Design And Construction Of Heterologous System For Isoprenoids Biosynthesis In Escherichia Coli

Plants derived isoprenoids have been an important source of drugs in human history. Since the yield of most rare isoprenoids in native plants is extremely low, methods such as direct extraction from plants or semi-synthesis from their intermediates are low efficient and expensive, and always waste natural resources. Synthetic biology introduces the principles and methodologies of engineering into biological studies, and it emphasizes the rational design of biological parts, devices, modules and systems to build new lives with a specific function. Consequently, using microorganisms with simple genetic manipulation as chassis cells,and by means of reconstructing the isoprenoid biosynthetic pathways, engineering the precursor supply and product exportation, it can achieve the large-scale production of important,isoprenoids. To design and construct efficient isoprenoid producer using E. coli, this study attempts to achieve the rational design of regulatory parts, catalytic parts of2-C-methyl-D-erythritol4-phosphate（MEP） pathway mining and their application in MEP pathway engineering, heterologous pathway assembly, globe cellular networks and isoprenoid exportation system engineering, and glucose feeding optimization on bioreactor scale.Regulatory parts are the basic elements for the design of functional devices and systems in synthetic biology research. Based on the back propagation artificial neural network （BP-ANN） algorithm with powerful non-linear mapping property, this study successfully constructed a computational platform for precisely predicting the strength of regulatory elements. To achieve this, a Trc Prom&RBS regulatory element library containing100mutated sequences with a relative strength from0to3.559was constructed. Based on this library, a prediction methodology was developed for precisely predicting the strength of regulatory parts using a back propagation neural network algorithm （BP-ANN）, which has a powerful non-linear mapping property. The best model NET90₁9₅76has a high correlation coefficiency of0.98both for model training and testing dataset. Based on the NET90₁9₅76model,16novel regulatory parts with an expected relative strength were designed and synthesized. The experimental results confirmed that the relative of these novel parts meets the expected values very well. Eventually, the novel regulatory parts were successfully applied for the expression of small peptide from Buthus martensi Karsch, finely tuning the activity of MEP pathway and heterologous resveratrol synthetic pathway. This methodology for rational design of regulatory element lay a solid foundation for synthetic biology research.Besides for rational design, directly mining biological parts from nature was also an important source for biological parts development. MEP pathway is the main precursor pathway for isoprenoids biosynthesis in prokaryotic bacteria, and the catalytic parts of MEP pathway have great diversities on their enzymatic activity and transcription control. Thus, using lycopene and amorphadiene as two model compounds, we have mined heterologous MEP pathway genes from various bacteria including Bacillus, Streptomyces, Saccharopolyspora and Erwina, and introduced these genes into E. coli to improve MEP pathway activity. The results indicate that over-expressing dxs2from Streptomyces avermitilis （dxs2_SAV） improves lycopene and amorphadiene production with10.5-fold and8.3-fold compared to that of control strain with empty plasmid. For gene idi from Bacillus subtilis （idi_BS）, the production of lycopene and amorphadiene was4.4-fold and10.6-fold improved. When co-expressing the two best modules dxs2_SAV and idi_BS, the production of lycopene and amorphadiene reached20.57mg/L and331.90mg/L, which improved16.5-fold and15.5-fold. This study indicates that the diverse MEP pathway genes can serve as an invaluable catalytic parts pool for the E. coli MEP pathway engineering and isoprenoid synthetic biology research.Genome-scale based chassis cell engineering is also an important aspect for construction of efficient isoprenoid biosynthetic system. Based on a series of genome-scale genetic targets, which were previously predicted as potential targets for improving isoprenoids in E. coli by the flux distribution comparison analysis （FDCA） algorithm, here we experimentally identified five novel knockout targets （eutD, deoB, yhftv, yahl, pta） and four novel amplification targets （ompE, ompN, cmk, ndk） can effectively improve the synthesis of lycopene in E. coli. Through combinatorial engineering of the best two knockout targets （gdhA, eutD） and three amplification targets （ompE, ompN, tpiA）, the yield of lycopene in shake flask fermentation was improved174%compared to control strain only with improved MEP pathway.Engineering isoprenoid efflux system to enhance the exportation of toxic heterologous products in microbial cells can release the adverse effects on cell growth, gene transcription and enzyme activities exerted by some unknown interaction between cellular networks. Tripartite efflux pumps have broad substrate spectrum and widely distribute in gram negative bacteria. This study selected pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, and overexpressed each component of these pumps in E. coli to improve isoprenoid production. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g.,31and37%improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with70%improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC-TolC-AcrB improved the specific yield of amorphadiene with118%, and AcrA-TolC-AcrB improved that of kaurene with104%. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.Owing to the great differences on the control of cell growth and gene expression between bioprocesses respectively for protein high expression and production of chemicals, traditional high cell density fermentation control strategies for protein high expression are always unavailable for the heterologous biosynthesis of chemicals. This study used the strain co-expressing dxs2_SAV and idi_BS as model, and firstly established a fed-batch fermentation process, whose glucose feeding rate during stationary phase was maintained at6.05g/h. The results indicated that24h-72h was important for isoprenoid production and the eventual yield of amorphadiene reached2.5g/L. Then the glucose feeding rate during stationary phase was optimized, and9.152g/h was determined as the optimal choice leading to4.85g/L amorphadiene. However the time for product rapid accumulation was not extended. This must be due to the rapid strain growth in the earlier stage and decrease of strain activity after72h. Thus, when keeping the glucose feeding rate of stationary phase at9.152g/h and simultaneously reducing the glucose feeding rate during the exponential phase, the amorphadiene reached6.1g/L, eventually.