De Novo Biosynthesis Of Three Terpenoids From Glucose By Engineered Escherichia Coli

Terpenoids are the most abundant natural compounds in nature.They are compounds composed of isoprene（C₅ unit）as the basic unit,generally including semiterpenes（C₅）,monoterpenes(C₁₀)and sesquiterpenes(C₁₅),diterpenes(C₂₀),triterpenes(C₃₀),tetraterpenes(C₄₀)and polyterpenes(C_>40).So far more than 80,000 kinds of terpenoids have been discovered,which exist widely in animals,plants and microorganisms and have very wide applications in many fields such as energy,spices,chemicals,food and medicine.Traditionally terpenoids are obtained mainly from physical extraction from natural plants as well as chemical synthesis.The physical extraction of terpenoids from plants suffers from a number of problems such as high cost,inefficiency and environmental damage.and chemical synthesis is often complex and expensive due to the complex structure of most terpenoids,Therefore,the pursuit of environmentally friendly and renewable resources has attracted more and more attention of researchers.Synthetic biology is providing the possibility for the sustainable and efficient production of high value-added terpenoids in engineered microbial cell factories,and the biosynthesis of terpenoids by microorganisms is expected to replace traditional terpenoid production methods.In this paper,the work of constructing the metabolic pathways of three terpenoids in Escherichia coli and using glucose as raw material to biosynthesize the three terpenoids was carried out.The work is carried out as follows:Biosynthesis of two sesquiterpenes T-cadinol andβ-eduesmol in Escherichia coli.First,the MVA metabolic pathway of T-cadinol was constructed through eight genes,Mva E,Mva S,ERG12,ERG8,MVD1,Id I,Isp A,and CS.The MVA metabolic pathway ofβ-eduesmol was constructed through eight genes,Mva E,Mva S,ERG12,ERG8,MVD1,Id I,Isp A and ES.The fermentation culture and GC/MS analysis of the recombinant strain yielded 7.2±0.8 mg/L and 15.0±1.4 mg/L for T-cadinol andβ-eudesmol,respectively.Next,we evaluated the effect of replacing Mva SA110G and mva E with Ato B,ERG13 and t HMG1 in the upstream synthetic pathway on the final yields,and found that the final yields were increased to 24.4±2.6 mg/L,26.0±3.6 mg/L,respectively.We also increased the yield to 35.9±4.3 mg/L and38.2±5.2 mg/L by overexpression of Id I gene,respectively.Finally,in view of the volatility of T-cadinol andβ-eudesmol during fermentation,we used the constructed two-phase fermentation system to replace the original fermentation system to substantially increase the yields to 133.5±11.2 mg/L and 143.9±10.3 mg/L,respectively.Biosynthesis of diterpenoids nephthenol in Escherichia coli.First,based on previous work on two sesquiterpenes,we constructed the MVA metabolic pathway of nephthenol from nine genes,Ato B,ERG13,t HMG1,ERG12,ERG8,MVD1,Id I,GGPP and NS.The initial yield of nephthenol was obtained by fermentation culture of the recombinant strain and GC/MS analysis at 40.8±3.8 mg/L.We then increased the final yield to 61.3±5.8 mg/L by overexpression of the Id I gene.In this study,the biosynthesis of three terpenoids,T-cadinol,β-eudesmol and nephthenol,was successfully completed for the first time through the expression of a constructed heterologous MVA pathway in Escherichia coli,providing valuable experience in the future biosynthesis of terpenoids using Escherichia coli and offering a possibility for future industrial applications.