Efficient Synthesis Of Eriodictyol Via Functional Expression Of A P450Flavonoid Hydroxylase In Metabolic Engineering Escherichia Coli

The health benefits of flavonoids in humans are increasingly attracting attention. As theextraction of high purity flavonoids from plants presents a major obstacle and chemicalsynthesis is associated with toxic byproducts and requires extreme reaction conditions,interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoidwith anti-inflammatory and antioxidant activities. Its efficient synthesis has been hamperedby two factors: the poor expression of cytochrome P450and low intracellular malonyl-CoAconcentration in E. coli. To address these issues, a truncated plant P450flavonoid3’hydroxylase (tF3’H) was functionally expressed as a fusion protein with a truncated P450reductase (tCPR) in Escherichia coli. This allowed the metabolically engineered E. coli toproduce eriodictyol from L-tyrosine by simultaneously coexpressing the fusion protein withtyrosine ammonia lyase (TAL),4-coumarate-CoAligase (4CL), chalcone synthase (CHS) andchalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance theavailability of malonyl-CoA and balance the relative gene expression to enhance theeriodictyol accumulation. Main results were described as following:(1) Through metabolic engineering to enhance the availability of malonyl-CoA, theconcentration of malonyl-CoA in the recombinant strain was increased to3.98±0.16μmol g-1DCW. Compared with the WT strain, the concentration of malonyl-CoA increased1630%. These results demonstrated that the metabolic engineering could increaseintracellular malonyl-CoA concentration.(2) Analysis of the secondary structure of F3’H from Gerbera hybrid using thesecondary structure prediction program revealed a helix motif at the N-terminal region, whichwas identified as a membrane anchor sequence. The membrane anchor sequence was cut byPCR. The truncated plant P450flavonoid3’ hydroxylase (tF3’H) was functionally expressedas a fusion protein with a truncated P450reductase (tCPR) from Catharanthus roseus in E.coli. Biosynthesis of eriodictyol from L-tyrosine was successfully achieved by solubleexpression of flavonoid3’ hydroxylase (F3’H) in E. coli.(3) The proper fermentation optimization was taken to increase the final yield oferiodictyol. Different temperatures were chosen for the induction phase. IPTG was added torecombinant strains at different concentrations and at different OD600. Through the properfermentation optimization, the highest eriodictyol production of strains BF1, BF2, BMF1andBMF2reached35.2mg L-1,42.6mg L-1,86.4mg L-1and107mg L-1, respectively.