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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Aims:We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. Methods and Results: Yarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2-hydroxyacetophenone (2-HAP) to optically pure (S)-1-phenyl-1,2-ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6Á96 U mg À1 (99Á8% e.e.) and 7Á85 U mg À1 (99Á9% e.e.), respectively, and showed moderate heat resistance at 40-50°C and acid tolerance at pH 5Á0-6Á0. An efficient whole-cell two-phase system was established using reductase-expressing recombinant Escherichia coli. The conversion of 2-HAP (20Á0 g l À1 ) conversion with the solvent of dibutyl phthalate was approximately 70-fold higher than in water. Furthermore, the two recombinant E. coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E. coli BL21 (DE3)/pET-28a-yacrII showed a broad substrate spectrum. Conclusions: A new whole-cell recombinant E. coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. Significance and Impact of the Study: We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.

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