Construction And Fermentation Optimization Of A Escherichia Coli For The Production Of Trans-4-Hy Droxyproline Without Extra Addition Of L-proline

Trans-4-hydroxy-L-proline,the hydroxylation products of L-proline, is widely used in many industries. Combination of L-proline biosynthetic pathway and the hydroxylation reaction catalyzed by proline 4-hydroxylase could produce trans-4-hydroxyproline directly from the conversion of glucose without extra addition of L-proline, which could also save the costs and make full use of the microbial resources.Firstly, a recombinant E. coli with enhanced L-proline biosynthesis was constructed. E143 A, K145 A mutations were performed in glutamate kinase to get the mutant pro BA2 by PCR of the whole plasmid. A tryptophan tandem promoter was fused with the mutated gene pro BA2 and a recombinant plasmid p ET28a-Ptrp2-pro BA2 was constructed. The E. coli strain containing the recombinant plasmid was cultured to verify the output of L-proline, which reached 1.4 g/L after fermented 24 h. Compared to the original strain, the accumulation of L-proline increased 26-fold.Then, the recombinant E. coli capable of producing trans-4-hydroxypline without the extra addition of L-proline was constructed. Two plasmids, p ET28a-Ptrp2-pro BA2 and p UC19-Ptrp2-hyp containing the two genes pro BA2 and hyp,respectively, were used to construct the co-expression systems. Two single-plasmid co-expression systems and a double-plasmid co-expression system were constructed. Co-expression of the two genes resulted in a continuous conversion from glucose to trans-4-hydroxyproline. The recombinant E. coli could be cultured in the medium with glucose as the single carbon source and without exogenous L-proline. Among the three constructed co-expression systems, the trans-4-hydroxypline production of the recombinant E. coli with a co-expression system based on the plasmid p UC19 was the highest. After 24 h of shaking-flask fermentation, the production of trans-4-hydroxyproline was 98.9 mg/L, increased 1-fold compared with the original strain.Finally, the fermentation medium was optimized, which was used to produce trans-4-hydroxypline without the extra addition of exogenous L-proline of the recombinant E. coli. After the optimization by single factor and orthogonal experimental analysis, the optimum medium was followed: glucose 10 g/L, tryptone 15 g/L, ferrous sulfate 3 mmol/L, magnesium sulfate 1 g/L, dipotassium phosphate 3 g/L, calcium chloride 0.015 g/L, initial p H 7.0. The recombinant E. coli was cultured with the optimized medium for 24 h. The production of trans-4-hydroxyproline reached 220.0 mg/L, a 1.25-fold increase compared with the original strain.