Production Of Tyrosol Glycosides In Engineered Escherichia Coli

Phenylethanoid glycosides,the natural products of plants,are composed of phenylethyl alcohol or its derivates(tyrosol,methoxyphenylethanol)as aglycones and glucose or rhamnose as sugar donors.Phenylethanoid glycosides possess various pharmaceutical activities including immunoregulation,anti-cancer,anti-fatigue,anti-aging,and neuroprotection,and are used in manufacturing health care products,environmental adaptation drugs and cosmetics.Due to the low yield of extraction from plant tissues and the complicated chemical processes,the production of phenylethanoid glycosides by constructing microbial cell factory has become a promising method with strategy of synthetic biology.In this thesis,biosynthetic pathway of tyrosol glycoside,the basic derivate of phenylethanoid glycoside,was designed to heterologous expression in E.coli.Tyrosol was produced from 4-hydroxyphenylpyruvic acid of the L-tyrosine biosynthetic pathway via the catalysis of decarboxylase and dehydrogenase.UDP-glucose as the sugar donor was transferred to tyrosol by glycosyl transferase,resulting in two different forms of tyrosol glycosides,the salidroside and phenylethanoid ?-D-glucoside.The full length genes encoding pyruvate decarboxylases from chromosomes 3 and 4 of Pichia pastoris GS115(synkdc3 and synkdc4,repectively),and from genome of Saccharomyces cerevisiae S288c(synaro10)were designed and chemically synthesized.When they were heterologously expressed in E.coli,synkdc4 was the most effective to produce tyrosol.The competiting gene feaB of tyrosol synthetic pathway was knocked out in high-yielding tyrosine chassis E.coli strain BAK10,generating strain BMT18.synkdc4 was then introduced into BMT18,generating to high-yielding tyrosol chassis strain BMT19,with a titer of 916.22mg/L for 96 h by fed-batch fermentation.Using ? Red recomnation method,feaB and ushA were deleted with the intergation of pgm and gulU in E.coli BL21(DE3)chromosome,generating chassis BMS10 for for the efficient supply of UDP-glucose.Glycosyltransferase genes synugt72b14 and synugt73b6 from Rhodiola sachalinensis,and synyjic from Bacillus licheniformis synyjic were designed and chemically synthesized with codon optimiztion.synyjic Production of salidroside and phenylethanoid ?-D-glucoside from tyrosol was achieved,and synyjic showed a yield of 0.53mol/mol for 24 h fermentation.After deletion of gene xylA,xylose deficient strain was generated.Thus,Escherichia coli-E.coli coculture system was constructed to produce salidroside and phenylethanoid ?-D-glucoside.By optimizing inoculation ratio,glucose-xylose ratio of the coculture system,763.27mg/L of salidroside was achieved in 72 h by fed-batch fermentation.