Editing Yeast Fermentation (section)

=== Construction and enzyme activity of the substrates ===

Recombinant ''P''. ''pastoris'' strains expressing endo-1, 4-β-mannosidase from ''Aspergillus niger'', DNase I from ''Bos taurus'', phytase from ''E''. ''coli'' and ''Dpn''I from ''Diplococcus pneumoniae'' were constructed in our lab. The recombinant strains expressing substrates were generated with the similar strategy as Endo H-P expressing ''P''. ''pastoris''. The coding sequences of these genes were optimized according to the codon usage preference of ''P''. ''pastoris'' and synthesized with overlapping PCR. The newly synthesized ORFs were sequenced and inserted into pHBM905A vectors, followed by digesting with ''Sal''I and transforming into ''P''. ''pastoris'' GS115. The recombinants were screened on MD plates without histidine and identified with PCR [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362766/#pone.0120458.ref016 16]]. The expressions of the foreign proteins were identified with SDS-PAGE.
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