The Study Of Microbial Synthesis Of Germacrene A The Precursor Of β-elemene

(3-elemene was extract from Curcuma wenyujin, an important herbal plant in traditional medicine of China, is a kind of sesquiterpene that has clear antitumor activity for a variety of tumor types. The extracting β-elemene from the plant was raw materials waste and costly.However, the chemical synthesis of β-elemene was has no industrial applicational value because of strict conditions and high-pollution. So discovering a new production technical route with economical, viable and large-scale will be of great significance.From cDNA library of leaves of Curcuma wenyujin a new IPP isomerase gene (GenBank:GU724874) which is key enzyme of tepenoid biosynthesis was cloned, expressed and characterized using function competation in E.coli. However, no gene of germacrene A synthase which catalyze FPP into Germacrene A, the precursor of β-elemene was obtained. So germacrene A synthase from cyanobacteria was chosen to produce germacrene A in miroorgansim due to its high activity and prokaryotic orgin.The titers of germacrene A in recombinant E.coli and S.cerevisiae harbouring germacrene A synthase were128μg/L and9.5μg/L. So E.coli express system was chosen to carry out further research.Synthetic biology combined metabolic engineering of microbes could be used to produce high valuable metabolites that are hard to extract or too expensive or complex to produce by chemical synthesis. On the base of above technology a heterologous MVA pathway was constructed which was composed of all eight genes. GAS and MVA pathway were co-expressed in E.coli to produce germacrene A. Combining induce conditions optimization, flask medidum optimization and solid-phase microextraction conditions optimization the titer of germacrene A reached to6325.5μg/L from the initial128μg/L using GC analysis. The yield had increased by54times.In this study, the yield of germacrene A was still too low to applied industrial production. Inclusion bodies that constituted76%of the total cell protein content in GAS expression in E.coli is a key restricting factor. Error-prone PCR and DNA shuffling techniques were used to introduce random mutates into GAS gene.An highthrough screening method based on LacZa structural complementation was used to screen mutants with soluble expression. This research is in progress.