Synthesis Of Sialylated Oligosaccharides Based On Yeast CTP Regeneration

The oligosaccharides on the cell surface participate in a series of life activities in the cell,and the oligosaccharides containing terminal N-acetylneuraminic acid（Neu5Ac）residues play an important role in cell adhesion,tumor migration,brain nerve development and viruses successfully invasion of the body.It has now become one of the key areas of research in the sugar life sciences.Obtaining enough sialylated oligosaccharides becomes the basis and key to explore its function.At present,although the method of chemically synthesizing sialylated oligosaccharides has been basically established,due to the need for complicated group protection and removal steps,the stereoselectivity is poor,and the synthesized oligosaccharides are expensive.Although enzymatic synthesis of sialylated oligosaccharides is highly stereoselective and regioselective,it requires expensive CTP and CMP-Neu5Ac as substrates.The recently developed whole-cell synthesis technology,although it can synthesize oligosaccharides in cells,has a very low yield due to the accumulation of synthetic sialylated oligosaccharides in the cells.Because of this,how to establish inexpensive synthesis of CTP and CMP-Neu5Ac and extracellular catalytic synthesis of sialylated oligosaccharides is the key to achieving industrial production of sialylated oligosaccharides.Firstly,a yeast that efficiently catalyzes the CMP to CTP was screened and the whole yeast cell-free catalytic system was optimized for efficient synthesis from inexpensive CMP to CTP.S.cerevisiae S189,which has the strongest ability to synthesize CTP by CMP,was screened by screening different kinds of yeasts.Based on this,shake flask optimization including CMP concentration,glucose concentration,pH and temperature was performed.The optimized reaction system was determined as:CMP concentration 40 mmol·L^-1,glucose concentration 120 mmol·L^-1,pH 8,temperature 37°C,final conversion rate 84%,and 53%increase before optimization.Secondly,we established a reaction system for optimizing the high-level expression of CMP-Neu5Ac synthetase and CMP-Neu5Ac catalytic synthesis.The coding gene neuA of CMP-Neu5Ac synthetase（NeuA）was ligated into the vector pET28a and pFLAG respectively to obtain recombinant plasmids pET28a-neuA and pFLAG-neuA,which were transformed into E.coli BL21（DE3）and induced by IPTG.The synthesis of CMP-Neu5Ac was analyzed to evaluate the specific activity of the NeuA enzyme expressed in different vector systems,and then the catalytic reaction system was optimized.Finally,the recombinant srain E.coli BL21（DE3）-pFLAG-neuA at an initial IPTG induction concentration of 0.5mmol·L^-1 and cultured at 30°C for 15 h with the highest enzyme activity.The specific activity of expressed NeuA enzyme was 13.5 U·mg^-1,E.coli BL21（DE3）-pET28a-neuA was 7.5U·mg^-1.The optimum temperature and pH for synthesis of CMP-Neu5Ac were 38°C and 9,obtained by optimizing the synthesis of CMP-Neu5Ac,the optimal reaction time was 3 h.And the final yield was 2.6 mmol·L^-1,which was 23%higher than that before optimization.Finally,we established a double yeast coupled catalyzed sialylated oligosaccharide system.The recombinant plasmid pYD1-ST23 was constructed on the surface of S.cerevisiae EBY100,and the recombinant EBY100-pYD1-ST23 was successfully selected by tryptophan-free SD medium and colony PCR.YNB-CAA-Glc was used as the fermentation medium.Induce expression under the condition of 25°C,and the resulting bacterial solution was mixed with the crude enzyme solution of recombinant strain E.coli BL21（DE3）-pFLAG-neuA and S.cerevisiae S189 yeast in a ratio of 1:1:1（v:m:v）.And mix the mixed broth with the reaction solution(CMP 10 mmol·L^-1,Neu5Ac 10 mmol·L^-1,Lactose10 mmol·L^-1)in a volume ratio of 1:1.After reacted at 30°C for 2 h,the formation ofα-2,3-sialylated oligosaccharides was successfully detected by thin layer chromatography（TLC）.