Construction Of An Engineered Strain For The Synthesis Of Lycopene Using Palmitic Acids As Carbon Source

Lycopene is a terponid that is used in the pharmaceutical,nutraceutical,cosmetic,and food industries.The engineered Escherichia coli is one of the excellent lycopene producers due to the high yield of lycopene and easy cultivation.However,there are many problems in the process of synthesizing lycopene by using glucose as a raw material,such as low theoretical conversion rate,high production cost,and difficulty in increasing production.Therefore,looking for the cheap alternative carbon sources is one of the effective ways.In this study,palmitic acids were selected as the carbon source to synthsis lycopene,the main advantage is that palmitic acids can produce acetyl-CoA through ?-oxidation and acetyl-Co A is the sole precursor of the MVA pathway.MVA pathway is a popular strategy for lycopene overproduction.so palmitic acids can be used as the sole carbon source for producing lycopene.Palmitic acid can provide sufficient precursor of acetyl-CoA for the MVA pathway.The purpose of this study is to construct a high-yield and stable engineered strain that can use palmitic acids as carbon source to produce lycopene.By simplifying the strain system and optimizing the metabolic pathways,finally,a highly productive and stable strain of lycopene can be obtained.In this study,combining the palmitic acid metabolism module with the lycopene producing module,to verify that the palmitic acid can be as the sole carbon source for the production of lycopene.Two strains FA01 and FA02 which can use palmitic acids have been constructed in our laboratory.In this basis,three plasmids(pLESKs,pSKPMIc and pLY116)that carrying the genes of lycopene synthesis pathway were transformed.When detecting the production of lycopene,only found that lycopene was produced.It indicated that palmitic acids could be used as the sole carbon source to produce lycopene.To facilitate the rational study of expression of heterologous pathway genes in E.coli,a new chromosomal integration method,named NRMI(Nigri recombinase mediated integration),was established in this experiment.NRMI is a new novel and rapid approach of large fragment gene integration for metabolic pathway optimization.Studies have shown that Nigri-nox,a tyrosine-site-specific system,can occur the ntegration reaction.To verify the integration reaction of Nigri-nox,we first inserted the nox sites on chromosomes and plasmids.Under the action of Nigri enzyme,plasmid can be integrated into the chromosome,thereby realizing the integration of large fragement genes.To expand its scope of application,continuous integration of large segments of genes was achieved with the help of two other tyrosine site-specific systems,including Cre-lox and Dre-rox.Therefore,the method can be used for the rational optimization of metabolic pathways.In order to improve stability of strain and yield of lycopene,the genes of MVA pathway were integrated into the chromosome.The MVA pathway was divided into upstream and downstream modules,the upstream module is ESK and the downstream module is PM.In order to obtain the final lycopene strain FMA02,the two modular genes were integrated into the chromosome in turn,and then optimizing the culture conditions,finally,the lycopene yeild of FMA02 reached to 65.44mg/g DCW.In this study,the method of optimitic MVA pathway will provide new information for further study of metabolic engineering.The use of palmitic acids as the only carbon source for the production of metabolites,with good industrial production prospects.