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.