Trans‐4‐Hydroxy‐l‐proline (trans‐Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Bioconversion of l‐proline using recombinant strain with proline‐4‐hydroxylase (P4H) is a preferred biocatalytic process in the economical production of trans‐Hyp. In this study, a recombinant E. coli overexpressing hydroxylase (P4H), γ‐glutamyl kinase and glutamate‐semialdehyde dehydrogenase (ProBA) genes were constructed by knocking out the key genes in the metabolism. These key genes contained putA encoding proline dehydrogenase (PutA) in the l‐proline metabolism and other catalytic enzyme genes, sucAB encoding α‐ketoglutarate dehydrogenase (SucAB), aceAK encoding isocitratelyase (AceA) and isocitrate dehydrogenase kinase/phosphatase (AceK) in the TCA cycle. This recombinant strain coupled the synthetic pathway of trans‐Hyp with TCA cycle of the host strain. It inhibited the consumption of l‐proline completely and promoted the accumulation of 2‐oxoglutarate (2‐OG) as a co‐substrate, which realized the highest conversion of glucose to trans‐Hyp. A fed‐batch strategy was designed, capable of producing 31·0 g l−1 trans‐Hyp from glucose. It provided a theoretical basis for commercial conversion of glucose to trans‐Hyp. Significance and Impact of the Study:Trans‐4‐Hydroxy‐l‐proline (trans‐Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans‐Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans‐Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short‐cut and efficient trans‐Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans‐Hyp. Significance and Impact of the Study:Trans‐4‐Hydroxy‐l‐proline (trans‐Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans‐Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans‐Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short‐cut and efficient trans‐Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans‐Hyp.