Photoautotrophic Production Of P-Coumaric Acid Using Genetically Engineered Synechocystis Sp.Pasteur Culture Collection 6803

In view of the environmental problems associated with climate change and greenhouse gas emissions,the potential of carbon sequestration has emerged as a research hotspot.Cyanobacteria can use solar energy and carbon dioxide to produce organic molecules bypassing the need for carbohydrate feedstock.This valuable trait has spearheaded its use in the field of bioproduction and ultimately metabolic engineering.As a natural phenolic compound,p-Coumaric acid is widely present in plants.Like other secondary metabolites,they are mainly produced to protect the plants against stress.p-Coumaric acid has many antioxidant and anti-inflammatory properties that are of importance to the pharmaceutical,food,cosmetic,and agricultural industries as precursors of active ingredients.This study aims to express the phenylalanine ammonia-lyase(PAL)from Rhodotorula glutinis and the cinnamic acid 4-hydroxylase(C4H)exogenous gene from the brassica Arabidopsis thaliana in the cyanobacterium Synechocystis sp.PCC6803 for the production of p-Coumaric acid.First and foremost,a plasmid was designed to harbor the PAL/C4H exogenous gene under the cyanobacteria Pcpc560 promoter to target the neutral docking site of the cyanobacterium Synechocystis sp.PCC6803.The plasmid was expressed in the wild-type Synechocystis and the mutant strains(Syn407-PAL/C4H)were cultivated in freshly prepared BG11 solution at 30? under white light illumination of 50?mol photons m^-2 s^-1 and ambient atmospheric air.After 7days of photoautotrophic growth,73 mg/L of p-Coumaric acid was produced.To further increase the production titer,investigations into the shikimate pathway led to the identification of the arogenate dehydrogenase which is encoded by the slr2081 locus in the cyanobacterium Synechocystis sp.PCC6803 genome.Arogenate is a precursor for Tyrosine production which is the competing pathway for phenylpropanoid synthesis.A new plasmid was designed to knock out the arogenate dehydrogenase from the wild-type Synechocystis.The plasmid also inculcated the PAL/C4H exogenous genes under the Pcpc560 promoter.After expressing the plasmid in the wild-type Synechocystis,the mutant strains(Syn001-PAL/C4H)were cultivated in freshly prepared BG11 solution at 30? under white light illumination of 50?mol photons m^-2 s^-1 bubbled with 1%(vol/vol)CO₂-enrich air at a flow rate of 50 sccm.197 mg/L of p-Coumaric acid was produced after 7 days of photoautotrophic growth.In conclusion,the unicellular cyanobacterium Synechocystis sp.PCC6803 was successfully engineered for the photoautotrophic production of p-Coumaric acid.The deletion of the competing metabolic route further increased the production titer.To provide a leveled ground for effective comparison between the two mutant Synechocystis strains designed in the experiments,both strains were grown in the augmented growth conditions.Syn001-PAL/C4H produced approximately 67mg/L of p-Coumaric acid more than Syn407-PAL/C4H.