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.

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