Recombinant Escherichia Coli For Tyrosol Fermentation Optimization And Metabolic Modification

Tyrosol is an important phenolic compound naturally occurring in nature.Because of its natural antioxidant activity,it is widely used in food,chemical and pharmaceutical fields.In the preliminary work of this research group,Escherichia coli MG1655 was metabolically engineered to construct a recombinant strain YMG5A*R（E.coliΔfea BΔphe AΔtyr BΔtyr R lac I::ARO10*trp E::ARO10*pab B::ARO10*pab A::ARO10*pyk F::ARO10*）.This thesis explores ways to increase the yield and fermentation speed of tyrosol from the two directions of fermentation process and construction of new strains.The specific research results are as follows:For the recombinant strain YMG5A*R,the medium composition and fermentation conditions were optimized at the shake flask level by a single factor method,and the response surface method was used to further optimize the results of the single factor experiment to obtain the optimal fermentation conditions for E.coli YMG5A*R.The ingredients of the shake flask medium are:on the basis of M9Y medium,the concentration of Ca²⁺is 1.63mmol·L^-1,ammonium sulfate is 3.28 g·L^-1,and the optimum fermentation temperature is27℃.Under the optimal conditions,the fermentation time was 60 h,and the tyrosol yield was 2.80 g·L^-1.The fermentation level of the shake flask under the optimized conditions was80%higher than the fermentation level of recombinant E.coli tyrosol under the starting conditions.According to the fermentation conditions of shake flasks,the optimal fermentation and feeding method of YMG5A*R under the conditions of 5 L fermenter was further explored.Through the comparison of batch fermentation,constant-rate flow-addition fermentation,and constant-p H index-flow-addition fermentation,methods to improve the yield of tyrosol were explored.The experimental results show that the constant p H index flow feeding method can better meet the requirements of strain growth and tyrosol fermentation.Under optimized conditions,after 48 h of fermentation,the OD₆₀₀reaches about 36,and the tyrosol yield reaches 4.53 g·L^-1,compared with batch fermentation,the yield of tyrosol increased by81.2%.Aiming at the cytotoxicity of tyrosol,the method of organic solvent extraction for two-phase fermentation was explored,and octanol was finally selected as the organic extraction solvent.Using two-phase fermentation,after 48 h of fermentation in a 5 L fermentor,the tyrosol output reached 5.62 g·L^-1.In view of the long fermentation time of existing experimental strains in tyrosol fermentation,a method of screening vigorously growing E.coli host bacteria from nature was explored to increase the biomass of recombinant bacteria and further accelerate the speed of tyrosol fermentation.197 E.coli strains were screened from the feces of various animals in nature,and the key enzyme producing tyrosol,phenylpyruvate decarboxylase gene ARO10*was transferred,and high-biomass recombinant bacteria producing tyrosol were screened,and finally high-expressing ARO10*was screened.E.coli host strain YEC166 with higher growth rate and higher biomass is still maintained after.Compared with YMG5A*R,the recombinant strain YEC166/p KK223-3-ARO10*constructed with YEC166 as the host strain has a greater increase in cell density.The wild-type host strain YEC166 was metabolically engineered to knock out the tyrosol-producing competition pathway genes fea B,phe A,tyr B and the aromatic regulatory repressor gene tyr R to obtain the recombinant strain YEB166Δfea BΔphe AΔtyr BΔtyr R,which was further transformed into a high-expressing tyrosol-producing key gene The recombinant plasmid p KK223-3-ARO10*was used to obtain the recombinant strain E.coli YC166.The cell density of the recombinant strain shake flask reached OD₆₀₀to 13.2,and the tyrosol yield was 2.13 g L^-1.Although the yield increased insignificantly,the fermentation time was shortened to 24 h.In a 5 L fermentor,fed-feed fermentation with constant p H index flow,cell density OD₆₀₀reached about 31,tyrosol accumulation reached 3.1 g·L^-1,and fermentation time was 24 h.The fermentation conditions of YEC166 were further optimized and maltose was determined to be the best carbon source.Under constant p H-exponential fed-batch fermentation conditions for 24 h,the final OD₆₀₀of YC166 fermentation reached 40 and the tyrosol yield was 4.28 g·L^-1.Compared with YMG5A*R,although the yield is slightly reduced,the fermentation time is shortened by 50%.