Cell-surface Display Of UGT76G1from Stevia Rebaudiana In Pichia Pastoris

Steviol glycosides, a natural sweetener which have high sweetness potency, are widelyused in the field of food, beverage, wine, medicine, daily chemical industry because of itsexcellent properties. However, Steviol glycosides have a bitter or licorice aftertaste.AndSteviol glycosides are complex molecules that makes it hard to manage. Those prevent steviolglycosides from becoming more widely marketed. Stevioside and rebaudioside A are two ofthe most component of Steviol glycosides. Stevia rebaudiana Bertoni leaf‘s total dry weight,stevioside is5-10%and rebaudioside A is2–4%. Rebaudioside A is the sweetest. It is morestable, more soluble and less bitter than stevioside. So rebaudioside A has great commercialvalue. There are several methods to produce high-purity rebaudioside A product, such asbreeding new varieties of stevia yielding rebaudioside A highly, Purifying by absorption resinand recrystallization, modifying Steviol glycosides by some enzymes like Cyclodextringlucanotransferase. However those methods have some disadvantage like complicated process,high production costs, poor modification, and so on. It has been reported that Steviarebaudiana Bertoni UGT76G1can catalyse the glucosylation of stevioside which hassignificant bitter aftertaste to form rebaudioside A. Display UGT76G1on the Pichia pastoriscell-surface through genetic engineering technology, which can help the industrial massproduction of rebaudioside A by enzyme in the future.In this study, we get uridine diphosphate glycosyltransferase gene from Steviarebaudiana Bertoni by gene synthesis after being optimized as the Pichia pastoris preferredcodons. The UGT76G1was displayed on the cell surface by using Pichia pastoris cell wallprotein Gcw61p. When the recombinant Pichia pastoris GS115/pGCW61-KAS1under shakeflask conditions, we found that the maximum level of its cell‘s UGT76G1activity was0.126U/g（Dry cell weight） after120h by induced with methanol. In the preliminary study,we found that the UGT76G1displayed on the cell surface has an optimum pH of9.0and anoptimum temperature of40℃.After verifying UGT76G1after can be displayed on the cell surface of Pichia pastorissuccessfully, we constructed recombinant plasmid with multi-copy expression cassettes to form multi-copy recombinant Pichia pastoris, in order to develop the level of UGT76G1activity. Base on pHKA-K61recombinant plasmid, we successfully constructed plasmids thathave2copies expression cassettes and4copies expression cassettes respectively:pHKA-K61×2; pHKA-K61×4; pHKA-(K21-K61)×2. Using electrotransformation, we gotmulti-copy strains. The copy number of target gene KAS1in each strains:GS115/pGCW61-KAS1（1copy）; GS115/pHKA-K61×2（2copies）; GS115/pHKA-K61×4（4copies）; GS115/pHKA-(K21-K61)×2（5copies）. When these four recombinant strainsunder shake flask conditions120h by induced with methanol, we found that in multi-copystrains, the more copy number of target KAS1it had, the higher level of UGT76G1activitywould be. And the harder Growth inhibition of strain will came out. Recombinant strainGS115/pHKA-(K21-K61)×2（5copies）had the highest level of UGT76G1activity，its highestactivity is0.479U/g （Dry cell weight） at72h. It was almost3fold than that ofGS115/pGCW61-KAS1（1copy）. However the serious growth I nhibition ofGS115/pHKA-(K21-K61)×2（5copies）appears, its OD600just arrived5~6. At72h by inducedwith methanol，GS115/pHKA-(K21-K61)×2（5copies） cells were selected to have a reaction.There is probably1.361mg/mL RA product, and the convert ratio is about70.37%（actualyield/theoretical yield）.