Optimization Of Fermentation Conditions For The Production Of Isoprene By The Recombinant Escherichia Coli YJM23

Isoprene is the simplest monomer substances of the terpene family, and itspolymers have been widely used in the rubber, food, spices, aviation fuel, organicpesticides, medicine and other fields. In view of the shortcomings of the currentindustrial production of isoprene, such as the depletion of oil resources, the high oilresources pollution, the biological production of isoprene has received an everincreasing interest. While it is low concentrations, difficult to collect and affected by theclimate and growth cycle by the natural plant and microbial production, it is verypromising by microorganism producting isoprene using genetic engineering. E. coli wasto selected as the host strain to product isoprene with the clear genetic background,simple operation and other advantages. It is very important to improve the isopreneproduction that study E. coli fermentation process and the optimization of fermentationconditions.In this thesis the following have been studied:Molecular identification of plasmid and the genetic characteristics of the doubledigestion in engineering E. coli; Separation and purification of fermentation products;Structure identification of target product using nuclear magnetic resonance technology;Establish quantitative methods of the product. DNA gel electropHoresis results showedthat plasmid size and fragment size and the corresponding theoretical value areconsistent, and the engineering bacteria were successfully constructed. Nucler magneticresonance（NMR） results showed that of the product chemical structure is isoprene, andquantitative analysis method by meteorological chromatograpHy and a standard curveare established: Y=747.80+38651.65X.Fermentation medium were optimized in shake flask level at the early stage offermentation. firstly, Important carbon and nitrogen sources and organic acids werescreened, glucose, beef extract and citric acid with great influence on the isopreneproduction were selected. Secondly, the basic scope of the various components’ optimalvalue was determined: Beef extract6g/L-8g/L, Glucose15g/L-25g/L, MgSO₄0.2mL/L-0.8mL/L, FeC₆H₅O₇NH4OH0.2g/L-0.4g/L, C6H8O7H2O1.1g/L-3.1g/L, Traceelement solution0.7mL/L-1.1mL/L, K₂HPO₄3H₂O5g/L-15g/L. Plackett-Burmandesign of eight factors and two levels was used to evaluate the effect of each mediumcomponent, and the significant factors affecting isoprene production were beef extract,trace elements mixture and dipotassium hydrogen pHospHate. And then steepest ascentprocedures were applied to define optimal response region for three key factors: Beefextract:6.5g/L-8.5g/L, Trace element mixture:0.047mL/L-0.153mL/L, K₂HPO₄3H₂O: 9.8g/L-13.4g/L. Finally, the ternary quadratic was determined adopting response surfacemethod based on central composite design, and the best ratio of medium were identified:K₂HPO₄3H₂O10.9g/L, Glucose20g/L, Beef extract7.7g/L, C₆H₈O₇H₂O6.3g/L,FeC₆H₅O₇0.3g/L, MgSO₄0.5mmol/L and （NH₄）₂SO₄1g/L. isoprene production afteroptimazation of the fermentation medium increased from140.16mg/L to935.02mg/L,increased by567.11%compared with the starting medium.It is investigated that adjusting pH by ammonia and sodium hydroxide in the shakeflask level, and the ammonia as the optimum pH regulator was determined. Inducerconcentration is optimized and the optimal induction concentration is0.05mmol/L.In the process of5L fermentor level optimization, the growth curve of the seedcells was draw, and the best vaccination time is about12h for the seed culture. The cellgrowth curve in the fermentation tank was drawed, and the optimal induction time isdetermined: OD600for12or so. Contrast to LB medium, M9medium and optimized M9culture medium, results showed that the optimized M9medium are best suited for theisoprene procuction of E.coli. Through optimization of the different inductiontemperature and dissolved oxygen level, the induction temperature of30℃anddissolved oxygen level of20%are most suitable for producing isoprene for engineeringE. coli fermentation.