Abstract: In order to produce trans-4-hydroxyproline directly from the conversion of glucose without extra addition of L-proline,site-directed mutagenesis and gene co-expression were used in this experiment to construct the recombinant E. coli. Firstly,two mutations E143 A,K145A were made on glutamate kinase,which was the key enzyme of L-proline biosynthetic pathway,to enhance the L-proline biosynthesis. Then,proline-4-hydroxylase gene was inserted to co-expressed with the mutated glutamate kinase gene. Co-expression of the two genes resulted in a continuous conversion from glucose to trans-4-hydroxyproline,without the extra addition of exogenous L-proline. At the shaking-flask stage,output of L-proline by the biosynthetic capacity enhanced strain reached 1.4 g/L. Yield of trans-4-hydroxyproline by the co-expression strain of pro BA2 and hyp was 96.9 mg/L,which doubled than the original strain. After the fermentation optimization,the optimum medium was:glucose 10 g/L,tryptone 15 g/L,ferrous sulfate 3 mmol/L,magnesium sulfate 1 g/L,dipotassium phosphate3 g/L,calcium chloride 0.015 g/L. The recombinant E. coli was cultured with the optimized medium for 24 h.The output of trans-4-hydroxyproline was achieved at 220.0 mg/L,a 1.3-fold increase than the original strain.