Directional Improvement Of Glycosidic Aroma Precursor Production Efficiency Of Engineering Bacteria Based On UDPG In-situ Regeneration And Random Mutation Technology

Aroma glycosides(glycosidically bound aroma)are widely exist in the natural plants,consisting of a sugar linked to a small volatile molecule.Compared with free aromatic substances,aromatic glycosides are more stable and have important biological functions and great commercial value.Aroma glycosides are usually catalyzed by UDP-glycosyltransferase(UGTs),in which the sugar donor is uridine diphosphate glucose(UDPG).A new type of tea glycosyltransferase CsUGT85A53 was discovered by our team earlier.It can synthesize corresponding glycosides from benzyl alcohol,phenylethanol and other alcoholic aromas in vitro.Phenylethanol is the material basis of flowers and fruits in tea plants.The study of phenylethanol can improve the aroma of tea.However,the high price of UDPG limits its application in industrial production.Some studies showed that the sucrose synthase(SUS)gene can convert UDP into the activated UDPG under certain conditions to regenerate UDPG in situ.UDP is a by-product of glycosylation reaction involving UGTs.In situ regeneration of UDPG not only reduces the cost of UDPG,but also avoids the inhibitory effect of UDP on the target product.In this paper,the random mutation of CsUGT85A53 by error-prone PCR was attempted to study the effect of mutation on the activity of CsUGT85A53.At the same time,the SUS gene of tea plant was cloned,the enzyme activity was analyzed,the engineering bacteria containing SUS gene was constructed,and the in-situ regeneration system of UDPG was established to realize the efficient production of aromatic glycosides.The main results are as follows:1)A random mutant library of CsUGT85A53 gene was constructed by error-prone PCR.By transforming the competent cells of E.coli BL21(DE3),some engineering bacteria contain the mutants of CsUGT85A53 were obtained,and the mutation rates of amino acids were between 1%and 2%.A mutant with a significant increase for the utilization of n-octanol was obtained by comparing the enzymatic activities of the recombinant proteins of mutants compared with the wild type CsUGT85A53.2)Two sucrose synthase(SUS)genes were cloned and obtained.Real-time quantitative PCR analysis showed that these two SUS genes(named and could respond significantly to low temperature stress in tea plants.The expression of CsSUS2 gene increased significantly in low temperature stress,while the expression of CsSUS6 gene decreased significantly.It was speculated that these two SUS genes played different physiological roles.3)These two SUS genes were inserted into pET-32a(+)vector for prokaryotic expression.The sizes of CsSUS2 and CsSUS6 were 91 KDa and 89 KDa,respectively.When SUS reacted with CSUGT85A53 protein,the formation of phenylethanoid glycosides could be successfully detected without UDPG,which confirmed that both CsSUS2 and CSSUS6 can regenerate UDPG from UDP and sucrose in situ.Further,the yield of phenylethanolic glycosides increased significantly by 1.67 and 1.3 times,respectively,when CsUGT85A53 was incubated with CsSUS2 or CSSUS6 compared with that with only CsUGT85A53.Further analysis showed that CsSUS2 and CSSUS6 could also change the glycosylation site preference of kaempferol.CsSUS2 could promote the formation of kaempferol diglycoside,while CsSUS6 could significantly increase the glycosylation of kaempferol at 3-OH site.The in situ regeneration of UDPG can significantly improve the ability of phenylethanoid glycosides synthesized by known functional glycosyltransferases,and also change the glycosylation mode of kaempferol to produce glycosides,thus promoting the synthesis of kaempferol disaccharides.