Efficient Transformation Of Trehalose By Recombinant Corynebacterium Glutamicum

Trehalose is a functional sugar that is heat-resistant,acid and alkali resistant,and does not have reducibility,widely used in pastry baking,fruit preservation,medical beauty,organ and tissue preservation,and other fields.At present,the main source of trehalose is enzymatic synthesis,and whole-cell biocatalysis is a type of enzymatic synthesis,which has the characteristics of stable catalytic effect,high reuse rate,and simple product recovery.This study constructed two effective conversion systems for whole cell catalytic synthesis of trehalose.Firstly,the trehalose synthase gene was cloned and expressed in Corynebacterium glutamicum,and its whole cell synthesis conditions were optimized.We have achieved efficient expression of trehalose synthase in host bacteria.Then,by fusing trehalose synthase and ankyrin genes,two surface displayed recombinant strains were constructed as immobilized whole cell catalysts for efficient trehalose synthesis.（1）The trehalose synthase gene Sc TreSK246A from Streptomyces coelicolor was connected with the p XMJ19 plasmid and transformed into E coli JM109.After amplifying the plasmid in E.coli JM109,the plasmid was extracted and transformed into C.glutamicum to construct a recombinant strain C.glutamicum ATCC13032/p XMJ19-Sc TreSK246A.Through SDS-PAGE and HPLC,it was confirmed that Sc TreSK246A could be expressed efficiently and has the transformation ability.Under the optimal conditions,the conversion rate of 300g·L^-1maltose reached 65.7%.After immobilization and 5 times reuse,the trehalose conversion rate is still above 80%.After 7 times reuse,the conversion rate of trehalose is still above 70%.（2）Due to the thick cell wall of C.glutamicum ATCC13032,2‰Triton X-100 need to be added in the transformation process for smooth progress.In this study,two recombinant C.glutamicum strains,C.glutamicum ATCC13032/p XMJ19-Por H-SctreSK246A,and C.glutamicum ATCC13032/p XMJ19-NCgl1337S-SctreSK246A were constructed using two anchors,Por H and NCgl1337,to anchor trehalose synthase from Streptomyces coelicolor on the cell surface,so that it can directly synthesize trehalose from maltose on the cell surface.After studying the activity of cells per unit wet weight,it was found that C.Glutamicum ATCC13032/p XMJ19-NCgl1337S-SctreSK246A contains higher enzyme activity.Compared with free enzymes,the thermal stability of anchored enzyme NCgl1337S-Sc TreSK246A was significantly improved,and the optimal temperature and p H did not change.（3）After optimizing the conditions,the whole-cell conversion substrate concentration in a 5 L fermentation tank was 300 g·L^-1,and the yield of trehalose reached 69.5%.The relative conversion rate of C.glutamicum ATCC13032/p XMJ19-NCgl1337S-SctreSK246A remained above 80%after 7 reuse and above 70%after 9 times reuse.