Engineering Of Escherichia Coli Coculture System For Efficient Production Of Tyrosol Glycosides

Glycosides are one type of the most important natural products derived from the secondary metabolism with various physicochemical and biological properties,making glycosides more attractive compounds that are used worldwide as therapeutic drugs,food additives and nutraceuticals in health care industry.Glycoside biosynthesis is typically derived from two divergent precursors and it is usually unefficient using monoculture because of the restriction of aglycone and activated sugars.Here,taking efficient production of salidroside and icariside D2 as examples of glycosides,we design and construct a syntrophic Escherichia coli-E.coli coculture composed of the phenylalanine deficient aglycone(AG)strain and the tyrosine deficient glycoside(GD)strain,which convergently accommodate biosynthetic pathways of tyrosol and glycosides,respectively.We studied the construction of the pathway and the regulation and optimization of the coculture system.We construct the constitutive expression vector of kdc4 and get the tyrosol strain BMT21 which produced 1.46g/L tyrosol in 36 h by batch fermentation.The specific glycosyltransferase gene synugt85a1 from Arabidopsis thaliana is codon optimized and overexpressed,yielding BMS23,which produces 927.04mg/L from 500mg/L tyrosol.AG and GD strain form a syntrophic coculture system by the crossfeeding of phenylalanine and tyrosine.To release the carbon competence,we engineer the AG strain to utilize xylose preferentially by deleting the manZ gene and adopt BMS10 which consume glucose exclusively to construct GD strain.We explored the ratio of glucose to xylose and the ratio of inoculation to optimize the fermentation parameters.Under the optimized condition,the cocluture system produce 670.58 mg/L salidroside by batch fermentation and 6.03 g/L salidorside by fed-batch fermentation withiout tyrosol accumulated.We adopt the non-specific glycosyltransferase YjiC from Bacillus licheniformis to verify the flexibility of the coculture system.It produce 403.45mg/L salidoside and 409.34 mg/L icariside D2 in the optimized conditions by batch fermentation.Distributing the synthetic pathways of aglycone and activated sugars to two strains and constructing a coculture system is a potential strategy for glycoside production and can be also used for the synthesis of other complicated natural products.