Biosynthesis Of D-tagatose And Surface Display Based On Pichia Pastoris

D-tagatose is a novel low-calorie sweetener with a 92%sweetness of sucrose,but less than 1/3 of sucrose in calories.D-tagatose has considerable application value in the fields of food and medicine because of its execllent physiological functions,such as preventing obesity,reducing blood sugar,resisting dental caries,and improving intestinal flora.Compared with chemical synthesis,the biosynthesis of D-tagatose has drawn much attention due to its advantages of environmental friendliness,fewer by-products,and high catalytic efficiency.Currently,studies on the conversion of D-galactose to D-tagatose by the whole-cell harboring L-arabinose isomerase（L-AI）have been widely reported.However,problems such as single expression system,lack of co-expression,and substrate transport obstruction still hinder the industrialization of D-tagatose.Thus,this study aims to develop an eukaryotic expression system for the production of D-tagatose,optimize co-expression ofβ-galactosidase（β-GAL）and L-AI,and utilize surface display technology to efficiently convert lactose to D-tagatose.The main research contents and results are as follows:（1）The genes lac LM and ara A encodingβ-GAL and L-AI,respectively,were cloned from Lactobacillus reuteri ATCC 53608 and Lactobacillus plantarum CY.6.The recombinant plasmids p GAPZB-lac LM and p GAPZB-ara A were successfully constructed and respectively transformed into Pichia pastoris GS115.The recombinant strains P.pastoris GS115/p GAPZB-lac LM and P.pastoris GS115/p GAPZB-ara A were constructed.The target proteins were successfully expressed and show catalytic activity.The study on the conversion conditions of two engineering strains showed that the optimal conditions of lactose hydrolysis with P.pastoris GS115/p GAPZB-lac LM were55℃,p H 6.0,3 m M Mn²⁺,60 g/L lactose,and 30 g/L cell concentration,reaction for48 hours.The optimal conditions for conversion of D-galactose to D-tagatose with P.pastoris GS115/p GAPZB-ara A were 60℃,p H 6.8,5 m M Mn²⁺,160 g/L D-galactose,and 60 g/L cell concentration,reaction for 12 hours.Further,co-production of two strains showed that the highest D-tagatose production was achieved with P.pastoris GS115/p GAPZB-lac LM and P.pastoris GS115/p GAPZB-ara A mass ratio of 5:1,total cell concentration of 70 g/L,50℃,and p H 7.0.The production of D-tagatose reached6.47 g/L.（2）The recombinant plasmids p GCW14-lac LM,p GCW14-ara A,p GAPZB-ara A-P_GCW14-lac LM,and p GAPZB-lac LM-P_GCW14-ara A were successfully constructed and transformed into P.pastoris GS115.Four engineering strains have been verified to successfully express the target proteins.Promoter studies have shown that P_GAP is superior to P_GCW14 in single gene expression.Compared with P_GAP-lac LM-P_GCW14-ara A in duel gene co-expression,the combination of P_GAP-ara A-P_GCW14-lac LM increased the activity of lactose hydrolysis by 70.56%and the activity of converting D-galactose to D-tagatose by 51.97%.The optimization results show that the optimal conditions of conversion from lactose to D-tagatose for P.pastoris GS115/p GAPZB-ara A-P_GCW14-lac LM were 55℃,p H 8.0,2 m M Mn²⁺,180 g/L lactose,and 60 g/L cell concentration,reaction for 48 h.The results of optimizing the carbon source in the culture medium showed the highest D-tagatose production was achieved when glucose and glycerol were used as carbon sources and the concentration was 4:16 g/L,with an increase from the initial 13.98 g/L to 19.88 g/L.（3）P.pastoris GS115/p GCW14SP-lac LM-Gcw14,the surface display strain ofβ-GAL,was successfully constructed using GCW14p as anchor protein.Compared to intracellular expression,the catalytic activity of the surface display strain was improved.While glycerol was used as carbon source of culture medium,the overall catalytic activity of the surface display strain increased by 68.44%.P.pastoris GS115/p GCW14SP-lac LM-Gcw14 and P.pastoris GS115/p GAPZB-ara A were used to co-produce D-tagatose.The results showed that the highest yield of D-tagatose was achieved in strains mass ratio of 4:2,total cell concentration of 50 g/L,52.5℃,and p H6.0,with a production of 16.3 g/L D-tagatose.Finally,D-tagatose production increased to 30.6 g/L through fed-batch,temperature-change and cell-repalced strategies.