptide synthetase pathway involved in quinolactacin A biosyn-
thesis from Penicillium by using heterologous reconstitution and in
vitro enzymatic synthesis. The N-desmethyl analog of quinolactacin
A was accessed through the construction of a hybrid bacterial and
fungi pathway in the heterologous host.
itrogen-containing heterocycles and fused ring systems
are among the most important structures in drug
acetylcholinesterase, α-glucosidase, and tumor necrosis factor,
and displays anticancer, antimicrobial, and insecticidal
activities.7 The N-methylated 4-quinolone is fused with a
five-membered lactam ring, which is derived from a tetramate
precursor. Quinolactacins can readily undergo C3-epimeriza-
tion under aqueous conditions and exist as a mixture of
diastereomers or enantiomers.7,8 The different alkyl groups in
the quinolactacins shown in Figure 1 correspond to the side
chains found in aliphatic amino acids, thereby suggesting that
the formation of the tetramate is likely catalyzed by an amino-
acid-activating enzyme, such as a nonribosomal peptide
synthetase (NRPS). The 4-quinolone portion of 1 has been
proposed by Nakagawa et al. to derive from L-tryptophan, as
labeled anthranilate was not incorporated.9 To investigate the
origin of the 4-quinolone moiety, we first performed labeled L-
Trp-(indole-d5) feeding to the producing host of Penicillium
citrinum DSM1997.9 A corresponding mass increase of +4 Da
was observed in 1 upon feeding (Figure S1), which confirms
that L-Trp is a precursor. The deuterium atom that was not
incorporated is most likely that connected to the C2 position
in L-Trp, which can be excised during the catabolism of L-Trp
to kynurenine 2 by indoleamine-2,3-dioxygenase (IDO) and
formamidase (Figure 2).10 We repeated the feeding with d5-N-
formyl-L-kynurenine, an intermediate in the breakdown of L-
Trp to 2, which led to the expected +4 Da increase in
molecular weight of 1 (Figure S2).
N
discovery.1 Natural products containing N-heterocycles sim-
ilarly display diverse biological activities and are attractive
targets for synthetic and biosynthetic investigations.2 Fungi is a
prolific producer of such natural products containing an
assortment of different N-heterocycles, such as pipecolate,
piperidine, pyrazine, pyridone, quinazoline, indole, and so on.3
A notable N-heterocycle structure is quinoline and the related
quinolone; both are found in important drugs such as a quinine
and fluoroquinolone. Our lab and others have investigated the
biosynthesis of fungal 2-quinolone compounds such as
penigequinolone and aspoquinolone.4 Here we report the
biosynthesis of quinolactacin A (1) from Penicillium, which
contains the 4-quinolone structure, as shown in Figure 1.5,6
Quinolactacin A (1) and related compounds are the only
fungal natural products that contain the 4-quinolone structure.
The unique structure of 1 endows the compounds with a broad
spectrum of biological activities, including the inhibition of
Guided by this evidence, we identified a potential
biosynthetic gene cluster (qlt) from the sequenced P. citrinum
genome (Figure 2A). The qlt cluster encodes two single-
Received: July 21, 2020
Figure 1. Representative 4-quinoline-containing natural products,
including quinolactacin A (1).
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Figure 2. Biosynthesis of quinolactacin A 1. (A) qlt BGC identified from P. citrinum DSM1997. Abbreviations: C0, truncated condensation; C,
condensation; A, adenylation; T, thiolation; R*, Dieckmann cyclization; N-MT, N-methyltransferase; IDO, indoleamine-2,3-dioxygenase; FMO,
flavin-dependent monooxygenase. (B) Proposed biosynthetic pathway of 1. GmdS is an amidase from the heterologous host A. nidulans A1145. (C)
LC−MS analysis of the in vitro biocatalytic synthesis of 1 using purified enzymes in the presence of SAM, FMN, ATP, MgCl2, and L-isoleucine. The
authentic standard of 1 was purified from P. citrinum.
module NRPSs (QltA and QltB), which may catalyze the
activation and condensation of two building blocks to form 1,
one of which is L-isoleucine; a flavin-dependent monoox-
ygenase (FMO) (QltD); a predicted N-methyltransferase (N-
MT) (QltE); and an IDO (QltC). Two homologous clusters
were found in Penicillium steckii and Aspergillus novofumigatus
(Figure S3). To confirm that the product of this cluster is 1, we
performed heterologous expression of the five genes in the
Aspergillus nidulans A1145 ΔEM strain (Supporting Informa-
tion).11 After 3 days of culture on Czapek Dox Starch
Tryptone (CDST) agar, the engineered strain produced a new
compound with the same retention time and mass as 1 with a
titer of ∼5 mg/L (Figure S4). Upon purification from a 4 L
culture, the compound was structurally confirmed to be 1 and
was found to exist as a diastereomeric pair (Table S4 and
Figures S14 and S17−S21).
Using the heterologous host, we next investigated the
minimal set of enzymes required to synthesize 1. Excluding any
qltA, B, D, and E completely abolished the formation of 1
(Figure S4). Excluding qltC that encodes an IDO, however, did
not lead to a significant change to the titer of 1. This hints that
QltC has the same function as the housekeeping IDO in A.
nidulans, and the qlt pathway can siphon 2 from primary
metabolism. To confirm this, purified QltC was assayed in the
presence of different indole-containing compounds (Figure
S5). The only IDO activity observed was the conversion of L-
Trp to N-formyl-L-kynurenine. Therefore, QltC is not an
essential enzyme for the biosynthesis of 1 and is encoded in
the pathway to provide additional flux of 2 in the native host.
The minimal set of enzymes required for formation of 1 is
therefore QltA, B, D, and E. To investigate the role of each
enzyme, we attempted the in vitro total enzymatic synthesis of
1 starting from 2 and in the presence of S-adenosyl methionine
(SAM), flavin mononucleotide (FMN), L-isoleucine, and
adenosine triphosphate (ATP).12,13 We were not able to
express the soluble form of P. citrinum QltB from either E. coli
or yeast. As a result, we cloned and expressed the homologue
from P. steckii (Figures S3 and S16). Unexpectedly, 1 was not
detected from in vitro reaction, and a new compound 6 with a
molecular weight of 175 was observed instead (Figure S6).
The scaled-up reaction followed by purification allowed the
identification of 6 to be N-methyl-2-hydroxyquinolone when
compared with an authentic standard (Figure 2B). The
formation of this shunt product suggested that a required
enzyme was missing in the in vitro reaction, and N-methylation
took place before the NRPS-catalyzed addition of L-isoleucine.
Indeed, the direct incubation of 2 with QltE and SAM led to
the formation of N-methyl-kynurenine 3, which was structur-
ally confirmed (Figure S7, Tables S5, and Figures S14 and
S22−S26). This demonstrates that QltE is the first
characterized kynurenine methyltransferase that can N-
methylate 2.
The addition of the FMO QltD to 3 led to the conversion to
a new compound 4, which gradually converted to the
previously observed 6 (Figure S8). A scaled-up in vitro
B
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Figure 3. Combinatorial biosynthesis of N-desmethyl quinolactacin 11. (A) LC−MS analysis of the in vitro assays of purified PqsA, PqsD, PqsE,
QltA, and QltB in the presence of anthranilic acid, CoA, malonyl-CoA, ATP, MgCl2, and L-isoleucine. (B) HPLC analysis of metabolites produced
by the heterologous host A. nidulans. (C) Combinatorial biosynthetic pathway to produce 11. PqsA, D, and E biosynthesize 9 from anthranilic acid.
reaction was performed to afford 4 for nuclear magnetic
resonance (NMR) characterization. Because 4 can sponta-
neously convert into 6, the purification of 4 was performed
using normal-phase high-performance liquid chromatography
(HPLC) to avoid any protic solvent. (See the Supporting
unit for condensation with L-isoleucine, the terminal amide in
4 must be hydrolyzed to the corresponding β-keto acid 5. This
is a reaction catalyzed by the amidase family of enzymes, which
are typically encoded as housekeeping enzymes in micro-
organisms. We hypothesize that an amidase encoded in A.
nidulans is responsible for this reaction. Three candidate
amidases were found in the A. nidulans genome, including
GmdS (accession number, AAL10515), which was shown by
whole-cell assays to hydrolyze benzamide and phenylaceta-
mide.18 We cloned and expressed GmdS and assayed its
activity in the presence of 4. The complete consumption of 4
and the rapid formation of 6 were observed in the presence of
GmdS, which is consistent with the hydrolysis to an amide,
followed by a much more facile intramolecular lactam
formation (Figure S15). We investigated the substrate scope
of GmdS and, surprisingly, found that it was also able to
hydrolyze β-keto esters into the corresponding β-keto acids,
which can readily decarboxylate (Figure S15). This observa-
tion implicates GmdS as being the responsible enzyme for the
exogenous β-keto ester hydrolysis previously observed in A.
nidulans.3a
We then repeated the in vitro enzymatic assay by including
GmdS with QltA, B, D, and E. Gratifyingly, we were able to
observe the formation of 1 in the presence of all of the
enzymes and cofactors (Figure 2C). 6 remains a shunt product
in the assay due to the competing nonenzymatic intra-
molecular cyclization of 5. The total enzymatic synthesis of 1
led us to propose the full biosynthetic pathway, as shown in
Figure 2B. Following the formation of β-keto acid 5, the
tridomain NRPS QltB is proposed to activate the carboxylate
as an adenylate and is loaded onto the T domain. The tetra-
domain NRPS activates and loads L-isoleucine onto its T
domain, followed by C-domain-catalyzed condensation with 5-
S-QltB to yield the β-keto amide-thioester adduct 7. The
terminal domain (R*) of QltA contains a conserved aspartate
(Figure S11), which is a signature for R* domains that catalyze
intramolecular Dieckmann condensations.19 We therefore
1
Information.) A comparison of H and 13C NMR data (Table
S6 and Figures S14 and S27−S31) of 4 with those of 3
confirmed that a methine group in 3 is absent, and the
methylene group of [δH 3.29, m and 3.64 (dd, J = 3.3, 18.0
Hz)] in 3 is replaced by a methylene singlet (δH 3.75) in 4,
which was supported via the HMBC correlations of H2-2 (δH
3.75) with C-4 (δC 116.8), C-3 (δC 196.7), and C-1 (δC
168.9). Combined with the observed molecular weight of 192,
the structure of 4 was assigned to be the β-ketoamide 3-(2-
(methylamino)phenyl)-3-oxopropanamide. Purified 4 can
undergo nonenzymatic intramolecular cyclization to afford 6
(Figure 2B), which suggests that the one-pot enzymatic
synthesis was mostly likely stalled at 4. QltD requires the
preinstallation of an N-methyl group to function, as the direct
reaction of QltD with 2 did not yield 2-amino-benzoylaceta-
mide (Figure S8). QltD was copurified with FMN (Figure S9)
and required molecular oxygen for catalysis, as no conversion
of 3 to 4 can be detected under anaerobic conditions. QltD,
however, does not require NADPH for reduction of the flavin
cofactor, which indicates that this is an internal monoox-
ygenase14 similar to lactate 2-monooxygenase15,16 (47%
sequence homology to QltD) and tryptophan-2-monooxyge-
nase.17 Therefore, we propose that QltD catalyzes the two-step
conversion of 3 to 4. First, 3 is oxidized to the imine while
reducing the FMN cofactor to FMNH2; this is followed by the
formation of flavin peroxide in the presence of molecular
oxygen, which can catalyze the decarboxylation to give 4 and
oxidized flavin (Figures S10).
The identification of 4 as a product of QltD and QltE
explains why the in vitro enzymatic synthesis using QltA, B, D,
and E could not produce 1 and instead accumulated 6. For the
NRPS enzyme to load an N-methyl-2-amino-benzoylacetate
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propose that R* catalyzes the cyclization and release of the
tetramate 8 from QltA. Intramolecular cyclization followed by
dehydration then yields the final product 1. The QltA A
domain was confirmed to prefer L-leucine, L-isoleucine, and L-
valine over the other proteinogenic amino acids (Figure S12),
which is consistent with the structures of isolated quinolactacin
analogs from different fungi (Figure 1). Interestingly, both the
native P. citrinum and the heterologous A. nidulans hosts
predominantly produced 1, and only trace amounts of
compounds with molecular weights corresponding to the
other quinolactacins could be detected (Figure S13). This may
be attributed to the C domain of QltA having more strict
specificity toward the aminoacyl moiety than the A domain
displayed in the activation assays.
Having established the biosynthetic pathway of 1, we turned
our attention to the generation of an analog through
engineered biosynthesis in A. nidulans. Our target analog was
N-desmethyl quinolactacin A 11, of which the biosynthesis
requires the desmethyl β-keto acid intermediate 2-amino-
benzoylacetate (2-ABA, 9) (Figure 3). The difficulty in
accessing 9 using the qlt pathway is the prerequisite of N-
methylation by QltE for QltD to perform decarboxylation.
Compound 9 is an intermediate in the biosynthesis of the
quorum-sensing signaling molecule 2-heptyl-4(1H)-quinolone
(HHQ) in Pseudomonas aeruginosa, which is also a 4-quinolone
compound.20,21 The formation of 9 in bacteria requires three
enzymes (Figure 3), a CoA-ligase PqsA that activates
anthranilate to anthraniloyl-CoA; a ketosynthase PqsD to
condense anthraniloyl-CoA and malonyl-CoA to yield 2-ABA-
CoA; and a thioesterase PqsE for the hydrolysis of thioester to
give 9.20 We reasoned that transplanting this bacterial pathway
in A. nidulans should provide the precursor 9 for conversion
into 11. To verify that 9 can be accepted by the NRPS, we
performed a one-pot enzymatic reaction with PqsA, D, and E
together with QltA and QltB. In the presence of the necessary
cofactors and substrates for the enzymes, we observed the
formation of two compounds (Figure 3A): the spontaneously
cyclized compound 2-hydroxyquinolone 10, which was
confirmed by comparing to a commercial standard, and a
new compound with identical UV absorbance to that of 1 and
the expected mass of 11 (Figure S14). We then constructed a
recombinant A. nidulans strain that expressed the three pqs
genes together with qltA and qltB, which, upon culturing,
produced 11 (Table S7 and Figures S32−S36) at a titer of ∼5
mg/L (Figure 3B). Therefore, this combinatorial biosynthesis
approach using a hybrid fungal/bacterial pathway afforded the
desired desmethyl analog of quinolactacin A.
AUTHOR INFORMATION
■
Corresponding Authors
Yi Tang − Department of Chemical and Biomolecular
Engineering and Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095, United
States; orcid.org/0000-0003-1597-0141; Email: yitang@
ucla.edu
Masao Ohashi − Department of Chemical and Biomolecular
Engineering and Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095, United
States; Email: gph422001@ucla.edu
Author
Mengting Liu − Department of Chemical and Biomolecular
Engineering and Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095, United
States; Hubei Key Laboratory of Natural Medicinal Chemistry
and Resource Evaluation, School of Pharmacy, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02426
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH 1R35GM118056 to Y.T.
M.L. is supported by a fellowship from the China Scholarship
Council (no. 2019060160162).
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02426.
Detailed experimental details and spectroscopic data
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