Bioactive natural products have evolved to inhibit specific cellular targets and have
served as lead molecules for health and agricultural applications for the last century
. The post-genomics era has brought a renaissance in natural product discovery using
synthetic biology tools
. However, compared to traditional bioactivity-guided approaches, genome mining of
natural products with specific and potent biological activities remains challenging
. Here we present the discovery and validation of a highly potent herbicide lead that
targets a critical metabolic enzyme that is required for plant survival. Our approach
is based on the coclustering of a self-resistance gene in the natural product biosynthetic
, which serves as a window to potential biological activity of the encoded compound.
We targeted dihydroxyacid dehydratase (DHAD) in the branched-chain amino acid (BCAA)
biosynthetic pathway in plants, the last step in this pathway often targeted for herbicide
. We showed the fungal sesquiterpenoid aspterric acid (AA) discovered using this method
is a submicromolar inhibitor of DHAD and is effective as an herbicide in spray applications.
The self-resistance gene astD was validated to be insensitive to AA and was deployed
as a transgene in establishment of plants that are resistant to AA. This herbicide-resistance
gene combination complements urgent efforts in overcoming weed resistance
. Our discovery demonstrates the potential of using a resistance-gene directed approach
in mining of bioactive natural products.
Weeds are a major source of crop losses, and the evolution of herbicide resistance
in weeds has led to an urgent need for new herbicides with novel modes of action
. The BCAAs biosynthetic pathway is essential for plant growth
. It is not present in animals and is therefore a validated target for highly specific
weed control agents
. The BCAA biosynthetic pathway in plants is carried out by three enzymes: acetolactate
synthase (ALS), acetohydroxy acid isomeroreductase (KARI), and DHAD (Fig. 1a). Given
the success of targeting ALS for herbicide development
, it is surprising that no herbicide that targets either of the other two enzymes
has been developed. DHAD which catalyzes β-dehydration reactions to yield α-keto acid
precursors to isoleucine, valine and leucine, is an essential and highly conserved
enzyme among plant species
(Extended Data Fig. 1a and Supplementary Fig. 1). Efforts toward synthetic DHAD inhibitors
resulted in compounds with submicromolar K
i; however, the compounds have no in planta activity
(Extended Data Fig. 1b).
Filamentous fungi are prolific producers of natural products (NPs), many of which
have biological activities that aid the fungi in colonizing and killing plants
. Therefore, fungal NPs represent a promising source of potential leads for herbicides.
The abundance of sequenced fungal genomes enables genome mining of new NPs with novel
. Although no NP inhibitors of DHAD are known to date, we reason a fungal NP with
this property might exist, given the indispensable role of BCAA biosynthesis in plants
To identify NP biosynthetic gene clusters that may encode a DHAD inhibitor, we hypothesized
that such cluster must contain an additional copy of DHAD that is insensitive to the
inhibitor, thereby providing the required self-resistance for the producing organism
to survive. The presence of a gene encoding a self-resistance enzyme is frequently
found in microbial NP gene clusters, as highlighted by the presence of an insensitive
copy of HMGR or IMPDH in the gene clusters for lovastatin (that targets HMGR) or mycophenolic
acid (that targets IMPDH), respectively (Extended Data Fig. 1c)
. This phenomenon has been used to predict molecular targets of NPs, as well as to
identify gene clusters of NPs of known activities
To identify possible self-resistance enzymes, we scanned sequenced fungal genomes
to search for colocalizations of genes encoding DHAD with core biosynthetic enzymes,
such as terpene cyclases, polyketide synthases, etc
. We identified a well-conserved set of four genes across multiple fungal genomes
(Fig. 1b), including the common soil fungus Aspergillus terreus that is best known
to produce lovastatin. The conserved gene clusters include genes that encode a sesquiterpene
cyclase homolog (astA), two cytochrome P450s (astB and astC), and a homolog of DHAD
(astD). Genes outside of this cluster are not conserved across the identified genomes
and are hence unlikely to be involved in NP biosynthesis. AstD is the second copy
of DHAD encoded in the genome, and is ~70% similar to the housekeeping copy that is
well-conserved across fungi (Supplementary Fig. 2). Therefore, AstD is potentially
a self-resistance enzyme that confers resistance to the encoded NP. Like a majority
of biosynthetic gene clusters in sequenced fungal genomes, the ast cluster has not
been associated with the production of a known NP
To identify the NP encoded by the ast cluster, we heterologously expressed astA, astB,
and astC genes in the host Saccharomyces cerevisiae RC01
. New compounds that emerged were purified and their structures were elucidated with
NMR spectroscopy (Supplementary Fig. 3 and Supplementary Table 5). RC01 expressing
only astA produced a new sesquiterpene (1), which was confirmed to be (−)-daucane
(Supplementary Fig. 4). RC01 expressing both astA and astB led to the biosynthesis
of a new product that was structurally determined to be the α-epoxy carboxylate (2)
(Fig. 1c). When astA, astB and astC were expressed together, a new compound (3) became
the dominant product (~ 20 mg/L). Full structural determination revealed the compound
to be the tricyclic aspterric acid (AA), which is a previously isolated compound (Fig.
. The biosynthetic pathway for AA is therefore concise: following cyclization of farnesyl
diphosphate by AstA to create the carbon skeleton in 1, AstB catalyzes oxidation of
1 to yield the epoxide 2. Further oxidation by AstC at carbon 15 yields an alcohol,
which can undergo intramolecular epoxide opening to create AA (Fig. 1d).
Upon its initial discovery, AA was shown to have inhibitory activity towards Arabidopsis
thaliana, however, the mode of action was not known
. Our resistance-gene directed approach led to rediscovery of this compound with DHAD
as a potential target. We first confirmed that AA is able to potently inhibit A. thaliana
growth in an agar-based assay (Fig. 2a, and Supplementary Fig. 5). AA was also an
effective inhibitor of root development and plant growth when applied to a representative
monocot (Zea mays) and dicot (Solanum lycopersicum) (Fig. 2b). To test if AA indeed
targets DHAD, we expressed and purified housekeeping DHAD from both A. terreus (XP_001208445.1,
fDHAD) and A. thaliana (AT3G23940, pDHAD), as well as the putative self-resistance
enzyme AstD (Supplementary Fig. 6). Both housekeeping DHAD enzymes converted dihydroxyisovalerate
to ketoisovalerate (pDHAD: k
cat = 1.2 sec−1, K
M = 5.7 mM) as expected. The enzyme activities, however, were inhibited in the presence
of AA (Extended Data Fig. 2). The IC50 values of AA towards fDHAD and pDHAD were 0.31
μM and 0.50 μM at an enzyme concentration of 0.50 μM, respectively (Extended Data
Fig. 3). AA was further determined to be a competitive inhibitor of pDHAD with a K
i = 0.30 μM (Extended Data Fig. 3). AA displayed no significant cytotoxicity towards
human cell lines up to 500 μM concentration, consistent with the lack of DHAD in mammalian
cells (Supplementary Fig. 7).
AstD catalyzes the identical β-dehydration reaction as DHAD, albeit with a significantly
more sluggish turnover rate (k
cat = 0.03 sec−1, K
M = 5.4 mM). However, the enzyme was not inhibited by AA, even at the solubility limit
of 8 mM (Extended Data Fig. 3). To determine if AstD can confer resistance to AA-sensitive
strains, we developed a yeast based assay. The genome copy of DHAD encoded by ILV3
was first deleted from Saccharomyces cerevisiae strain DHY ΔURA3, which resulted in
an auxotroph that requires exogenous addition of Ile, Leu and Val to grow. We then
introduced either fDHAD or astD episomally, both of which allowed the strain to grow
in the absence of the three BCAAs (Extended Data Fig. 4). However, yeast expressing
fDHAD was approximately 100 times more sensitive to AA (IC50 of 2 μM) compared to
yeast expressing AstD (IC50 of 200 μM) (Fig. 2c). Collectively, the biochemical and
genetic assays validated AA as the first natural product inhibitor of fungal and plant
DHAD; and AstD serves as the self-resistance enzyme in the ast biosynthetic gene cluster.
The (R)-α-hydroxyacid and (R)-configured β-ether oxygen moieties in AA mimic the (2R,
3R)-dihydroxy groups present in natural substrates such as dihydroxyisovalerate. The
β-ether oxygen in AA is in position to coordinate to the 2Fe-2S cluster that is a
required cofactor in both fungal and plant DHAD
. To understand potential AA mechanism of action, we determined the crystal structure
(2.11 Å) of the pDHAD complexed with 2Fe-2S cluster (holo-pDHAD) (Fig. 2d, Extended
Data Fig. 5, and Extended Data Table 1). We identified a binding chamber at the homodimer
interface, similar to that found in the holo bacterial L-arabinonate dehydratase
(Fig. 2d). The interior of the chamber is positively charged (2Fe-2S and Mg2+) while
the entrance is lined with hydrophobic residues. The modeled binding mode of α,β-dihydroxyisovalerate
and AA predicted by computational docking are shown in Fig. 2e. The pocket is sufficiently
spacious to accommodate the bulkier AA, and provide stronger hydrophobic interactions
than the native substrate with a 5.3 ± 0.3 kcal/mol gain in binding energy (Fig. 2e).
Base on the holo-pDHAD structure, we constructed a homology model of AstD to determine
potential mechanism of resistance (Extended Data Fig. 5 and 6). Comparison of pDHAD
and the modeled AstD structures shows that while most of the residues in the catalytic
chamber are conserved, the hydrophobic region at the entrance to the reactive chamber
in AstD is more constricted as a result of two amino acid substitutions (V496L and
I177L). Narrowing of the entrance could therefore sterically exclude the bulkier AA
from binding in the active site, while the smaller, natural substrates are still able
to enter the chamber.
To explore the potential of AA as an herbicide, we performed spray treatment of A.
thaliana with AA. We added AA into a commercial glufosinate formulation known as Finale®
at a final AA concentration of 250 μM
. We then sprayed AA solution onto glufosinate resistant A. thaliana. Finale® alone
had no observable inhibitory effects on plant growth, but adding AA severely inhibited
plant growth (Extended Data Fig. 7). In addition, A. thaliana plants treated with
AA before flowering failed to form normal pollen, which was also observed previously
. We found that the pistil of treated plants could still be successfully pollinated
using healthy pollen from the untreated A. thaliana, indicating that AA preferentially
affects pollen but not egg formation (Extended Data Fig. 8 and 9). This effect was
also observed with a lower concentration of AA (100 μM). Thus, in addition to its
herbicidal properties, AA could potentially be used as a chemical hybridization agent
for hybrid seed production
We next investigated whether plants expressing astD are resistant to AA. This was
motivated by the successful combination of glyphosate and genetically modified crops
that are selectively resistant to glyphosate (Roundup Ready®)
. The A. terreus astD gene was codon optimized and the N-terminus was fused to a chloroplast
localization signal derived from pDHAD. Wild type or astD transgene-expressing A.
thaliana was then grown on media that contained 100 μM AA. In the presence of AA,
the growth of wild-type plants was strongly inhibited, and arrested at the cotyledon
stage (Fig. 3a). In contrast, the growth of astD transgenic plants was relatively
unaffected by AA, as indicated by the normally expanded rosette leaves, elongated
roots, and whole plant fresh weight (Fig. 3a and b). The expression of AstD was verified
by western blot (Supplementary Fig. 8). A spray assay was also performed using T2
astD transgenic A. thaliana plants, which showed no observable growth defects under
such treatment (Fig. 3c). In contrast, the control plants carrying the empty vector
showed a strong growth inhibitory phenotype when treated with AA (Fig. 3c). Quantitative
measurements of plant height showed AstD effectively confers AA resistance to A. thaliana
In summary, resistance-gene directed discovery of NPs in the fungus A. terreus led
to the discovery of a natural herbicide AA and the determination of its mode of action.
In addition, introducing astD as a transgene or editing the sequence of the plant
DHAD endogenous gene could be used to create AA-resistant crops. We suggest that AA
is a promising lead for the development as a broad spectrum commercial herbicide.
General materials and methods
Biological reagents, chemicals, media and enzymes were purchased from standard commercial
sources unless stated. Plant, fungal, yeast and bacterial strains, plasmids and primers
used in this study are summarized in Supplementary Tables 3, 4 and 5. DNA and RNA
manipulations were carried out using Zymo ZR Fungal/Bacterial DNA Microprep™ kit and
Invitrogen Ribopure™ kit respectively. DNA sequencing was performed at Laragen, Inc.
The primers and codon optimized gblocks were synthesized by IDT, Inc.
Expression of ast genes in Aspergillus nidulans for cDNA isolation
Plasmids pYTU, pYTP, pYTR digested with PacI and SwaI were used as vectors to insert
. A gpda promoter was generated by PCR amplification using primers Gpda-pYTU-F and
Gpda-R with pYTR serving as template. Genes to be expressed were amplified through
PCR using the genomic DNA of Aspergillus terreus NIH2624 as a template. A 4.5 kb fragment
obtained using primers AstD-pYTU-recomb-F and AstA-pYTU-recomb-R was cloned into pYTU
together with a gpda promoter by yeast homologous recombination to obtain pAstD+AstA-pYTU.
Yeast transformation was performed using Frozen-EZ Yeast Transformation II Kit™ (Zymo
Research). A 2.4 kb fragment obtained using primers AstB-pYTR-recomb-F and AstB-pYTR-recomb-R
was cloned into pYTR by yeast homologous recombination to obtain pAstB-pYTR. Similarly,
a 2.3 kb fragment obtained using primers AstC-pYTP-recomb-F and AstC-pYTP-recomb-R
was cloned into pYTP by yeast homologous recombination to obtain pAstC-pYTP.
All three plasmids (pAstD+AstA-pYTU, pAstB-pYTR and pAstC-pYTP) were transformed into
A. nidulans following standard protocols to result in the A. nidulans strain TY01
. TY01 was cultured in liquid CD-ST medium (20 g/L starch, 20 g/L peptone, 50 mL/L
nitrate salts and 1 mL/L trace elements) at 28°C for 3 days. Total RNA of TY01 was
extracted with the Invitrogen Ribopure™ kit, and total cDNA of TY01 was obtained using
the SuperScript III reverse transcriptase kit (Thermo Fisher Scientific). The cDNA
fragment of astA was PCR amplified using primers AstA-xw55-recomb-F and AstA-xw55-recomb-R.
The cDNA fragment of astB was PCR amplified using primers AstB-xw06-recomb-F and AstB-xw06-recomb-R.
The cDNA fragment of astC was PCR amplified using primers AstC-xw02-recomb-F and AstC-xw02-recomb-R.
The cDNA fragment of astD was PCR amplified using primers AstD-pXP318-F and AstD-pXP318-R.
All the introns were confirmed to be correctly removed by sequencing.
Construction of Saccharomyces cerevisiae strains
Plasmid pXW55 (URA3 marker) digested with NdeI and PmeI was used to introduce the
. A 1.3 kb fragment containing astA obtained from PCR using primers AstA-xw55-recomb-F
and AstA-xw55-recomb-R was cloned into pXW55 using yeast homologous recombination
to afford pAstA-xw55. The plasmid pAstA-xw55 was then transformed into Saccharomyces
cerevisiae RC01 to generate strain TY02
Plasmid pXW06 (TRP1 marker) digested with NdeI and PmeI was used to introduce the
. A 1.6 kb fragment containing astB obtained from PCR using primers AstB-xw06-recomb-F
and AstB-xw06-recomb-R were cloned into pXW06 using yeast homologous recombination
to afford pAstB-xw06. The plasmid pAstB-xw06 was then transformed into TY02 to generate
Plasmid pXW06 (LEU2 marker) digested with NdeI and PmeI was used to introduce the
. A 1.6 kb fragment containing astC obtained from PCR using primers AstC-xw02-recomb-F
and AstC-xw02-recomb-R were cloned into pXW02 using yeast homologous recombination
to afford pAstC-xw02. The plasmid pAstC-xw02 was then transformed into TY03 to generate
URA3 gene was inserted into ilv3 locus of Saccharomyces cerevisiae DHY ΔURA3 strain
to generate UB01. A 879 bp homologous recombination donor fragment with 35–40 bp homologous
regions flanking ilv3 ORF was amplified using primers ILV3p-URA3-F and ILV3t-URA3-R
using yeast gDNA as template. The PCR product was gel purified and transformed into
Saccharomyces cerevisiae DHY ΔURA3, and selected on uracil dropout media to give UB01.
The resulting strain was subjected to verification by colony PCR with primers ILV3KO-ck-F
and ILV3KO-ck-R and the amplified fragment was sequence confirmed.
The URA3 gene inserted into ilv3 locus of Saccharomyces cerevisiae DHY ΔURA3 was deleted
from UB01 using homologous recombination to generate UB02. A 150 bp homologous recombination
donor fragment with 75 bp homologous regions flanking ilv3 ORF was amplified using
primers ILV3KO-F and ILV3KO-R, gel purified and transformed into UB01, and counterselected
on 5-fluoroorotic acid (5-FoA) containing media to give UB02. The resulting strain
was subjected to verification by colony PCR with primers ILV3KO-ck-F and ILV3KO-ck-R
and the amplified fragment was sequenced confirmed.
The empty plasmid pXP318 (URA3 marker) was transformed into UB02 to generate TY05
Plasmid pXP318 digested with SpeI and XhoI was used as vector to introduce gene encoding
. The cDNA of Aspergillus terreus NIH 2624 served as template for PCR amplification.
A 1.7 kb fragment obtained using primers fDHAD-pXP318-F and fDHAD-pXP318-R were cloned
into pXP318 using yeast homologous recombination to afford fDHAD-pXP318. Then, fDHAD-pXP318
was transformed into UB02 to generate TY06. fDHAD was driven by a constitutive promoter
Plasmid pXP318 digested with SpeI and XhoI was used as vector to introduce astD gene
. The cDNA isolated from TY01 served as the template for PCR amplification. A 1.8
kb fragment obtained using primers AstD-pXP318-F and AstD-pXP318-R were cloned into
pXP318 using yeast homologous recombination to give AstD-pXP318. A FLAG-tag was also
add to the N-terminal of AstD. Then, AstD-pXP318 was transformed into UB02 to generate
TY07. AstD was driven by a constitutive promoter TEF1.
Fermentation and compound analyses and isolation
A seed culture of S. cerevisiae strain was grown in 40 mL of synthetic dropout medium
for 2 d at 28°C, 250 rpm. Fermentation of the yeast was carried out using YPD (yeast
extract 10 g/L, peptone 20 g/L) supplement with 2% dextrose for 3 d at 28°C, 250 rpm.
HPLC-MS analyses were performed using a Shimadzu 2020 EVLC-MS (Phenomenex® Luna, 5μ,
2.0 × 100 mm, C-18 column) using positive and negative mode electrospray ionization.
The elution method was a linear gradient of 5–95% (v/v) acetonitrile/water in 15 min,
followed by 95% (v/v) acetonitrile/water for 3 min with a flow rate of 0.3 mL/min.
The HPLC buffers were supplemented with 0.05% formic acid (v/v). HPLC purifications
were performed using a Shimadzu Prominence HPLC (Phenomenex® Kinetex, 5μ, 10.0 × 250
mm, C-18 column). The elution method was a linear gradient of 65–100% (v/v) acetonitrile/water
in 25 min, with a flow rate of 2.5 mL/min. GC-MS analyses were performed using Agilent
Technologies GC-MS 6890/5973 equipped with a DB-FFAP column. An inlet temperature
of 240°C and constant pressure of 4.2 psi were used. The oven temperature was initially
at 60°C and then ramped at 10°C/min for 20 min, followed by a hold at 240°C for 5
To isolate compound 1, the fermentation broth of TY02 was centrifuged (5000 rpm, 10
min), and cell pellet was harvested and soaked in acetone. The organic phase was dried
over sodium sulfate, concentrated to oil form, and subjected to silica column purification
with hexane. To isolate compound 2, the fermentation broth of TY03 was centrifuged
(5000 rpm, 10 min), and supernatant was extracted three times with ethyl acetate.
The organic phase was dried over sodium sulfate, concentrated to oil form, and then
and subjected to HPLC purification. To isolate compound AA, the fermentation broth
of TY04 was centrifuged (5000 rpm, 10 min), and supernatant was extracted three times
with ethyl acetate. The organic phase was dried over sodium sulfate, concentrated
to oil form, and subjected to HPLC purification.
Structure determination of compounds
Compound 1, colorless oil readily dissolved in hexane and chloroform, had a molecular
formula C15H24, as deduced from EI-MS [M]+
m/z 204, and showed
(n-hexane; c = 0.1). GC-MS 70 eV, m/z (relative intensity): 204 [M]+ (42), 189 (5),
161 (35), 136 (100), 133 (10), 121 (70), 119 (25), 107 (20), 105 (27), 93 (21), 91
(26), 79 (13), 77 (15), 69 (20), 55 (12), 43 (12), 41 (13), 38 (21); 1H NMR (500 MHz,
CDCl3): δ 5.37 (1H, m), 2.20-2.10 (5H, m), 2.10-2.00 (2H, m), 1.95 (1H, d, 15.3),
1.75 (3H, s), 1.71 (3H, q, 1.7), 1.61 (3H, brs), 1.44 (1H, dd, 11.4, 7.2), 1.36 (1H,
m), 1.31 (1H, dd, 11.3, 2.6), 0.73 (3H, s); 13C NMR (125 MHz, CDCl3): δ 138.4, 138.3,
122.4, 122.2, 57.4, 42.6, 41.4, 40.3, 34.5, 29.6, 27.3, 25.0, 23.3, 20.6, 19.2. Both
of the NMR and MS spectrums are identical to a known compound (+)-daucane, however,
the optical rotation is opposite which led to the assignment of 1 to be (−)-daucane
Compound 2, colorless oil readily dissolved in ethyl acetate and chloroform, had a
molecular formula C15H22O3, as deduced from LC-MS [M+H]+
m/z 251, [M-H]−
m/z 249. 1H NMR (500 MHz, CDCl3): δ 8.09 (1H, brs), 3.25 (1H, t, 7.4), 2.71 (1H, dd,
14.6, 6.5), 2.48 (1H, dd, 14.8, 6.3), 2.36 (1H, dd, 14.0, 6.6), 2.26 (1H, m), 2.15
(1H, dd, 16.3, 8.9), 2.08 (1H, d, 12.0), 1.84 (1H, q, 13.1), 1.73 (3H, d, 2.3), 1.59
(3H, d, 2.2), 1.48~1.35 (3H, m), 1.31 (1H, td, 11.5, 9.0), 0.86 (3H, s). 13C NMR (125
MHz, CDCl3): δ 176.0, 135.8, 123.2, 60.1, 59.8, 59.4, 44.1, 40.5, 38.8, 30.6, 29.3,
24.9, 23.8, 20.6, 17.8.
Compound 3 is a colorless oil readily dissolved in acetone and chloroform, had a molecular
formula C15H22O4, as deduced from LC-MS [M+H]+
m/z 267, [M-H]−
m/z 265. 1H NMR (500 MHz, CDCl3): δ 4.29 (1H, d, 8.5), 3.92 (1H, d, 8.3), 3.48 (1H,
d, 8.3), 2.42 (1H, dd, 14.9, 7.3), 2.37~2.28 (2H, m), 2.25 (1H, dd, 13.0, 4.4), 2.20~2.17
(1H, m), 2.12 (1H, d, 13.4), 2.01 (1H, m), 1.80~1.65 (2H, m), 1.71 (3H, s), 1.64~1.54
(1H,m), 1.60 (3H, s), 1.50 (1H, m); 13C NMR (125 MHz, CDCl3): δ 178.2, 134.5, 125.2,
82.9, 76.3, 75.6, 55.4, 53.0, 36.6, 36.2, 33.8, 32.2, 23.6, 23.4, 20.9. 3 is identical
to aspterric acid (AA) as reported
Protein expression, purification and biochemical assay
To express and purify pDHAD, primers pDHAD-pET-F and pDHAD-pET-R were used to amplify
a 1.7 kb DNA fragment containing pdhad (AT3G23940). The PCR product was cloned into
pET28a using NheI and NotI restriction sites. The resulting plasmid pDHAD-pET was
transformed into E.coli BL21 (DE3) to give TY08. To express and purify fDHAD (XP_001208445.1),
primers fDHAD-pET-F and fDHAD-pET-R were used to amplify a 1.6 kb DNA fragment containing
fdhad. The PCR product was cloned into pET28a using NdeI and NotI restriction sites.
The resulted plasmid fDHAD-pET was transformed into E. coli BL21 (DE3) to obtain TY09.
To express and purify AstD (XP_001213593.1), primers AstD-pET-F and AstD-pET-R were
used to amplify a 1.6 kb DNA fragment containing astD. The PCR product was cloned
into pET28a using NdeI and NotI restriction sites. The resulted plasmid AstD-pET was
transformed into E. coli BL21 (DE3) to obtain TY10. All DHADs fused a 6×His-tag with
a molecular weight ~62 kD were expressed at 16°C 220 rpm for 20 h after 100 μM IPTG
induction (IPTG was added when OD600 = 0.8). Cells of 1 L culture were then harvested
by centrifugation at 5000 rpm at 4°C. Cell pellet was resuspended in 15 mL Buffer
A10 (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 8% glycerol, 10 mM imidazole). The cells were
lysed by sonication, and the insoluble material was sedimented by centrifugation at
16000 rpm at 4°C. The protein supernatant was then incubated with 3 mL Ni-NTA for
4 h with slow, constant rotation at 4°C. Subsequently the Ni-NTA resin was washed
with 10 column volumes of Buffer A50 (Buffer A + 50 mM imidazole). For elution of
the target protein, the Ni-NTA resin was incubated for 10 min with 6 mL Buffer A300
(Buffer A + 300 mM imidazole). The supernatant from the elution step was then analyzed
by SDS-PAGE together with the supernatants from the other purification steps. The
elution fraction containing the recombinant protein was buffer exchanged into storage
buffer (50 mM Tris-HCl pH 7.2, 50 mM NaCl, 10 mM MgCl2, 10% glycerol, 5 mM DTT, 5
In vitro activity assays were carried out in 50 μL reaction mixture containing storage
buffer, 10 mM (±)-sodium α,β-dihydroxyisovalerate hydrate (4) and 0.5 μM of purified
DHAD enzyme. The reaction was initiated by adding the enzyme. After 0.5 h incubation
at 30°C, the reactions were stopped by adding equal volume of ethanol. Approximately
0.1 volume of 100 mM phenylhydrazine (PHH) was added to derivatize the product 3-methyl-2-oxo-butanoic
acid (5) into 6 at room temperature for 30 min. 20 μL of the reaction mixture was
subject to LC-MS analysis. The area of the HPLC peak with UV absorption at 350 nm
were used to quantify the amount of 6. (Extended Data Fig. 2).
The inhibition percentage of AA on DHADs determined using in vitro biochemical assays
are calculated by following equation:
Growth inhibition assay of S. cerevisiae on plates or in the tubes
S. cerevisiae was grown in isoleucine, leucine and valine (ILV) dropout media (20
g/L glucose, 0.67 g/L Difco™ Yeast Nitrogen Base w/o amino acids, 18 mg/L adenine,
arginine 76 mg/L, asparagine 76 mg/L, aspartic acid 76 mg/L, glutamic acid 76 mg/L,
histidine 76 mg/L, lysine 76 mg/L, methionine 76 mg/L, phenylalanine 76 mg/L, serine
76 mg/L, threonine 76 mg/L, tryptophan 76 mg/L, tyrosine 76 mg/L) to test growth inhibition
of AA on S. cerevisiae. S. cerevisiae was incubated at 28°C until OD600 of the control
strain without AA treatment reached about 0.8. The ratio of yeast OD600 in media with
AA treatment to yeast OD600 in media without AA was calculated as the percentage of
growth inhibition. The inhibition curve was plotted as percentage of inhibition versus
AA concentrations. To further prove AA affects BCAA biosynthesis, isoleucine, leucine
and valine was also complemented to the media with or without treatment of AA. The
growth curves of TY05, TY06 and TY07 were also plotted in Extended Data Fig. 4. The
OD600 was recorded for every 20 min over a total of 50 h. Percent inhibition. The
growth inhibition percentage of AA on S. cerevisiae strain is calculated by dividing
the cell density (OD600) of the AA-treated strain to the corresponding untreated strains
when OD600 reaches ~ 0.8 using following equation:
in which 0.8 is the OD600 of untreated strain.
Growth inhibition assay of plants on plates or in the tubes
MS (2.16 g/L Murashige and Skoog basal medium, 8 g/L sucrose, 8 g/L agar) media was
used to test the growth inhibition of AA on A. thaliana, Solanum lycopersicum, and
Zea mays. A. thaliana, S. lycopersicum, and Z. mays were grown under long day condition
(16/8 h light/dark) using cool-white fluorescence bulbs as the light resource at 23°C.
AA was dissolved in ethanol and added to the media before inoculating strains or growing
plants. The media of control treatment contains the same amount of ethanol, but without
Plant growth inhibition assay by spraying
AA was firstly dissolved in ethanol and then added to solvent (0.06 g/L Finale® Bayer
Inc. + 20 g/L EtOH). The control plants were treated with solvent containing ethanol
only. A. thaliana that are resistant to glufosinate (containing the bar gene) were
grown under long day condition (16/8 h light/dark) using cool-white fluorescence bulbs
as the light resource at 23°C. Spraying treatments began upon the seed germination,
and was repeated once every two days with approximately 0.4 mL AA solution per time
Structure determination of holo-pDHAD
The gene encoding pDHAD (residues 35–608) was cloned into pET21a derivative vector
pSJ2 with an eight histidine (8×His) tag and a TEV protease cleavage site at the N-terminus.
The following primers were used for cloning: the forward primer DHAD-F and the reverse
primer DHAD-R. The double mutant K559A/K560A for efficient crystallization was designed
using the surface entropy reduction prediction (SERp) server
. Mutations were generated by PCR using the forward primer K559AK560A-F and reverse
primer K559AK560A-R. All constructed plasmids were verified by DNA sequencing.
pDHAD purified under aerobic conditions was found to contain no iron-sulfur cluster
(apo form). Hence we performed [2Fe-2S] Cluster reconstitution under the atmosphere
of nitrogen in an anaerobic box. The protein was incubated with FeCl3 at the ratio
of 1:10 for 1 h on ice and then 10 equivalents of Na2S per protein was added drop-wise
every 30 min for 3 h. The reaction mixture was then incubated overnight. Excess FeCl3
and Na2S were removed using a SephadexTM G-25 Fine column (GE Healthcare)
The reconstituted holo-pDHAD was crystallized in an anaerobic box. The proteins (at
10 mg/mL) were mixed in a 1:1 ratio with the reservoir solution in a 50 μL volume
of 2 μL and equilibrated against the reservoir solution, using the sitting-drop vapor
diffusion method at 16°C. Crystals for diffraction were observed in 0.1 M sodium acetate
pH 5.0, 1.5 M ammonium sulfate after 5 d.
All crystals were flash-cooled in liquid nitrogen after cryo-protected with solution
containing 25% glycerol, 1.5 M ammonium sulfate, 0.1 M sodium acetate pH 5.0. The
data were collected at 100K and at the Beam Line 19U1 in Shanghai Synchrotron Radiation
Facility (SSRF). Diffraction data of holo-pDHAD was collected at the wavelength of
0.97774 Å. The best crystals diffracted to a resolution of 2.11 Å. The Ramachandran
plot favored (%), allowed (%) and outlier (%) are 98.05, 1.60, and 0.36 respectively.
All data sets were indexed, integrated, and scaled using the HKL3000 package
. The crystals belonged to space group P42212. The statistics of the data collection
are summarized in Extended Data Table 1.
The holo-pDHAD structure was solved by the molecular replacement method Phaser embedded
in the CCP4i suite and the L-arabinonate dehydratase crystal structure (PDB_ID: 5J83)
as the search model. All the side chains were removed during the molecular replacement
. The resulting model were refined against the diffraction data using the REFMAC5
program of CCP4i
. Based on the improved electron density, the side chains of holo-pDHAD protein, iron
sulfur cluster, water molecule, acetate ion, sulfate ions, and magnesium ion were
manually built using the program WinCoot
. The R
work and R
free values of the structure are 17.67% and 22.15%, respectively. The detailed refinement
statistics are summarized in Extended Data Table 1. The geometry of the model was
validated by WinCoot. Structural factor and coordinate of holo-pDHAD have been deposited
in the Protein Bank (PDB code: 5ZE4).
Homology modelling of AstD and docking of substrate or AA into active site of holo-pDHAD
The structure of holo-pDHAD was prepared in Schrodinger suite software under OPLS3
. Hydrogen atoms were added to reconstituted crystal structures according to the physiological
pH (7.0) with the PROPKA tool in Protein Preparation tool in Maestro to optimize the
hydrogen bond network
. Constrained energy minimizations were conducted on the full-atomic models, with
heavy atom coverage to 0.5 Å. The homology model was performed in Modeller 9.18
, using the crystal structure of holo-pDHAD solved in this work as a template. Sequence
alignment in Modeller indicated that AstD and pDHAD shared 56.8% sequence identity
and 75.0% sequence similarity (Extended Data Fig. 6). All the highly conserved residues
and motifs were properly aligned. A total of 2000 models were generated for each target
in Modeller with the fully annealed protocol. The optimal models were chosen for docking
studies according to DOPE (Discrete Optimized Protein Energy) score.
All ligand structures were built in Schrodinger Maestro software
. The LigPrep module in Schrodinger software was introduced for geometric optimization
by using OPLS3 force field
. The ionization state of ligands were calculated with Epik tool employing Hammett
and Taft methods in conjunction with ionization and tautomerization tools
. The docking of a ligand to the receptor was performed using Glide
. We included cofactors observed in crystal structure during the docking. Since both
water and SO4
2− occupied the catalytic site, they were excluded prior to docking. Cubic boxes centered
on the ligand mass center with a radius 8 Å for all ligands defined the docking binding
regions. Flexible ligand docking was executed for all structures. Ten poses per ligand
out of 20,000 were included in the post-docking energy minimization. The best scored
pose for the ligand was chosen as the initial structure for further study. The MM/GBSA
method was introduced to evaluate the ligand binding affinity based on the best scored
docking pose in Schrodinger software. Figures are prepared in PyMOL and Inkscape
. Both of native substrate α,β-dihydroxyisovalerate and AA were docked into the catalytic
site of pDHAD. The cross-section electrostatic surface map shows this unique catalytic
pocket has a positively charged internal and a hydrophobic entrance, which binds to
negatively charged “head” and hydrophobic “tail” of substrate or AA respectively.
Thus the negatively charged “head” can lead both of the substrate and AA into the
catalytic chamber. The bulky hydrophobic tricyclic moiety of AA, however, provides
stronger hydrophobic interactions to the entrance and blocks the entrance of active
site due to the hydrophobic residues at the entrance, including G68, A71, I72, I134,
A133, M141, V212, F215, M498 and P501. In contrast, the smaller “tail” of native substrate
provides less interactions to entrance because the smaller size limits efficient hydrophobic
contact to nearby residues. This implies that once AA binds to pDHAD, it can prevent
substrate approaching the active site. We also introduced molecular mechanics generalized
Born and surface area (MM/GBSA) continuum solvation method, an widely used approach
for relative binding energy calculation, to evaluate the relative binding affinity
for both ligands
. The MM/GBSA calculations had been done in Prime
(Schrödinger 2015 suite). The MM/GBSA energy was calculated using following equation:
E denotes energy and includes terms such as protein–ligand van der Waals contacts,
electrostatic interactions, ligand desolvation, and internal strain (ligand and protein)
energies, using VSGB2.0 implicit solvent model with the OPLS2005 force field. The
solvent entropy is also included in the VSGB2.0 energy model, as it is for other Generalized
Born (GB) and Poison–Boltzmann (PB) continuum solvent models.
MM/GBSA calculation shows that the relative binding energy for AA and α,β-dihydroxyisovalerate
is −18.6 ± 0.3 kcal/mol and −13.3 ± 0.2 kcal/mol respectively, which shows the binding
constant of AA to active site is about 6000 times greater than α,β-dihydroxyisovalerate.
This further confirms that AA is a competitive inhibitor of pDHAD.
Cytotoxicity assay of AA
Cell proliferation experiments were performed in a 96-well format (five replicates
per sample) using melanoma cell line A375 and SK-MEL-1. AA treatments were initiated
24 h postseeding for 72 h, and cell survival was quantified using CellTiter-GLO assay
Cross experiment of A. thaliana
To make male sterile A. thaliana, AA was added to chemical hybridization agent (CHA)
formulation (250 μM AA, 2% ethanol, 0.1% Tween-80, 1% corn oil in water), which has
less inhibition effect on the growth of A. thaliana. Flowers of the AA treated col-0
were selected as the female parent. The non-treated A. thaliana containing a glufosinate
resistant gene were used as male parent to donate pollen. 2-week old F1 progeny resulting
from the cross were treated by Finale (11.3% glufosinate-ammonium) at 1:2000 dilution.
The results are summarized in Extended Data Fig. 9.
Construction of the transgenic plants
The coding sequence of AstD was codon optimized for A. thaliana. A chloroplast localization
signal (CLS) of 35-amino acid residues derived from the N-terminal of A. thaliana
DHAD (MQATIFSPRATLFPCKPLLPSHNVNSRRPSIISCS) was fused to N-terminus of the codon optimized
AstD. A 3×FLAG-tag was inserted between the CLS and the codon optimized AstD (Supplementary
Table 6). The gene block containing CLS, FLAG-tag and astD was synthesized and then
cloned into pEG202 vector using Gateway LR Clonase II Enzyme Mix (ThermoFisher scientific).
The original CaMV 35S promoter of pEG202 was substituted by Ubiquitin-10 promoter
to drive the expression of AstD. The construct was electro-transformed into Agrobacterium
tumefaciens strain Agl0 followed by A. thaliana transformation using the standard
floral dip method
. The A. thaliana Col-0 ecotype was transformed. Positive transgenic plants were selected
using the glufosinate resistance marker, and were tested for survival in presence
Protein expression verification with western blot
Approximately 0.5 gram of leaf tissue of transgenic A. thaliana was grounded in liquid
nitrogen. Proteins were homogenized in 2× SDS buffer followed by 5-min centrifuge
at 21,000 g to remove undissolved debris. The supernatant containing resolved proteins
were loaded onto a 4–12% Bis-Tris gel, and separated using MOPS running buffer. Transfer
was conducted using iBlot2 dry transfer device and PVDF membrane. The total proteins
were stained with Ponceau to demonstrate equal loading. Western blotting was performed
using Sigma monoclonal anti-FLAG M2-Peroxidase antibody, followed by detection using
Amersham ECL Prime detection reagent.
The data that support the findings of this study are available within the paper and
its Supplementary Information, or are available from the corresponding authors upon
Extended Data Figure 1
The rationale of resistance-gene directed discovery of a natural herbicide with new
mode of action
a, Phylogenetic tree of DHAD among bacteria, fungi and plants. The evolutionary history
was inferred by using the Neighbor-Joining method (MEGA7). Units represent the number
of amino acid substitutions per site. b, Representatives of small molecules that inhibit
DHAD in vitro, but fail to inhibit plant growth. c, Examples of co-localization of
biosynthetic gene clusters (BGCs) and targets. The biosynthetic core genes are shown
in blue and the self-resistance enzymes (SREs) are shown in red. The blockbuster cholesterol-lowering
lovastatin drug targets HMG-CoA reductase (HMGR) in eukaryotes. In the fungus Aspergillus
terreus that produces lovastatin, a second copy of HMGR encoded by ORF8 is present
in the gene cluster (top). BGC of the immunosuppressant mycophenolic acid from Penicillium
sp. contains a second copy of inosine monophosphate dehydrogenase (IMPDH), which represents
the SRE to this cluster (bottom).
Extended Data Figure 2
Biochemical assays of DHAD functions
a, Assaying DHAD activities in converting the dihydroxyacid 4 into the α-ketoacid
5. Formation of 5 can be detected on HPLC by chemical derivatization using phenylhydrazine
(PHH) to yield 6. b, LC-MS traces of the biochemical assays of A. thaliana DHAD (pDHAD).
Extracted ion chromatogram (EIC) of positive ion mass of [M+H]+=207 is shown in red.
i. The derivatization reaction was validated by using the authentic 5. ii. The bioactivity
of pDHAD in converting 4 into 5 was validated. iii. Addition of DMSO to pDHAD enzymatic
reaction mixture has no effect. iv. Addition of 10 μM AA to the reaction mixture abolished
pDHAD activity. The experiments were repeated independently for 3 times with similar
Extended Data Figure 3
Inhibition assay of different DHADs using AA
Three DHAD enzymes were assayed, including pDHAD (
lant DHAD from A. thaliana), fDHAD (
ungal housekeeping DHAD from A. terreus) and AstD (DHAD homolog within ast cluster).
IC50 and K
i values of AA were measured based on inhibition percentage at different AA concentrations.
Center values are averages, errors bars are s.d.; n = 3 biologically independent experiments.
a, Plot of the inhibition percentage of 0.5 μM fDHAD as a function of AA concentration.
b, Plot of the inhibition percentage of 0.5 μM pDHAD as a function of AA concentration.
c, Plot of the inhibition percentage of 0.5 μM AstD as a function of AA concentration.
d, Analysis of inhibitory kinetics of AA on pDHAD using the Lineweaver-Burk method
at different concentrations of AA (left). Linear fitting of apparent Michaelis constant
M,app) as a function of AA concentration yields the inhibition constant (K
i) of AA on pDHAD (right).
Extended Data Figure 4
Growth curve of S. cerevisiae ΔILV3 expressing AstD and fDHAD
The genome copy of DHAD encoded by ILV3 was first deleted from Saccharomyces cerevisiae
strain DHY ΔURA3 to give UB02. UB02 was then either chemically complemented by growth
on ILV (leucine, isoleucine and valine)-containing media or genetically by expressing
of fDHAD or AstD episomally (TY06 or TY07, respectively). The empty vector pXP318
was also transformed into UB02 to generate a control strain TY05. The optical density
of cell growth under different conditions were plotted as a function of time. Center
values are averages, errors bars are s.d.; n = 3 biologically independent experiments.
a, Growth curve in ILV dropout media with no AA. b, Growth curve in ILV dropout media
with 125 μM AA. c, Growth curve in ILV supplemented media; d, Growth curve in ILV
supplemented media with 250 μM AA.
Extended Data Figure 5
X-ray Structure of holo-pDHAD and homology model of AstD
a, Superimpositions monomer of holo-pDHAD (PDB: 5ZE4, 2.11 Å) and RlArDHT (PDB: 5J84).
The holo structure containing the 2Fe-2S cofactor and Mg2+ ion in the active site.
The structure of holo-pDHAD is in white; the crystal structure of RlArDHT is in cyan.
b, Superimpositions of holo-pDHAD and homology modeled AstD. The structure of AstD
was constructed by homology modeling based on the structure of holo-pDHAD. The structure
of holo-pDHAD is in white; the crystal structure of AstD is in green. c, The electron
density map of cofactors in the holo structure of pDHAD. White grid: 2Fo-Fc map at
1.2 σ level. Green grid: Fo-Fc positive map at 3.2σ level. Cyan sticks: acetic acid
molecule. d, Comparison of the active sites in the crystal structure of pDHAD and
the modeled structure of AstD. The cartoon represents superimposed binding sites of
pDHAD (white) and AstD (green). The shift of a loop in AstD, where L518 (correspond
to V496 in pDHAD) is located, coupled with a larger L198 residue (correspond to I177
in pDHAD) lead to a smaller hydrophobic pocket of AstD than that in pDHAD. e, The
surface of binding sites of AstD (left) and pDHAD (right). The smaller hydrophobic
channel in modeled AstD cannot accommodate the AA molecule (yellow balls-and-sticks).
Extended Data Figure 6
Sequence alignment between pDHAD and AstD
The sequence identity between pDHAD and AstD is 56.8%, whereas the similarity between
them is 75.0%. Residues were colored according to their property and similarity.
Extended Data Figure 7
Spray assay of AA on A. thaliana
Glufosinate resistant A. thaliana was treated with (right) or without (left) AA in
the solvent, which is a commercial glufosinate based herbicide marketed as Finale®.
To improve the wetting and penetration, AA was firstly dissolved in ethanol and then
added to solvent (0.06 g/L Finale® Bayer Inc. + 20 g/L ethanol) to make 250 μM AA
spraying solution. The control plants were treated with solvent containing ethanol
only. Spraying treatments began upon the seeds germination, and were repeated once
every two days with approximately 0.4 mL AA solution per time per pot for 4 weeks.
The picture shown below is taken after one month of treatment. The application rate
of AA is approximately 1.6 lb/acre, which is comparable to the commonly used herbicide
glyphosate (0.75~1.5 lb/acre). The experiments were repeated independently for 3 times
with similar results.
Extended Data Figure 8
Specific inhibition of anther development in A. thaliana
Comparison of flower organs between the AA treated (a–c) and non-treated (d–f) Arabidopsis.
a compare to d, the AA treated flower shows abnormal pistil elongation due to the
lack of pollination. b compare to e, the AA treated flower is missing one stamen.
c compare to f, the AA treated anther is depleted of healthy and mature pollen. The
experiments were performed twice with similar results.
Extended Data Figure 9
Schematic illustration of results from the cross experiment
a, Wild type A. thaliana treated with 250 μM AA was pollinated with pollen from the
un-treated plant that carries the glufosinate resistant gene. Offspring was obtained,
and inherited the glufosinate resistance from the pollen donor. b, similar as in a,
except that the pollen donor was also treated with 250 μM AA. No offspring was obtained
from this cross. Similar results were obtained with the treatment of AA at 100 μM.
Extended Data Table 1
Data collection and refinement statistics (molecular replacement).
a, b, c (Å)
135.5, 135.5, 66.0
α, β, γ (°)
90, 90, 90
sym or R
Bond lengths (Å)
Bond angles (°)
Values in parentheses are for highest-resolution shell.