Study On The Strategies For Improving Terpenes Bioproduction In Escherichia Coli

Pinene is a natural dicyclo-monoterpene with wide applications.As its isomers,?-pinene and?-pinene can dimerize into a high energy-density compound that can be used as a renewable jet fuel.On an industrial scale,pinene is traditionally produced as a byproduct of paper pulping or extracted from turpentine,which requires a large expense of raw materials and has a low yield.To reduce the cost and improve the yield of pinene production,novel synthesis strategies are necessary to be developed.Recently,heterologous synthetic pathway of pinene has been introduced into Escherichia coli,leading to a titer of 32 mg/L^[1].To further impove the titer,a novel synthetic biological strategy is demonstrated and applied in this study.Initially,to enhance the compability of pinene producing pathway for the host,and to overcome the problem of spontaneous pathway interruption that is caused by serious metabolic burden imposed by pinene synthesis,efforts have been made to stabilize the expression and replication of pathway producing plasmid,including substitutions of homologous scar sequences,shifting genetic elements and disrupting endogeneous recombination pathway.Next,through over-expressing enzymes of the heterologous mevalonate pathway and monitoring substrate levels,mevalonic kinase and mevalonate diphosphate decarboxylase have been identified as rate-limiting enzymes.Pinene titers are notably improved by overexpressing them.On the basis of that,a novel strategy of dymically repressing gene expressions in Escherichia coli has been developed.By using Lux quorum-sensing system from Vibrio fischeri to control the transcription of bacterial small RNA,the expression level of any targeted gene of interest can be repressed as the host grows.After optimizations,the repression of green fluorescent protein which is constitutionally expressed changes from 0 at the beginning of culturing to at most 90%during stationary phase.Moreover,joint repression of multiple genes is also developed to better regulate bacterial metabolic networks.Under the comparison of existing constantly repressing strategy,our strategy is able to improve pinene titers by strongly repressing the expression of essential genes with no detected influence on the host growth,which is a great advantage.After screening,joint repression of multiple gene candidates finally increases pinene titer to 128.4 mg/L.At last,a priority of demand for NADPH instead of acetyl-coenzyme A is concluded.We hope that this promising strategy can be further applied in other issues of metabolic engineering.