Production Of Limonene In Engineered Escherichia Coli

Limonene is an important monocyclic monoterpene which is widely used in the pharmaceutical,food,household chemicals,fragrance,and detergent.Traditionally,limonene is extracted from the fruits of citrus,but this preparation method is greatly affected by the raw materials,and as the application range of limonene is continuously expanding,the supply of limonene may be insufficient.Biosynthesizing limonene in E.coli by metabolic engineering would be an alternative production route to increase the supply.In this study,we constructed an efficient platform for producing limonene with heterologous MVA pathway in E.coli by combinatorial optimization strategy.The whole limonene synthesis pathway was divided into three modules: the upstream module containing mva E and mva S,the midstream module containing MK,PMK,PMD,IDI and the downstream module containing GPPS,LS.The ELIM17 strain harbored the three modules produced 11.71 mg/L of limonene.In order to improve the production of mevalonate,the wildtype mva S was replaced by mutated mva SA100 G and the production of mevalonate was improved to 485.99 mg/L,which increased 2.7 times compared with wildtype mva S.Then expression pattern of the mva E and mva S from Enterococcus faecalis was screened by RBS engineering,and the ELIM47 strain produced 1304.36 mg/L.After optimizing the upstream module,the yield of limonene increased to 84.97 mg/L,however,a large amount of mevalonate was also detected in the strain ELIM65.In order to reduce the accumulation of mevalonate,the MK gene derived from Methanosarcina mazei with higher catalytic activity was used and the T7 promoter was replaced with the constitutive promoter FAB80 in the middle module,the production of limonene was increased to 183.53 mg/L and the mevalonate was consumed.Deploying the NPP as the substrate to biosynthesize limonene leaded to further improvement of production of limonene(694.61 mg/L).The conditions of two-phase fermentation were optimized to further increase the production of limonene.By using the optimized conditions,a fedbatch fermentation experiment was carried out,and 19.92 g/L of glucose was consumed in 84 hours,the yield of limonene was 1.29 g/L.It is the first time to synthesize limonene in E.coli using NPP as a substrate.At the same time,the strategy used in this study can be used to improve the production of other monoterpenes.