Construction Of Genetically Engineered Bacteria For Modifying Phenolic Compounds To Synthesize Phenolic Derivatives

Phenolic compounds including 4-hydroxybenzoic acid and vanillin etc.are widely used in people's daily life.The yields and natural resources for many of these compounds are limited.In order to make up for the shortage of natural resources to meet the needs of the market and people's lives,some phenolic compounds are produced by chemical syntheses.However,in some fields such as food,cosmetic and pharmaceutical,people prefer the natural products or the products from biosynthesis.Therefore,the construction of a microbial cell factory using the modern biotechnology to produce the phenolic derivatives has broad prospects.Apart from the CO2 fixation from photosynthetic organisms,the utilization of CO2 by heterotrophic organisms for the syntheses of some valuable chemicals is also an interesting thing.This study reported unprecedented microbial biosynthesis of 4-hydroxybenzyl alcohol,vanillin and salicylaldehyde in E.coli from phenol and guaiacol using three artificial pathways.In this work,three novel synthetic pathways for the syntheses of 4-hydroxybenzyl alcohol,vanillin and salicylaldehyde were created in E.coli.In the recombinant E.coli strain,the genes encoding a carboxylic acid reductase(CAR)from Nocardia sp.strain NRRL 5646?phosphopantetheine transferase(PPTase)Sfp from Bacillus subtilis,Salicylic acid decarboxylase(Sdc)from yeast Trichosporon moniliiforme WU-0401,endogenous alcohol dehydrogenases(ADHs)from E.coli,and a set of genes named as yclBCD encoding 4-hydroxybenzoate decarboxylase,which belongs to the hydroxyarylic acid DC/phenol carboxylase family in the B.subtilis subsp.subtilis 168 genome were introduced.As a cell factory,it combined the process of CO2 combination with the phenolic substrate and the further reduction of the resulted carboxyl group,with which the hydroxyl benzyl alcohol can be obtained in one fermentation step directly.With phenol and CO2 as the substrates under aerobic condition,the fermentations with the existence of the recombinant E.coli harboring plasmid pETDuet-yclBCD-car-sfp was used to produce 4-hydroxybenzoic acid from phenol and CO2(supplied by NaHCO3)in culture medium,as well as to produce 4-hydroxybenzyl aldehyde and 4-hydroxybenzyl alcohol from the reduction of 4-hydroxybenzoic acid.The supernatant composition of the fermentation broth of recombinant E.coli strains was analyzed by HPLC and HPLC-MS,which confirmed the production of 4-hydroxybenzoic acid,4-hydroxybenzyl alcohol,and phenol.The recombinant E.coli harboring plasmid pETDuet-yclBCD-car-sfp produced 11.331 ± 1.882 mg/1 4-hydroxybenzyl alcohol with phenol and CO2 in 12h of flask cultivation.However,With guaiacol and CO2 as the substrates under aerobic condition,the fermentations with the existence of the recombinant E.coli harboring plasmid pETDuet-yclBCD-car-sfp was analyzed by HPLC,there are no peaks of the product of vanillin,We found the decarboxylase can,t catalyze the regioselective carboxylation of guaiacol to form vanillic acid.The supernatant composition of the fermentation broth of he recombinant E.coli harboring plasmid pETDuet-sdc-car-sfp was analyzed by HPLC,there are no peaks of the product of Salicylaldehyde,we found a salicylic acid decarboxylase that reversibly catalyze the regioselective carboxylation of phenol to form salicylic acid,but the salicylic acid was unaffected by this carboxylic acid reductase(CAR).