Results
Biodegradation
metabolites of 2,4-DNT and DNTS transformed by NfsI in vitro
We initially conducted codon optimization forEnterobater
cloacae NfsI to improve its expression in switchgrass (Fig.
S1 ). Thereafter, we produced the recombinant NfsI in E.coli and
examined the biodegradation metabolites of 2,4-DNT by LC-PDA/ESI-MS/MS.
Our results showed that the NfsI
transformed 2,4-DNT in
2/4-hydroxyl
amino-4/2-mononitrotoluene (HAMNT, Peak 1) through a two-electron
reduction (Fig. 1A, Fig. S2A, E ). Furthermore, HAMNT was
reduced to 2/4-amino-4/2-mononitrotoluene (AMNT, Peak 2) or formed a
novel compound by autonomous polymerization (Peak
3) (Fig. 1A, B, Fig. S2B,
C, F, Fig. S3 ). In addition, we analyzed the
biodegradation metabolites of DNTS
and found that the NfsI only transformed DNTS into 4-hydroxyl
amino-4/2-mononitrotoluene-3-SO3-(HAMNTS, Peak 4) (Fig. 1C,
D, Fig. S2D, G ).
NfsI had higher catalytic efficiency toward2,4-DNT than DNTS in
vitro
To evaluate the catalytic
efficiency of NfsI toward 2,4-DNT and DNTS, we next examined NfsI enzyme
kinetic characterization in vitro . Compared with DNTS,
the NfsI had lower Kmtoward 2,4-DNT (256.9 ± 68.9 μM vs. 2814.1 ± 586.9 μM), while it had
higher Vmax toward 2,4-DNT (183.0 ± 18.3
μM·s-1·mg-1 vs. 19.4 ± 1.6
μM·s-1·mg-1) (Table 1 ).
Moreover, the NfsI had higher Kcat/Km toward
2,4-DNT (6.83 ± 0.29) than DNTS (0.07 ± 0.22), suggesting that the NfsI
is highly efficient at detoxifying 2,4-DNT rather than DNTS in
vitro (Table 1 ). Thus, we selected 2,4-DNT as the NfsI
substrate for further studies.
Effects of pH value and
NADPH concentration on NfsI enzymatic activity
Both pH value and NADPH concentration had significant effects on NfsI
enzymatic activities in vitro . Therefore, we next decided to
examine the optimum pH and NADPH concentration for NfsI toward 2,4-DNT.
Our results showed that NfsI enzymatic activities initially increased
and then decreased from pH 4.0 to 9.0. The optimum pH was around 6.0 at
which the NfsI reached its highest activity (Fig. 2A ). We
further studied the effect of NADPH concentration on NfsI enzymatic
activity at pH 6.0. The NfsI activity was dramatically elevated as the
NADPH concentration increased from 50 μM to 200
μM until a limiting rate was
reached (Fig. 2B ).
Generation and identification ofNfsI overexpressing
transgenic switchgrass plants
To improve uptake efficiency of
2,4-DNT and DNTS in plants, we overexpressed the codon optimizedNfsI in switchgrass plants.
All transgenic lines were produced from a single genotypic embryogenic
switchgrass callus line through Agrobacterium -mediated
transformation, which excluded the potential influence of the genetic
background of switchgrass on 2,4-DNT and DNTS tolerance as well as
biodegradation. Eighteen independent positive transgenic switchgrass
lines were identified by genomic PCR with NfsI specific primers.
The control plants were generated with the pANIC6B empty vector which
was used as the backbone for constructing NfsI -overexpressing
vector. There was no obvious morphological or developmental difference
between transgenic and control switchgrass plants in our greenhouse
condition (Fig. 3A ).
Overexpression
of NfsI in switchgrass enhanced plant tolerance against 2,4-DNT
We next decided to study if engineeringNfsI in switchgrass has
potential for remediation of 2,4-DNT. Two transgenic lines
NfsI_OE-02 and -14 with highestNfsI transcript abundance were selected for further investigation
(Fig. 3B ). Firstly, we regenerated numerous
plantlets from wild type, control,
and transgenic switchgrass plants by immature inflorescence-derived
callus cultures. These uniform plants were employed for examining the
tolerance to 2,4-DNT. Our results showed that 2,4-DNT had significant
impacts on the growth and development of wild type switchgrass plants.
They exhibited obvious etiolation and wilting symptoms after 14 days
when incubated with more than 5 mg·L-1 2,4-DNT
(Fig. S4A ). In addition, as 2,4-DNT concentration increased
from 2 to 40
mg·L-1, their
root length decreased gradually, while the ROS content increased
(Fig. S4B, C ). Furthermore, we measured the root length and ROS
content ofNfsIoverexpressing transgenic switchgrass plants. Compared with control
plants, the root length of transgenic switchgrass plants increased by
32.2-46.1%, and their ROS contents decreased by 21.8-35.6% at a high
2,4-DNT level (20 mg·L-1) (Fig. 4 ).
Therefore, our results indicate that the NfsI overexpressing
transgenic switchgrass plants have a higher capacity to tolerate 2,4-DNT
than control plants.