Preliminary Study On The Production Of Squalene And Artemisinic Acid By Pseudomonas Nitroreducens Jin1 Metabolic Engineering

From the perspective of current food production,terpenoids are widely used.This additive can replace flavors,sweeteners,etc.its excellent antioxidant,antibacterial and anticancer functions have been recognized by everyone.In the synthesis of terpenoids,the potential of biosynthesis is great.In this study,Pseudomonas nitroreducens Jin1,a prokaryote that has the application prospect of engineering bacteria for food and can make full use of eugenol(or isoeugenol)as carbon source,was selected as the host strain for genetic transformation,in order to study the synthesis of terpenoids in Pseudomonas.The main results are as follows:(1)The whole genome of Pseudomonas nitroreducens Jin1 was sequenced and the genome function was annotated.It was found that there were many terpene related genes annotated,and the terpene skeleton metabolic pathway was combed to determine the synthetic target products artemisinic acid and squalene.(2)After codon optimization,Amorpha fruticosa-4,11-diene synthase(ADS)gene and P450 monooxygenase(CYP71AV1)gene were synthesized.The synthetic gene was connected to the double expression vector prsfduet1 and introduced into Pseudomonas nitroreducens Jin1 and Escherichia coli for double expression.The yield of artemisinic acid was measured under the same conditions.Under the same conditions,the yield of artemisinic acid was 61.659 ?g/L,42.357 ?g/L,Pseudomonas nitroreducens Jin1 was1.46 times higher than Escherichia coli under the same conditions.(3)After codon optimization,squalene synthase(SQS)was synthesized,and the overexpression vector prsfduet1 SQS was constructed.It was introduced into Pseudomonas nitroreducers Jin1 and E.coli BL21(ED3).The squalene content was detected under the same conditions,which were 961.01 ?g/L and 345.83 ?g/L.Similarly,the yield of squalene in Pseudomonas nitroreducens Jin1 is much higher than that in E.coli BL21(ED3).The squalene yield of Pseudomonas nitroreducens Jin1 SQS was 2.78 times that of E.coli BL21(ED3)-SQS.In conclusion,Pseudomonas nitroreducens Jin1 strain is more suitable for the transformation of engineering bacteria in terpene production than the common engineering strain E.coli BL21.