Results
Cloning and bioinformatics analysis of EGT synthase
gene
Cloning and homology analysis of EGT synthase
gene
Based on the amino acid sequences of NcEgt2 and GfEgt2from N. crassa and G. frondosa , three hypothetical
proteins with certain similarity were obtained by Protein Blast in the
protein database of P. eryngii . The specific protein information
is as follows: PLP-dependent transferase [P.
eryngii ](KAF9497900.1), PLP-dependent transferase [P.
eryngii ](KAF9497887.1), PLP-dependent transferase [P.
eryngii ](KAF9493115.1). The corresponding genes were named asPeEgt2a , PeEgt2b and PeEgt2c , which were 1290 bp,
1290 bp and 1245 bp, respectively. The coverage/similarity ofNcEgt2 amino acid sequence alignment with N. crassa was
94%/36.95%, 93%/37.78% and 96%/36.64%, respectively. The
coverage/similarity of GfEgt2 amino acid sequence alignment withG. frondosa was 94%/53.65%, 94%/50.99% and 93%/50.56%,
respectively. Our previous studies have mined and clonedPeEgt1 [30] (MAZY01000061.1) in the EGT
synthesis pathway of P. eryngii .
Because P. eryngii is a eukaryote, most of the DNA sequence
corresponding to its expressed proteins have introns. When
heterologously expressed in other model species, whole exon genes should
be used. Therefore, it is necessary to extract the total RNA of P.
eryngii (Supplementary data:
Fig. S1). DNase was not used to
process the sample during RNA extraction, so there will be a shallow
band above the gel electrophoresis
picture for genomic DNA. In the subsequent reverse transcription
process, gDNA Remover will be added to remove genomic DNA. Then, the
cDNA was obtained by reverse transcription with total RNA as template,
and then the potential EGT synthase genes were amplified with cDNA as a
template. The results are shown in Fig. S1, and the bands ofPeEgt2a , PeEgt2b and PeEgt2c were about 1300 bp,
which were consistent with the expected band size. The target genes were
recovered by cutting gel.
After adding A to the end of the recovered products, they were connected
to the T vector using the pMD19-T cloning vector kit, and then
transformed into E. coli DH5α. The transformants were selected
and identified by colony PCR using the universal primers M13-47/RV-M.
The correct transformants were cultured and the plasmids were extracted
for sequencing. The sequencing results showed that the lengths ofPeEgt2a and PeEgt2b are 1290 bp, and the length ofPeEgt2c is 1245 bp, both of which are complete CDS.
In P. eryngii , the nucleotide similarity of PeEgt2a ,PeEgt2b and PeEgt2c was 72.57 %, and the amino acid
similarity was 69.86 %, as shown in Fig. S2 and Fig. S3. The nucleic
acid sequence and amino acid sequence of Egt1 gene of P.
eryngii and Pleurotus ostreatus and Pleurotus nebrodensiswere highly homologous. The nucleic acid sequence homology was 97.03 %,
and the amino acid sequence homology was as high as 97.93 %[30].
Physicochemical properties analysis and structure
prediction of EGT
synthase
The physicochemical properties were analyzed by ExPASy ProtParam as
follows: PeEgt2a , PeEgt2b and PeEgt2c were composed
of 429, 429 and 414 amino acids, respectively, and the molecular weights
were 47.99, 47.55 and 46.12 kDa, respectively. The isoelectric points
were predicted to be 6.6, 6.53 and 6.06, respectively. The total
negatively charged residues (Asp + Glu) were 44, 41 and 46,
respectively, which were higher than the total positively charged
residues (41, 38 and 38, respectively). The predicted instability
indexes were 33.08, 39.48 and 40.25, respectively, indicating thatPeEgt2a was relatively stable. In yeast, the estimated half-life
was greater than 20 h. The total fat indexes were 89.77, 90.44 and
85.05, respectively. The average values of hydrophilicity were -0.179,
-0.09 and -0.138, respectively. The hydrophilicity of the protein was
analyzed by ExPASy ProtScale. PeEgt2a , PeEgt2b , andPeEgt2c were the most hydrophobic between the 150-200 amino
acids, located at 181 and 182, 181, 161, respectively. As shown in Fig.
S4, the greater the ordinate positive value, the stronger the
hydrophobicity; the smaller the negative value, the stronger the
hydrophilicity, and the hydrophilic region in the figure is larger than
the hydrophobic region. According to the predicted average
hydrophilicity, it can be seen that PeEgt2 belongs to hydrophilic
protein.
PredictProtein was used to predict the secondary structure of EGT
synthases, as shown in Fig. S5. PeEgt2a , PeEgt2b andPeEgt2c were mainly random coil, accounting for 48.72%, 53.15%
and 48.79% respectively. Followed by α-helix, accounting for 37.76%,
34.27% and 37.44% respectively. The β-sheet was the least, accounting
for 13.52%, 12.59%, and 13.77%, respectively.
Through CD-SEARCH prediction analysis of domains and superfamilies,PeEgt2a , PeEgt2b , and PeEgt2c all contain CsdA
domains and belong to CsdA superfamily members. They are described as
selenocysteine lyases or cysteine desulfurases that mainly play a role
in amino acid transport and metabolism in cells. Using SWISS-MODEL, the
protein tertiary structure was constructed by searching homologous
templates, as shown in Fig. S6.
Expression of EGT synthase in S.
cerevisiae
Construction of Egt1 single-gene
engineered
strains
The integration site of PeEgt1 was selected as XI-5, and the
corresponding knockout plasmid was pQc009. After extracting IMX581
genome and plasmid pQc009, the product was tested by 1%
agarose gel electrophoresis, as
shown in Figure 1. There is a band at the top of the lane 1 and 2, which
is the genome of S. cerevisiae , and the bottom diffuse band is
degraded RNA. Using S. cerevisiae genomes as template, the
amplification results showed that
upstream homologous arm Ⅺ-5-us was 663 bp, promoter TEF1p was 412 bp and
downstream homologous arm Ⅺ-5-ds was 468 bp; using pYES2 as template,
the amplification result showed that terminator CYC1t was 248 bp; the
length of PeEgt1 was about 2600 bp, which was amplified by
primers plus their homologous arms. As shown in Figure 1, the PCR
products corresponded with the expected band sizes. After the above
bands were cut and recovered, the five fragments were assembled into the
homologous repair fragment XI-5-us-TEF1p-PeEgt1 -CYC1t-XI-5-ds of
the PeEgt1 expression frame by overlap extension PCR, as shown in
Figure 1, the length is about 4500 bp. There are many miscellaneous
bands below the target band, because the annealing temperature is
uncertain during the overlap extension, so the method of adding 0.5°C
per cycle is adopted. There may be some base pairing between the five
short segments. The high temperature leads to duplex unwinding, and the
low temperatures, these complementary fragments may be mismatched; in
addition, the primers may also have individual bases combined with the
template, resulting in base mismatch, so it is necessary to cut the gel
to recover the purified product.
The knockout plasmid pQC009 and
homologous repair fragment XI-5-us-TEF1p-PeEgt1 -CYC1t-XI-5-ds
were co-transformed into S.cerevisiae competent cells. After
resuscitation, they were evenly coated on Sc-Ura auxotrophic medium for
screening. The results are shown in Figure 2. Multiple single colonies
grow on the transformation plate, and the negative control did not grow.
Because the transformed strain contained the plasmid pQC009 and the
expression frame of Ura3, it could grow on the Sc-Ura plate, while the
control strain did not contain the knockout plasmid, it was still a Ura
auxotrophic strain and could not grow on Sc-Ura auxotrophic plates.
Using homologous arm primers on the genome, single colonies were
selected for PCR identification to ensure successful transformation of
the engineered strain.
The protein of the engineered strain IMX581-PeEgt1 and the
original strain IMX581 were extracted respectively. The results of
SDS-PAGE were shown in Figure 2. The protein size of PeEgt1 was
96.8 kDa [30]. There was no significant difference
in the expression protein between the engineered strain and the original
strain, which may be due to the low expression of the protein. It is
necessary to verify the activity of PeEgt1 by detecting EGT by
HPLC.
By HPLC detection, as shown in
Figure 3, the peak of EGT standard substance appeared at 21.077 min.
Control strain IMX581 did not produce EGT without the integration of EGT
synthetase gene. The peak of IMX581-PeEgt1 indicated thatS. cerevisiae could synthesize EGT only by integratingPeEgt1 , which proved that PeEgt1 was active. After 7 days
of fermentation, the EGT yield of IMX581-PeEgt1 was 2.63±0.05
mg/L.
Construction of double-gene engineered
strains
The three potential PeEgt2were integrated into the IMX581-PeEgt1 which had lost the
knockout plasmid to construct the double-gene engineered strains. X-2 of
IMX581 was selected as the integration site of PeEgt2 gene
expressing strain, and the corresponding knockout plasmid was pQc029.
The homologous recombinant fragments were amplified, and the five
fragments were assembled by overlap extension PCR. Three
X-2-us-TDH3p-PeEgt2 -TEF1t-X-2-ds five-fragment homologous repair
templates were constructed, respectively, as shown in Fig. S7.
The above homologous repair
templates and the knockout plasmid pQc029 were co-transformed into the
IMX581-PeEgt1 competent cells by electroporation. After
resuscitation, they were coated on the Sc-Ura auxotrophic medium. The
transformants were picked up and the integration of the two genes was
verified by colony PCR with homologous arm primers. The successfully
transformed double-gene engineered strains were IMX581-PeEgt1 -2a,
IMX581-PeEgt1 -2b , IMX581-PeEgt1 -2c . After 7
days of fermentation, as shown in Fig. S8, the yield of EGT was
increased to 4.04±0.23 mg/L and 4.30±0.23 mg/L by integratingPeEgt2a and PeEgt2b , respectively, which increased by
53.61% and 63.50%. After integrating PeEgt2c , the yield of EGT
was 2.63±0.09 mg/L, which was not significantly different from that of
IMX581-PeEgt1 . The results showed that PeEgt2c had no
enzyme activity, while PeEgt2a and PeEgt2b had enzyme
activity.
PeEgt2a , PeEgt2b andPeEgt2c were separately integrated into the S.cerevisiaeIMX581 to construct the IMX581-PeEgt2 single-gene engineered
strains. EGT was not detected after fermentation, indicating that only
the single-gene engineered strains containing PeEgt2 could not
synthesize EGT.
Construction of multi-gene engineered
strains
In fungi, the synthesis pathway of
EGT only requires the participation of two genes Egt1 andEgt2 , but three potential PeEgt2 genes are obtained by
bioinformatics mining in P. eryngii . Based on the above
construction of double-gene engineered strains, we speculate thatPeEgt2a and PeEgt2b have enzyme activity, andPeEgt2 contains at least two isozymes. Therefore, we further
constructed a triple-gene engineered strain to explore the significance
of the presence of multiple PeEgt2 genes. The XI-3 was selected
as the knockout site, and the corresponding knockout plasmid was pQc006.PeEgt2b was integrated into IMX581-PeEgt1 -2a, which had
lost the knockout plasmid, to obtain triple-gene engineered strain
IMX581-PeEgt1 -2a -2b . After 7 days of fermentation,
the EGT yield was 4.45±0.15 mg/L, which was not statistically
significant compared with the yield of double-gene engineered strains. A
total of eight engineered strains were constructed, and their EGT
production was summarized in Table 1.