Enzymatic synthesis of 2-hydroxy-4H -quinolizin-4-one scaffolds by integrating coenzyme a ligases and a type III PKS from Huperzia serrata

Article abstract of DOI:10.1039/d0ra04133e

2-Hydroxy-4H-quinolizin-4-one scaffolds were enzymatically synthesized by integrating three enzymes including phenylacetate-CoA ligase (PcPCL) from an endophytic fungus Penicillium chrysogenum MT-12, malonyl-CoA synthase (AtMatB) from Arabidopsis thaliana, and a type III polyketide synthase (HsPKS3) from Chinese club moss Huperzia serrata. The findings paved the way to produce these kinds of structurally interesting alkaloids by engineered microorganisms. This journal is

Full text of DOI:10.1039/d0ra04133e

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Enzymatic synthesis of 2-hydroxy-4H-quinolizin-
4-one scaffolds by integrating coenzyme a ligases
and a type III PKS from Huperzia serrata†
Cite this: RSC Adv., 2020, 10, 23566
Juan Wang,‡ Ning Ding,‡ Yun Wu, Xiaoping Shi, Bowen Qi, Xiao Liu,
Xiaohui Wang,
*
Jun Li, Pengfei Tu and Shepo Shi
2-Hydroxy-4H-quinolizin-4-one scaffolds were enzymatically synthesized by integrating three enzymes
including phenylacetate-CoA ligase (PcPCL) from an endophytic fungus Penicillium chrysogenum MT-
12, malonyl-CoA synthase (AtMatB) from Arabidopsis thaliana, and a type III polyketide synthase (HsPKS3)
from Chinese club moss Huperzia serrata. The findings paved the way to produce these kinds of
structurally interesting alkaloids by engineered microorganisms.
Received 8th May 2020
Accepted 15th June 2020
DOI: 10.1039/d0ra04133e
rsc.li/rsc-advances
coenzyme A (CoA) thioesters as starters to perform iteratively
decarboxylative condensation reactions with malonyl-CoAs to
Introduction
produce a vast array of molecules with diverse structures and
biological activities.6 Remarkably, some plant specic type III
PKSs represented as acridone synthase (ACS) and quinolone
synthase (QNS) could efficiently employ bulky N-
methylanthraniloyl-CoA as a starter to synthesize acridone and
quinolone alkaloids, where the formation mechanism involved
a canonical C–N bond forming reaction (Scheme 1A and 1B).7
Accordingly, non-physiological substrates containing nitrogen
atoms have been applied to mimetically generate complex
polyketide-alkaloid hybrids with important bioactivities.8
However, restricted by the limited knowledge on the promis-
cuous substrate tolerance and the uncontrollable cyclization
modes of the polyketide intermediates, synthesis of designated
molecules using type III PKSs is currently a great challenge. On
the other hand, reactions catalyzed by type III PKSs require
commercially unavailable or extremely expensive CoA thioesters
Quinolizinones represent an important group of heterocyclic
molecules with a bridgehead nitrogen atom, displaying unique
physicochemical attributes, such as polar zwitterionic character
and low log P value, and a broad spectrum of bioactivities,
including selective activation of M1 muscarinic receptor, inhi-
bition of HIV integrase and phosphoinositide-3-kinase (PI3K),
and anti-ulcerative and anti-allergic activities.1 Noteworthily,
a
number of quinolizinones, being the bioisosteres of
quinolone-type antibiotics, also indicate promising potential to
be new antibacterial agents (Fig. 1).2 In addition, quinolizinones
tend to be readily reduced to partially- or fully-saturated
heterocyclic systems,3 generally termed as quinolizidines
which widely occur in the plant kingdom, particularly in the
families of Leguminosae, Lycopodiaceae, Huperziaceae and so
on, exhibiting great bioactivity diversity (Fig. 1).4 Encouraged by
the signicant importance of quinolizinones in new drug
research and development, strategies to construct quinolizi-
none scaffolds have been extensively reported.3c,5 However, their
chemical syntheses always involved multistep and environ-
mentally harmful reactions. Accordingly, investigations on the
enzymatic syntheses of quinolizinone scaffolds are denitely
meaningful and interesting, which would also pave the way to
produce pharmaceutically important quinolizinones by engi-
neered microorganisms.
It has been well reported that type III polyketide synthases
(PKSs), a class of architecturally simple homodimeric proteins
belonging to chalcone synthase superfamily, could utilize
Modern Research Center for Traditional Chinese Medicine, Beijing University of
Chinese Medicine, Beijing 100029, China. E-mail: shishepo@163.com
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra04133e
Fig. 1 Examples of synthetic quinolizinones (1–3) and natural prod-
ucts containing quinolizidine ring system (4–6).
‡ These authors contributed equally to this work.
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Scheme 1 Enzymatic synthesis of alkaloids by plant specific type III PKSs. (A) Acridone scaffold by ACS; (B) quinolone scaffold by QNS, and (C)
quinolizinone scaffolds by integrating PcPCL, AtMatB, and HsPKS3.
as substrates, which also greatly connes the application of type PKSs library comprising ten proteins (Fig. S1†). Only two type III
III PKSs as ideal biocatalysts.
PKSs (HsPKS1 and HsPKS3) from H. serrata,11,12 as a result,
In the present study, enzymatic synthesis of 2-hydroxy-4H- could expectedly catalyze the generation of a product with same
quinolizin-4-one scaffolds by integrating three enzymes molecular weight m/z 162.0552 ([M + H]+), consistent with an
including phenylacetate-CoA ligase from an endophytic fungus empirical molecular formula of C9H7NO2 (calc for C9H8NO2,
Penicillium chrysogenum MT-12 (PcPCL),9 malonyl-CoA synthase 162.0555). Evaluated by the peak area showed on the HPLC
from Arabidopsis thaliana (AtMatB),10 and a type III PKS from charts, the crude yield of the product generated by HsPKS3 is
Chinese club moss Huperzia serrata (HsPKS3) was described about 2.5 times that of HsPKS1, the temperature and pH optima
(Scheme 1C).11
were 30–40 C and 7.0–8.0, respectively. In order to unambig-
ꢀ
uously determine the structure of the enzymatic product, large-
scale co-incubation of 2-pyridylacetyl-CoA, malonyl-CoA, and
the recombinant enzyme HsPKS3 was performed, and the
enzymatic product was puried by prep-HPLC (2.3 mg, yield
ꢁ72%). The NMR spectroscopic data showed nine carbon
resonances at dC 168.9, 161.8, 144.8, 131.2, 127.4, 125.6, 114.6,
96.5, and 95.2, six proton resonances at dH 6.01 (1H, s), 6.39 (1H,
s), 6.92 (1H, t, J ¼ 6.0 Hz), 7.34 (1H, t, J ¼ 8.0 Hz), 7.44 (1H, d, J ¼
9.0 Hz), and 8.81 (1H, d, J ¼ 7.0 Hz). Comprehensive analysis of
the 1D- and 2D-NMR spectra (Table S1 and Fig. S2–S5†) allowed
unambiguous assignments of all the proton and carbon reso-
nances. Accordingly, the structure of the product was
unequivocally determined as 2-hydroxy-4H-quinolizinone-4-one
(P1) (Scheme 1C), a very useful scaffold for facile access to
quinolizione and quinolizine skeletons with great structural
complexity and biological diversity.13
Results and discussion
Retrosynthetic analysis suggested that enzymatic synthesis of 2-
hydroxy-4H-quinolizin-4-one scaffold might be achieved via
intramolecular cyclization of the diketide intermediate
produced by a certain type III PKS from the condensation of 2-
pyridylacetyl-CoA and one molecule of malonyl-CoA (Scheme 2).
The great challenge is to screen out a suitable type III PKS which
could readily accept 2-pyridylacetyl-CoA as a starter to perform
the aforementioned condensation and intramolecular cycliza-
tion reactions. Accordingly, 2-pyridylacetyl-CoA was chemically
synthesized and used as a probe for the screening of a type III
Next, we keep going forward to seek for the right CoA ligases
which could efficiently convert commercially available 2-
(pyridine-2-yl) acetic acid and malonic acid to 2-pyridylacetyl-
CoA and malonyl-CoA. At4CL, an A. thaliana originated 4-cou-
marate coenzyme A ligase (4CL) with the versatile capabilities of
employing diverse phenylpropanoic acid analogues to form
their corresponding CoA thioesters,9b,14 was rstly selected as
a candidate. Unfortunately, the puried At4CL could not
Scheme 2 Retrosynthetic analysis of the enzymatic synthesis of 2-
hydroxy-4H-quinolizin-4-one scaffold.
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convert 2-(pyridine-2-yl)acetic acid to its CoA thioester. It has revealed that the olenic proton resonated at dH 6.01 (1H, s) was
been reported that phenylacetate-CoA ligase from P. chrys- replaced by a methyl group [dH 2.01 (3H, s), dC 10.1], demon-
ogenum (PcPCL) could produce phenylacetate-CoA from phe- strating the successful synthesis of 2-hydroxy-3-methyl-4H-
nylacetic acid.9 Considering the high similarity of 2-(pyridine-2- quinolizinone-4-one (P2) (Table S1 and Fig. S12–S13†). When
yl)acetic acid and phenylacetic acid, we assumed that PcPCL malonic acid was replaced by the bulky ethylmalonic acid or
might accept 2-(pyridine-2-yl)acetic acid to produce its CoA allylmalonic acid, 2-hydroxy-3-ethyl-4H-quinolizinone-4-one
thioester. Thus, PcPCL was cloned from P. chrysogenum MT-12, (P3) and 2-hydroxy-3-allyl-4H-quinolizinone-4-one (P4) could
an endophytic fungus we previously isolated from H. serrata.15 also be produced but with poor yields (Fig. S14 and S15†), which
Expectedly, the recombinant PcPCL could also efficiently accept should be caused by the facts of that the bulky malonic acid
2-(pyridine-2-yl)acetic acid, besides phenylpropanoic acid derivatives could not be efficiently accepted by AtMatB.10
analogues such as p-coumaric acid, ferulic acid, and 4-phenyl- Notably, when 2-(pyridine-2-yl)acetic acid was replaced by 2-(5-
propanoic acid, to generate 2-pyridylacetyl-CoA (52 mg, yield uoro-pyridine-2-yl)acetic
acid,
7-uoro-2-hydroxy-4H-
ꢁ58%) (Fig. S6–S8†). Notably, PcPCL could also accept 2-(5-F- quinolizin-4-one (P5) could be detected from the one-pot reac-
pyridine-2yl) acetic acid and 2-(6-F-pyridine-2yl) acetic acid to tion mixture by LC-MS (Fig. S16†). In contrast, (6-uoro-
produce their corresponding CoA thioesters (Fig. S9 and S10†). pyridine-2-yl)acetyl-CoA could not be accepted by HsPKS3 to
For the formation of the 2-pyridylacetyl-CoA derivatives, kinetic generate the expected quinolizinone scaffold. Large-to-small
analysis indicated that the values of KM and kcat are of the same substitutions of the amino residues lining the active-site
magnitude as those reported for p-coumaric acid (Table S2†).9 cavity of HsPKS3 revealed that a mutant, HsPKS3 N221G,
Regarding to the enzymatic synthesis of malonyl-CoA, a gene could obviously increase the yields of P3 and P4 (Fig. S17†). In
annotated as malonyl-CoA synthase (AtMatB) was amplied contrast, the other single-site mutants, F225L, F275L, and
from A. thaliana and heterologously expressed in E. coli S348G did not obviously change the yields of the end products.
according to our previous report (Fig. S11†).10
Noteworthily, the only one residue mutation (HsPKS3 N221G)
To avoid the tedious and time-consuming purication of also made the production of 2-hydroxypyrido[2,1-a]isoindole-
CoA thioester intermediates, one-pot synthesis of quinolizinone 4,6-dione, a known pyridoisoindole scaffold produced by
scaffolds was developed. Under the optimized reaction condi- HsPKS1 (Fig. S18†).8a
tion (pH 7.5 and temperature 37 ꢀC), the 2-hydroxy-4H-
With the merits of environmental friendly conditions, low
quinolizinone-4-one scaffold (P1) was successfully synthesized processing cost, and easy separation of the end products,
from 2-(pyridine-2-yl) acetic acid and malonic acid by the one- enzymes catalyzed one-pot multistep reactions have recently
pot incubation of PcPCL, AtMatB, and HsPKS3 (Fig. 2A). attracted signicant attention.16 The method reported here
When malonic acid was replaced by methylmalonic acid, the circumvented the separation of CoA thioester intermediates,
one-pot reaction generated P2 with molecular weight m/z which is tedious, time-consuming, and challenging due to the
176.0703 ([M + H]+) which is 14 Da larger than that of P1 high hydrophilicity and the easy degradation of CoA thioesters.
(Scheme 1C and Fig. 2B), suggesting the additional presence of Additionally, the one-pot reaction could also provide a recycling
a methyl group in P2. Large-scale reactions combined with system that coenzyme A released from the CoA thioesters by
HPLC purication allowed the accumulation of 8 mg (yield HsPKS3 could be reused by the CoA ligases (PcPCL and AtMatB).
ꢁ45%) sample for measuring the NMR spectra of P2. Compar- Predictably, this method would be more practical and economic
ison of the NMR spectroscopic data of P2 with those of P1 using an ATP regeneration system since ATP is the critical and
costly cofactor in the cascade reactions.17 Nevertheless, this
work sets the stage for the construction of an engineered
microorganism to produce 2-hydroxy-4H-quinolizin-4-one
scaffolds.
Experimental section
Chemicals
Chemicals and reagents were purchased from Sigma Aldrich (St.
Louis, MO, USA), J&K Scientic Ltd. (Beijing, China), BioDee
Biotechnology Co. Ltd. (Beijing, China), unless noted otherwise.
Restriction enzymes, DNA polymerase and DNA ligase were
purchased from Takara Biotechnology Co. Ltd. (Dalian, China).
Primer synthesis and DNA sequencing were performed at Invi-
trogen (Shanghai, China). Malonyl-CoA was purchased from
Sigma Aldrich (St. Louis, MO, USA). Chemical shis (d) were
Fig. 2 LC-MS charts for the one-pot formation of (A) 2-hydroxy-4H-
quinolizinone-4-one (P1) and (B) 2-hydroxy-3-methyl-4H-quinolizi-
none-4-one (P2) by PcPCL, AtMatB, and HsPKS3. The HPLC chro-
recorded with reference to solvent signals (1H NMR: CD3OD
3.31 ppm; DMSO-d6 2.50 ppm; 13C NMR: CD3OD 49.0 ppm;
matograms were measured at 230 nm.
DMSO-d6 39.5 ppm). HRESIMS analyses were performed with
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LCMS-IT-TOF system (Shimadzu, Kyoto, Japan) equipped with concentrated by millipore column and diluted ve-fold with
an electrospray ionization source.
20 mM KPB buffer (pH 7.8), containing 10% (v/v) glycerol and
100 mM NaCl. The puried protein was analyzed by SDS-PAGE,
and protein concentration was determined by the BCA method
(Protein Assay, BIOMIGA) with the bovine serum albumin as
standard.
For the preparation of malonyl-CoA synthase (AtMatB), the
plasmid encoding the known MatB from A. thaliana was con-
structed according to our previous report, and the expression
and protein purication were performed according to the same
procedure reported in the literature.22
Expression and protein purication of type III PKSs
We have previously reported three type III PKSs (AsCHS1,
AsPKS1, and AsPKS2) from Aquilaria sinensis,18 two type III PKSs
(HsPKS1 and HsPKS3) from Huperzia serrata,11,14 two quinolone
synthases (ErQNS1 and ErQNS2) from Euodia rutaecarpa,19 and
two type III PKSs (ClDCS and ZoCURS) from Curcuma longa and
Zingiber officinale.20 The preparation of the nine recombinant
proteins were performed according to our previous reports. For
the preparation of the recombinant protein HsPKS2, the
plasmid encoding the known HsPKS2 from H. serrata was
constructed according to the previous report, and the expres-
sion and protein purication were performed according to the
same procedure reported in the literatures.21
Chemical synthesis of 2-pyridylacetyl-CoA
2-Pyridylacetic acid (5 mmol) prepared from 2-pyridylacetic acid
hydrochloride via alkalization using sodium hydroxide and N-
hydroxysuccinimide (5 mmol) were dissolved in a ask con-
taining 90 mL anhydrous tetrahydrofuran (THF). Then, the
solution of 8 mmol dicyclohexyl carbodimide (DCC) in 10 mL
THF was dropped slowly into the ask. Aer being stirred for 5 h
Cloning, expression, and protein purication of coenzyme A
ligases
For the preparation of phenylacetate-CoA ligase (PCL), the gene under room temperature, the mixture was ltered to remove the
encoding PCL (AJ001540) was amplied via polymerase chain white precipitates. The ltrate was evaporated under reduced
reaction (PCR) from genomic DNA isolated from Penicillium pressure and crystallized in absolute ethanol. Then, the solu-
chrysogenum MT-12 using Pyrobest™ DNA Polymerase. The PCR tion of 0.12 mmol crystals in 5 mL acetone was dropped into the
mixture (50 mL) contained 25 ng of plasmid DNA, 5 mL of 10ꢂ ask containing 10 mL mixture of CoA (60 mmol) and NaHCO3
Pyrobest buffer, 4 mL dNTPmix (2.5 mM each), 50 pmol of each (0.3 mmol) solutions. Aer being stirred for 5 h at 0 ꢀC, the
primer (forward primer 50-CC GGAATTC ATGGTTTTTTTACCTCC- acetone was completely removed by vacuum evaporation. Then,
30 and reverse primer 50-CCCAAGCTT GATCTTGCTACCAG CC-30), ammonium acetate was added to the water phase to a nal
1.25 units of Pyrobest™ DNA Polymerase, and ddH2O 30.25 mL. concentration of 4%, and the mixture was loaded onto Sep-Pak
The gene was amplied using a Mastercycler nexus (Eppendorf, cartridge preconditioned with 4% ammonium acetate. The
Hamburg, Germany), with the following parameters: 94 ꢀC for column was eluted with 4% ammonium acetate solution to
1 min followed by 35 cycles of 98 ꢀC for 5 s, 54 ꢀC for 45 s, 72 ꢀC for completely remove the free CoA, and then 2-pyridylacetyl-CoA
1 min and 30 s. The amplied gene was cloned into a pMD19-T was obtained by elution with ddH2O. The structure was eluci-
1
vector and sequenced. Aer removal of the introns by over- dated by HRESIMS and H NMR techniques.
1
lapping PCR method, a full length of pcl gene was nally obtained 2-Pyridylacetyl-CoA was obtained as white powder. H NMR
using N- and C-terminal specic primers
50- (500 MHz, in CD3OD): dH 8.47 (1H, d, J ¼ 5.5 Hz), 7.81 (1H, td, J
CGCGGATCC ATGGTTTT TTTACCTCCAAAGG-30 (the BamHI site ¼ 7.5, 2.0 Hz), 7.43 (1H, d, J ¼ 7.5 Hz), 7.34 (1H, td, J ¼ 7.5, 5.5
is underlined) and 50-CCCAAGCTT TTAATGATGGTGATGGT- Hz), 4.27 (2H, s), 8.57 (1H, s), 8.20 (1H, s), 6.13 (1H, d, J ¼ 6.0
GATGGATCTTGCTACCGCCTTTC-3' (the Hind III site is under- Hz), 3.45 (2H, overlapped), 4.50 (1H, s), 4.08 (1H, s), 4.82 (1H, s),
lined). The amplied full-length pcl gene was digested with BamHI/ 4.27 (1H, s), 4.02 (1H, m), 3.58 (1H, m), 3.36 (4H, s), 3.02 (2H,
Hind III and cloned into the BamHI/Hind III sites of pMAL-C2X m), 2.39 (2H, m), 1.08 (3H, s), 0.83 (3H, s). HRESIMS: m/z [M +
vector. Aer conrmation of the sequence, the plasmid containing H]+ 887.1551, calc for C28H42N8O17P3S, 887.1596.
the full-length cDNA encoding PcPCL was transformed into E. coli
BL21(DE3). The cells harboring the plasmid were cultured to an
OD600 of 0.6 in LB medium containing 100 mg mLꢃ1 of Ampicillin
Enzymatic synthesis of 2-hydroxy-4H-quinolizin-4-one by
HsPKS3
ꢀ
at 37 C, and 0.5 mM isopropyl-1-thio-b-D-galactopyranoside was
then added to induce protein expression. The culture was incu- The reaction mixture contained 50 nmol of 2-pyridylacetyl-CoA,
bated further at 17 ꢀC for 16 h. The E. coli cells were harvested by 50 nmol of malonyl-CoA, 0.4 nmol of the puried recombinant
centrifugation at 7500g for 10 min using Ependorf 5810R centri- protein HsPKS3, in a nal volume of 500 mL of 100 mM potas-
fuge (Ependorf, Germany), and resuspended in TANG buffer sium phosphate buffer (pH 7.0). The reaction was incubated at
[50 mM Tris/HCl, pH 7.5, 200 mM NaCl, 0.002% sodium azide and 37 ꢀC for 12 h. The products were then extracted three times
10% (w/v) glycerol] containing 0.1% (v/v) PMSF. Cells were dis- with 800 mL of ethyl acetate. Aer removal of solvent by evapo-
rupted by sonication, and centrifuged at 8000g for 40 min. The ration, the residue was redissolved by MeOH and analyzed by
supernatant was loaded on a Ni2+ Sepharose 6 Fast Flow column LC-MS on an Agilent ZORBAX SB C18 Column (4.6 mm I.D. ꢂ
(Biohao). Aer washing the column with Tang buffer, the 250 mm, 5 mm) with a ow rate of 1.0 mL minꢃ1. The gradient
recombinant protein was subsequently eluted with TANG buffer elution for analyzing was performed with aqueous acetonitrile
containing 500 mM imidazole. The protein solution was then (MeCN). 0–5 min, 5–30% MeCN; 5–10 min, 30–40% MeCN; 10–
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15 min, 40% MeCN; 15–22 min, 40–60% MeCN; 22–25 min, 60– extracted three times with ethyl acetate. Aer removal of solvent
95% MeCN. by evaporation, the residue was redissolved by MeOH and
For large scale reaction: 20 mmol of 2-pyridylacetyl-CoA, 20 analyzed by HPLC and LC-MS on an Agilent ZORBAX SB C18
mmol of malonyl-CoA and 0.2 mmol of recombinant HsPKS3 was Column (4.6 mm I.D. ꢂ 250 mm, 5 mm) with a ow rate of 1.0
dissolved in 100 mL 100 mM potassium phosphate buffer (pH mL minꢃ1. The gradient elution for analyzing was performed
ꢀ
7.0) and incubated at 37 C for 12 h. The reaction mixture was with aqueous acetonitrile (MeCN): 0–5 min, 5–30% MeCN; 5–
extracted by 100 mL ethyl acetate for three times. Aer removal 10 min, 30–40% MeCN; 10–15 min, 40% MeCN; 15–22 min, 40–
of solvent under reduced pressure, the residue was dissolved in 60% MeCN; 22–25 min, 60–95% MeCN, 25–35 min.
0.5 mL of MeOH and puried by semi-preparative HPLC using
Agilent ZORBAX Eclipse XDB C18 Column (4.6 mm I.D. ꢂ 250 Construction of HsPKS3 mutants
mm, 5 mm). The structure of the puried product was deter-
mined by HRESIMS and NMR techniques.
HsPKS3 mutants (N221G, F225L, F275L, S348G) were constructed
using the Mutagenesis with In-Fusion HD Cloning Plus Kit (Takara)
following the standard protocol and a pair of complementary
mutagenic primers as follows (mutated codons are underlined);
N221G, forward primer: 50-CCCAAATCTCATAGGC TCTGCTT-30,
Enzymatic synthesis of 2-pyridylacetyl-CoA derivatives by
PcPCL and kinetic analysis
The reaction mixture contained 0.10 mmol acid, 62.5 mmol reverse primer: 50-AAGCAGAGGC TATGAGATTTGGG-30; F225L,
MgCl2, 1.6 mmol NaCl, 0.15 mmol CoA, 32 mmol ATP, and 10 forward primer: 50-TTCTGCTTTACTG GGCGATGGTGC-30, reverse
nmol of the puried PcPCL, in a nal volume of 10 mL of primer: 50-GCACCATCGCCCAG TAAAGCAGAA-30; F275L, forward
100 mM Tris–HCl buffer (pH 7.5). The reaction was incubated at primer: 50-AG CTGGGATGGTACTG TTACTGAT-30, reverse primer:
30 ꢀC for 6 h. The products were then puried by Sep-Pak 50-ATCAGTAACAG TACCATCCCAGCT-30; S348G, forward primer:
cartridge using the method mentioned in the section of 50-GGAAACATGGGC A GCCCTTCTGTG-30, reverse primer: 50-
synthesizing 2-pyridylacetyl-CoA. The enzymatic product was CACAGAAGGGCTGCC CATGTTTCC-30.
analyzed by LC-MS on an Shimadzu shim-pack XR-ODS II
(2.0 mm I.D. ꢂ 100 mm, 2.2 mm) column with a ow rate of 0.2
Conclusions
mL minꢃ1. The gradient elution for analyzing was performed
In summary, the present work describes the one-pot formation
of quinolizinone scaffolds by integrating phenylacetate-CoA
ligase (PcPCL), malonyl-CoA ligase (AtMatB), and a type III
polyketid synthase (HsPKS3). Here, both the formation of 2-
pyridylacetyl-CoA by PcPCL and the synthesis of quinolizinone
scaffold from 2-pyridylacetyl-CoA by HsPKS3 have never been
reported. The ndings not only expanded the biosynthetic
repertoire of type III PKSs but also paved the way to produce
pharmaceutically important quinolizinones by engineered
microorganisms.
with aqueous acetonitrile containing
5 mM ammonium
formate: 0–10 min, 5% MeCN; 10–20 min, 5–100% MeCN.
Kinetic parameters were determined according to the
method previously reported. By varying the concentration of the
acid at the xed concentration of ATP, CoA, and Mg2+, the
apparent kinetic parameters for 2-pyridylacetic acid derivatives
were determined by tting the initial rates of CoA thioesters
formation with the Michaelis–Menten equation.23
Enzymatic synthesis of malonyl-CoA by AtMatB
The reaction mixture contained 0.10 mmol malonic acid, 62.5
mmol MgCl2, 1.6 mmol NaCl, 0.15 mmol CoA, 32 mmol ATP, and
10 nmol of the puried AtMatB, in a nal volume of 10 mL of
100 mM potassium phosphate buffer (pH 7.0). The reaction was
Conflicts of interest
There are no conicts to declare.
ꢀ
incubated at 30 C for 6 h. The products were then puried by
Acknowledgements
Sep-Pak cartridge using the method mentioned in the section of
synthesizing 2-pyridylacetyl-CoA. The enzymatic product was
analyzed by LC-MS on an Shimadzu shim-pack XR-ODS II
(2.0 mm I.D. ꢂ100 mm, 2.2 mm) column with a ow rate of 0.2
mL minꢃ1. The gradient elution for analyzing was performed
This work was nancially supported by the National Natural
Science Foundation of China (No. 81573312, 81773842), the
Drug Innovation Major Project (No. 2018ZX09711001-006).
with aqueous acetonitrile containing
formate: 0–10 min, 5% MeCN; 10–20 min, 5–100% MeCN.
5 mM ammonium
Notes and references
1 (a) Y. Kitaura, T. Oku, H. Hirai, T. Yamamoto and
M. Hashimoto(Fujisawa Pharmaceutical Co., Ltd.), US Pat.
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0.1 mmol malonic acids, 62.5 mmol MgCl2, 60 mmol ATP,
0.45 mmol CoA, 10 nmol PcPCL, and 10 nmol AtMatB, in a nal
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HsPKS3 was added into the reaction mixture, and the reaction
was incubated at 33 ꢀC for another 6 h. The products were then
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