Improved And Applicated Of Glucaric Acid-responsive Promoter In Escherichia Coli

Gluconic acid(GA),a high-value bio-refined product,is widely used in food,chemical and medical fields.At present,most GA on the market is obtained through chemical synthesis methods.However,this method has the disadvantages of expensive catalysts,many by-products and pollution,which limits the expansion of the GA in industry.Cell-based GA biosynthesis has the advantages of high efficiency,pollution-free and low cost,and is a potential method to replace chemical methods in the synthesis of commercial GA in the future.The research of GA biosynthesis pathway has made rapid development in the past ten years.In 2008,the MIT scientific research team first done metabolic engineering transformation in E.coli to make it capable of synthesizing GA.Since then,a large number of synthetic biology methods have been applied to the GA synthesis pathway and the yield of GA has been improved.However,the current GA biosynthesis method has the following problems,which limit the further improvement of GA production:(1)Myo-inositol oxygenase(MIOX)is the key rate-limiting enzyme in the GA synthesis pathway,and the enzyme activity is weak(2)When E.coli is used as the host cell,GA influences the growth of the host cell due to its acid toxicity.Compared with E.coli,yeasts have the advantages of fast growth and high acid tolerance.It may be a better choice to use yeas as the host rather than E.coli.In addition,biosensors are widely used in synthetic biology research because they can effectively respond to the concentration of small molecules.The biosensor that responds to GA was first discovered in E.coli by the Church's team,but the working mechanism of GA-responsive promoter has not been reported yet.In the previous research of our group,we successfully analyzed the core region of GA-responsive promoter and made predictions about its working mechanism.Based on the core region of the GA-responsive promoter and its work mechanism,the thesis improved the GA-responsive promoter and applied the improved promoter to the improvement of GA production.The research content is as follows:(1)Based on the GA-responsive promoter F291-390,this thesis applies it to the construction of a screening tool for GA high-yielding strains.In this paper,three screening tools for GA high-yielding strains are designed according to the different screening principles.Put the tetracycline resistance gene(tetA),SDS resistance gene(tolC),and kana resistance gene(kanaR)as reporter genes downstream of the GA-responsive promoter,so that the transcription level of the resistance gene is regulated by the GA concentration.Therefore,the growth rate of the strain was coupled with the GA concentration to establish a linear relationship under a certain external screening pressure.When tetA is used as a screening gene for GA high-yielding strains,GA enters into the cell in large quantities,making the growth rate of bacteria slow due to acidosis;when tolC is used as a screening gene for GA high-yielding strains,the strains will agglomerate in a higher concentration of SDS solution.And some deaths are not conducive to subsequent subculture;when kanaR is used as a screening gene for GA high-yielding strains,it can effectively screen GA high-yielding strains,but the screening efficiency of this screening system is low,and a promoter with a stronger ability to respond to GA is needed.(2)Randomly mutation of the core region of the GA-responsive promoter was generated by using error-prone PCR and the diverse mutant promoters were respectively constructed on a plasmid expressing GFP and transferred to DH5?.By measuring the fluorescence intensity of the cell at different concentrations of GA,4 promoter mutants with different degrees of enhanced GA-responive ability were screened from 500 mutant strains.Among them,the dynamic range of the promoter mutant of F291-390-164del-165del-177G-188A response to GA was 7.2 times that before the mutation,and the sensitivity was increased by 9.2 times.(3)The GA-responsive mutant promoter of F291-390-164del-165del-177G-188A was used in the construction of GA high-yielding strain screening tool again.According to the discussion and analysis in the previous chapter,the kanamcillin resistance gene(kanaR)in this chapter is placed as a reporter gene downstream of the GA response mutant promoter of F291-390-164del-165del-177G-1 88A,so that the transcription level of the resistance gene is regulated by the GA concentration.Therefore,the growth rate of the strain is coupled with the GA concentration to establish a linear relationship under a certain external screening pressure.Through a comprehensive comparison of the effectiveness of the screening system,a GA high-yielding strain screening tool based on the kanamcillin resistance gene was successfully constructed,a screening system for GA high-yielding strains based on kanamycin resistance genes was successfully constructed,which provides an effective technology platform for subsequent high-throughput screening of key catalytic molecules and high-yielding strains.