The Study On Biosynthesis Of Saturated Organic Acids Via Enoate Reductases

Chemical and energy industries rely on fossil fuels.However,it is inevitable that fossil storage shrinks down,not mention the deteriorating enviroment.Synthetic biology supplied a new strategy to produce chemicals by engineering strains using cheap carbon sources.P-coumarate,cinnamate and muconic acid are important unsaturated acids with great values.P-coumarate and cinnamate can be reduced by chemical methods,and the products 3-(4-hydroxyphenyl)propionic acid(HPPA)and phenylpropionic acid(3PPA)are also valuable chemicals used in flavouring industry and pharmaceuticals.There are no reports about biosynthesis of HPPA and 3PPA by now.Adipic acid is a dicarboxylic acid for nylon synthesis and is produced via hydrogenation of muconic acid in industries.P-coumarate,cinnamate and muconic acid are ?-? unsaturated acids.To achieve the hydrogenated products,enoate reuctases were screened.Enoate reuctase from Clostridium acetobutylicum that can achieve HPPA from p-coumarate and 3PPA from cinnamate was discovered.Under aerobic condition,CaER has an initial enzyme activity as 1.26 mmol/h/gDCW for p-coumarate and an initial enzyme activity as 3.18 mmol/h/gDCW for cinnamate.Then feeding experiments of phenylalanine and tyrosine were carried out and the downstream modulars were optimized.The whole pathways of HPPA and 3PPA accumulation were completed.At 72 h,225.10 mg/L HPPA and 356.43 mg/L 3PPA were detected.The results showed the effeciency of CaER on p-coumarate and cinnamate hydrogenation,and provied the possibility of de novo production of HPPA and 3PPA.To get higher 3PPA production,several approaches were carried out to further optimize the expression strategy of CaER.NADH is a cofactor of CaER,meaning the reduction of oxidized NAD+ should be considered.With the reaction caused by fdhl,oxidized NAD+ was consumed and NADH was formed,with 394.74 mg/L 3PPA accumulation.Then,to balance the difference of oxygen requirements in the whole pathway,shaker speed was considered as a visible sign of dissolved oxygen level in medium and 403.30 mg/L 3PPA was accumulated at 200 rpm.To deepen the contrast of dissolved oxygen level in co-culture system,VHB was involved in oxygen accumulation of upstream pathway cells and 507.16 mg/L 3PPA was produced.Tac promoter and T7 promoter were compared to lac promoter and it was found that 467.98 mg/L 3PPA was produced with tac promoter.CaER has the ability to catalyze p-coumaric acid and cinnamic acid.To test its enzyme activity towards muconic acid,aerobic condition and microaerbic condition were chosen.To hydrogenize muconic acid,CaER was tested under aerobic condition,but no product was detected.Adipic acid was only detected under microaerobic condition by the titer of 74.23 mg/L at 24 h.After introducing a modular catalyzing salicylic acid into muconic acid,28.70 mg/L adipic acid was detected when salicylic acid was fed.By combination of all the parts,a novel pathway was synthesised and produced 12.42 mg/L adipic acid.Co-culture system provided an increased titer of adipic acid as 18.29 mg/L compared to single-culture system.With the help of VHB,28.22 mg/L adipic acid was accumulated.This study not only established novel biosynthetic pathways of HPPA and 3PPA for the first time,but also developed a new microbial pathway for adipic acid production via CaER.These strategies showed a broad prospects of metabolic engineering.