Conclusion
Combined with our previous study, a total of four EGT synthetases were
mined in P. eryngii , which were PeEgt1 , PeEgt2a ,PeEgt2b , and PeEgt2c . Using CRISPR/Cas9 technology, theEgt1 and Egt2 genes of P. eryngii were integrated
into the genome of S. cerevisiae IMX581, and eight engineered
strains of S. cerevisiae were successfully constructed, including
four single-gene engineered strains:
IMX581-PeEgt1 , IMX581-PeEgt2a , IMX581-PeEgt2b ,
IMX581-PeEgt2c ; three
double-gene
engineered strains:
IMX581-PeEgt1 -2a , IMX581-PeEgt1 -2b ,
IMX581-PeEgt1 -2c ; one triple-gene engineered strain:
Imx581-PeEgt1 -2a -2b . It has been found that EGT can
be generated when only the PeEgt1 expression box is integrated;
on this basis, the integration of PeEgt2a or PeEgt2bincreased EGT production by 53.61% and 63.50%, respectively, compared
with single-gene engineered strain. In this study, the EGT synthase
genes mined from P. eryngii was used to construct the synthesis
pathway of EGT in S. cerevisiae and verify its function, which
laid a foundation for further improving the synthesis pathway of EGT in
edible fungi and providing effective genes for the construction of
engineered strains with high expression of EGT.