Materials and methods
Materials
Strains and plasmid
P. eryngii (P. eryngii Pe821) was purchased from Shouguang
Institute of Edible Fungi, Shandong Province; DH5α-TOPO-PeEgt1engineered strain, containing PeEgt1 gene from P. eryngii ;E. coli DH5α, preserved in our laboratory; S. cerevisiaeIMX581, a Ura auxotrophic strain, has integrated Cas9 gene into its
genome; pMD19-T cloning vector, purchased from TAKARA; pRS42K is aS. cerevisiae expression vector, presented by Professor Christof
Taxis of the European Molecular Biology Laboratory, for amplification of
terminator TEF1p; the pYES2 vector was preserved in our laboratory and
used to amplify the terminator CYC1t.
Reagent and medium
Tool enzyme: 2 × Rapid Taq Master
Mix、Phanta Max Super-Fidelity DNA Polymerase
was purchased from Nanjing Vazyme
Biotech Co.,Ltd.. Test kits: HiPure PCR Pure Micro Kit, HiPure Plasmid
Micro Kit was purchased from
Magen Biological Co., Ltd.; TransScript One-Step gDNA Removal and cDNA
Synthesis SuperMix was purchased from Beijing TransGen Biotech Co.,
Ltd.; pMD™19-T Vector Cloning Kit was purchased from Takara Bio Inc..
The EGT standard was purchased from Tianjin Zhongkenuoshi Biotechnology
Co., Ltd..
YPD medium: 10 g peptone, 5 g yeast
extract, 10 g glucose, constant volume to 1 L; YPD solid medium was
supplemented with 2% agar powder. Sc-Ura medium: 0.1 g adenine,
arginine, cysteine, leucine, lysine, threonine, tryptophan, 0.05 g
aspartic acid, histidine, isoleucine, methionine, phenylalanine, serine,
tyrosine, proline, valine, 6.7 g YNB (Yeast nitrogen base), 20 g agar
powder, added 0.9 L distilled water, sterilized at 115°C for 15 min,
added 0.1 L sterilized 20% glucose solution before use; Sc-All medium:
on the basis of the above, 0.1g of uracil (Ura) was added.
Methods
Mining and cloning of EGT synthesis genes
in P.
eryngii
The reported EGT synthase NcEgt2 (NCU11365) from N. crassaand EGT synthase GfEgt2 (A0H81_07972) from G. frondosawere used as templates sequences to identified homologous proteins inP. eryngii through NCBI and GeneBank protein databases.
After the P. eryngii strains were inoculated in PDB (28℃, 150
rpm, 7 d), the fresh mycelial pellets were used to extract total RNA by
STE method [31]. Total RNA was reverse transcribed
into cDNA using the reverse transcription kit. Primers were designed and
synthesized using potential homologous proteins as template (The
specific primer information is shown in Supplementary data: Table S1).
The cDNA was used as a template for PCR amplification, and then the
products was digested and recovered
using the gel electrophoresis recovery kit. Since the target gene
cloning uses high-fidelity polymerase, the amplification products are
blunt end. In order to be able to TA cloning, 2 × Rapid Taq Master Mix
was used to add A to the end of the target gene, and then connected with
the pMD19-T cloning vector.
Then the constructed vector was transformed into E. coli DH5α
competent cells. After the transformants were picked on the plate for
colony PCR identification, the correct transformants were sent to
Beijing Tsingke Biotechnology Co., Ltd. for sequencing.
Construction of EGT single-gene engineered
strains
The Cas9 gene has been integrated
into the genome of S. cerevisiae IMX581, which can express Cas9
protein. After the method of Yueping Zhang et al.[32] was improved, the competent cells of S.
cerevisiae were prepared and the electrotransformation conditions were
referred to. Using CRISPR/Cas9 gene editing technology, the existing
knockout sites of IMX581 were selected to construct homologous repair
templates and knockout plasmids. The two were co-transformed into IMX581
by electroporation. Under the action of homologous recombination repair
mechanism, the target gene was integrated into the genome ofS. cerevisiae . Then, the
transformants were screened by Sc-Ura auxotrophic medium, and the
transformants were identified by colony PCR. The correct transformants
were selected and cultured in YPD to obtain the fermentation broth, and
the protein was extracted. SDS-PAGE gel electrophoresis was used to
identify whether EGT synthase was expressed in S. cerevisiae .
The integration sites were XI-5, X-2 and XI-3 of S. cerevisiaeIMX581, and the corresponding knockout plasmids were pQc009, pQc029 and
pQc006. Homologous repair template construction: using the genome ofS. cerevisiae IMX581 as a template, high-fidelity polymerase was
used to amplify the upstream and downstream homologous arms of the
integration site (XI-5-us and XI-5-ds, X-2-us and X-2-ds, XI-3-us and
XI-3-ds) and the promoters TEF1p, TDH3p, PGK1p; the terminators CYC1t
and TEF1t were amplified using pYES2 plasmid and pRS42K plasmid as
templates. The EGT synthase genes were amplified by using the correctly
sequenced cloning vector as templates. Five fragments of upstream
homologous arm, promoter, target gene, terminator and downstream
homologous arm were assembled by overlap extension PCR.
Construction of EGT double- and triple-gene engineered
strains
S. cerevisiaeIMX581 is a Ura auxotrophic strain
and cannot grow in Sc-Ura medium. The
URA3 gene is a gene on the yeast
chromosome V, which encodes orotidine 5-phosphate decarboxylase. If the
enzyme is inactivated, the yeast cannot grow unless the medium contains
uridine or uracil. If S. cerevisiae IMX581 was transferred to
knockout plasmid containing URA3 expression frame, the successfully
transformed strain could grow in Sc-Ura auxotrophic medium. At this
time, if 5-fluoroorotic acid (5-FOA) is added to the medium, the
orotidine 5-phosphate decarboxylase of the URA3 gene can convert 5-FOA
into toxic substances (such as 5-fluorouracil, which can cause cell
death), and strains containing knockout plasmids cannot grow; however,
the free plasmid has the probability of loss. If the successfully
transformed engineered strains can grow in the medium containing 5-FOA,
indicating that the plasmid has been lost, and the Ura auxotrophic
strain is obtained again, the same Ura auxotroph screening method can be
used to integrate the foreign gene into other sites of the genome.
The method of knockout plasmid loss was as follows: The single colonies
with successful transformation were inoculated in liquid YPD medium
containing 800 mg/L 5-FOA and cultured overnight (30°C, 200 rpm). The
above bacterial liquid was streaked on the Sc-All nutrient complete
solid medium containing 800 mg/L 5-FOA, and cultured at 30°C for 2 days.
Single colonies were picked and cultured on Sc-Ura and Sc-All plates at
30°C for 2 days. And if it could not grow on the Sc-Ura plate and grew
normally on the Sc-All plate, the plasmid could be initially judged to
be lost.
Then the primers at both ends of the homologous arm of the integration
site were used for colony PCR to identify whether the exogenous fragment
was successfully integrated, whether the knockout plasmid was
successfully lost. After the success, the preparation of competent cells
and the integration of exogenous genes were performed again.
The specific integration operation of double- and triple-gene engineered
strains was the same as that of single-gene engineered strain. After
successful integration, EGT was fermented and extracted, and its content
was detected by HPLC.
EGT extraction and
detection
The engineered strains and the
control strain were streaked on the YPD plate and cultured at 30°C for 2
days. Single colonies were picked up and cultured in 50 mL liquid YPD
medium overnight (30°C, 200 rpm) to obtain seed liquid. Then
the bacterial liquid was transferred
to a 50 mL centrifuge tube, centrifuged (5000 rpm, 2 min, 25°C),
discarded the supernatant, added an appropriate amount of
ddH2O to re-suspend the yeast, and adjusted the
OD600 value of the bacterial liquid to 1.0. After that,
1 mL of seed liquid was inoculated into 50 mL liquid YPD medium for 7
days (30°C, 200 rpm), three parallel.
Extraction of EGT: 1 mL fermentation broth was centrifuged in a 1.5 mL
centrifuge tube (5000 rpm, 5 min, 25°C). The supernatant was discarded,
1 mL 50% ethanol was added, and the cells were resuspended. After
overnight extraction at 4°C, the supernatant obtained by centrifugation
(5000 rpm, 5 min, 25°C) was EGT crude extract. Finally, the EGT yield
was detected by HPLC after filtration with 0.22 μm organic filter
membrane.
HPLC analysis of EGT:
chromatographic column: Ultimate® HILIC AmphionⅡ
(specification: 4.6 mm×150 mm, inner diameter: 5 μm); mobile phase: 80
% acetonitrile and 20 % ultrapure water; injection volume: 20 μL; flow
rate: 0.9 mL/min; detection wavelength: 254 nm; column temperature:
30°C.
Bioinformatics and statistical
software
DNAMAN 8 was used for DNA sequence
and amino acid sequence alignment. Analysis of protein primary structure
by ExPASy ProtParam. PredictProtein predicts protein secondary
structure. CD-SEARCH predicted the domain and superfamily of proteins.
SWISS-MODEL predicted the tertiary structure of the protein and
performed homology modeling. Excel 2019, Graphpad Prism 9 for data
processing and drawing.