Eiffcient Extracellular Expression Of Inuhn Fructotransefrase In Pichia Pastoris Under The Regulation Of Promoters

Difructose anhydride III (DFA III) is a novel low-caloric sweetener sweetener substitutewhich can promote mineral absorption, accelerate bone formation, diuretic action and preventtooth decay. It was an additive in baked foods, beverage, candy, as well as in pharmaceuticalformulations thanks to its characteristics of high sweetness and low energy value. Inulinfructotransferase (IFTase) can hydrolysis inulin into DFA III with biodegradation method.However, IFTase expressed by the wild-type strain or E. coli has the characteristics of lowextracellular production, which could not meet the needs of large-scale applications due to thecomplexity of the downstream process and greater capital investment. Therefore, the mainpurpose of this work is to obtain efficient extracellular expression of IFTase in Pichia pastoris(P. pastotis) system with inducible or constitutive promoters, which has importantsignificance for the industrial application of IFTase for DFA III production.In this paper, three expression strains harboring ift gene under the regulation of Alcoholoxidase1(AOX1), Formaldehyde dehydrogenase1(FLD1), or Glyceraldehyde-3-Pdehydrogenase (GAP) promoter were constructed, the impact of the ift copy numbers insertedinto GS115genome and shaking flask cultivation conditions of P. pastoris were also studied.Fed-batch fermentation of three kinds recombinants were implemented in3L fermenters withhigh cell density fermentations. Finally, IFTase achieved efficient secretion expression in P.pastoris. The main works and results are as follows:Ift gene was inserted into the vector pPIC9K containing an inducible promoter AOX1(PAOX1) or promoter FLD1(PFLD1), or the vector PGAPZαA containing a constitutivepromoter GAP (PGAP). These recombinant vectors were then electroporated into the genomeof P. pastoris GS115. Inducible recombinant yeast GS115-PAOX1-IFTase andGS115-PFLD1-IFTase with different ift copy numbers were screened using G418gradientconcentration method, while constitutive recombinant P. pastoris GS115-PGAP-IFTase werecarried out using antibiotic Zeocin. Notebly, the capacity to IFTase secretion ofGS115-PAOX1-IFTase and GS115-PGAP-IFTase were tremendous affected by ift copynumbers inserted into GS115genome, and strains with multicopy numbers exhibited a higheractivity; However, GS115-PFLD1-IFTase strain was weakly affected by the ift copy numbers,only strains with1mg/mL G418reactance showed slightly higher IFTase activity, indicatingthat protein activity and the copy numbers of the exogenous gene does not always exhibit apositive correlation.Shake flask level studies of recombinant P. pastoris showed: IFTase secreted byGS115-PAOX1-IFTase was affected by many factors with methanol induction. The optimumconditions were: initial biomass OD600=7,10%medium volume, supplementary of1.5% methanol of total volume per24h, cultivation medium, temperature and speed were pH6.5,30oC and250rpm, respectively. And after induced for96h, the highest IFTase activity ofGS115-PAOX1-IFTase reached11.92U/mL; Both methanol and methylamine hydrochloridecan be used as an inducer of recombinant P. pastoris GS115-PFLD1-IFTase for IFTaseexpression in four mediums: methanol-methylamine hydrochloride, methanol-ammoniumsulfate, glycerine-methylamine hydrochloride and glucose-methylamine hydrochloride, andIFTase activity were greatly affected by P. pastoris cell density. Unlike most of the literaturereported, using methylamine hydrochloride as the inducer, both glycerol and glucose can alsobe an effective carbon source for IFTase expression by PFLD1-controlled P. pastoris. Theinitial concentration of glycerol and glucose are required to strictly control in low range(respectively:0.5%-1.5%and1%-1.5%), as high concentrations can result in excessive cellgrowth and decrease the IFTase yields. Low Cell density is the reason for IFTase activity ofGS115-PFLD1-IFTase with double induction of methanol-methylamine hydrochloride. Addwith auxiliary nitrogen source of yeast extract/tryptone (Y/P) can effectively solve thisproblem; P. pastoris GS115-PGAP-IFTase can achieve secretory expression of IFTase inmediums using glycerol or glucose as carbon source without any inducer. Overgrowth ofthese constitutive recombinant yeast is the main reasons for low expression levels of IFTase,so the initial concentrations of glycerol and glucose should be controlled at2%-4%and4%-5%, respectively.Recombinant P. pastoris strains were cultivated in3L fermentor with fed-batchhigh-density fermentation strategy in mineral salt medium and achieved efficient extracellularexpression of IFTase. PAOX1-controlled GS115-PAOX1-IFTase showed the strongestcapacity for IFTase extracellular expression, with the highest IFTase activity of105.4U/mL,which was5.4times and1.3times higher than expressed by wild bacteria and E. coli system,respectively; Under the conditions of high density fermentation, the biomass of yeast cells ofGS115-PFLD1-IFTase was greatly improved. This PFLD1-controlled P. pastoris with doubleinduction of methanol-methylamine hydrochloride exhibited higher IFTase production thaninduced by single inducer, and the extracellular IFTase activity reached62.72U/mL whichwas3.2times of the wild enzyme. The IFTase activity of this recombinant yeast under carbonsource of glycerol or glucose in3L fermenter increased by4.3times and7.2times than inshake flasks cultivations; With phase-regulation and fed-batch strategy, PGAP-controlledGS115-PGAP-IFTase yeast achieved larger increase of IFTase extracellular expression whichwas7.7-fold and8.3-fold of shake flask levels, and was1.7times and2.2times of the wildenzyme, respectively.Crude IFTase enzymes secreted by inducible strains can be purified with ultrafiltrationand DEAE ion-exchange chromatography, and constitutive strain can be directly purified withNi-chromatography. All of the purification products exhibited single protein band. Studies of characterization of recombinant IFTase showed that its optimum temperature and pH were60oC and6.5, respectively. The stability of temperature and pH was also little difference with thewild enzyme. Our studies indicated that P. pastoris could be an efficient secretory system forIFTase extracellular expression.