An Escherichia coil recombinant strain producing trans-4-hydroxy-L-proline (Hyp) was constructed by introducing a proline 4-hydroxylase gene into an L-proline-producing E. coil. Plasmid pPF1, which contains a gene encoding feedback resistant gamma -glutamyl kinase (proB74), was constructed and introduced into E. coil W1485 putA. The recombinant E. coli W1485 putA/pPF1 strain produced L-proline (1.2 g/l). The proline production by W1485 putA/pPF1 was converted to Hyp production by introducing pWFH1 which contains a proline 4-hydroxylase gene. E. coil W1485 putA which harbors pWFP1 carrying the proline I-hydroxylase gene, proB74, and proA produced 25 g/l of Hyp in 96 h. A novel biosynthetic pathway of Hyp, which has not previously been produced in E, coil, was constructed in E. coil.