Cloning And Expression Of Maltooligosyltrehalose Trehalohydrolase In Pichia Pastoris From Micrococcus Roseus QS412

Trehalose has been widely used in the fields of cell,organ,food,cosmetic and medicine preservation.Several methods have been developed for the production of trehalose.Thereinto,a two-enzyme system consisting of MTSase and MTHase can directly transfer starch into trehalose and is a very promising method for trehalose production in large scale.In this study,we constructed recombinant expression vector pPICZαA-MTH and investigated the secretory expression of MTHase in Pichia pastoris GS115 and KM71 strains.First,according to the DNA sequence of TreZ gene,specific primers were synthesized for the amplification of target gene.After PCR reaction with Micrococcus roseus genome as template,we got a 1.9Kb DNA fragment.The fragment was ligated with pMD 18-T vector,and then transfered into E.coli DH5α. Pick the white colonies for colony PCR.The positive recombinants were further identified by DNA sequencing and the result was the same as reported. Then,the target fragment was digested by EcoRⅠand XbaⅠrestriction enzymes,and ligated directionally to pPICZαA plasmid which was digested by the same enzymes.The recombinant plasmid pPICZαA-MTH was transformed into Pichia pastoris GS115 and KM71 strains after linearization.The multi-copy recombinants were selected on the plates containing Zeocin of different concentrations.Finally,we got several colonies which could grow well on plate containing 1000μg/ml Zeocin.We conformed the phenotype of GS115 recombinants by observing the difference of growth rate on MM and MD plates,and found that they were all Mut~+.Further,we investigated the expression of foreign gene in these recombinants.We measured the growth curve of GS115/pPICZαA-MTH and KM71/pPICZαA-MTH in BMGY culture medium and determined the best time for transferring was 20h and 25h,respectively.We also measured the total protein variation curve with incubation time in BMMY medium,and determined the proper induction time was 72h.SDS-PAGE results showed that there was clear protein band of about 72.5KD for KM71/pPICZαA-MTH,while there was not for GS115/pPICZαA-MTH.Western blot result of KM71/pPICZAα-MTH sample showed one single band,while GS115/pPICZαA-MTH and control showed nothing which further MTHase has successfully expressed in Pichia pastoris KM71 strain,but failed in Pichia pastoris GS115 strain. In the end,we tried to explain the reason for the silence of TreZ gene in GS115 strain.RNA of GS115/PICZαA -MTH was extracted and then RT-PCR was performed.The result showed that TreZ gene has successfully transcribed in GS115/PICZAα-MTH.It is some other problems on the translation level that leads to the silence of TreZ gene.