Microbial Proline Racemase-Proline Dehydrogenase Cascade for Efficient Production of D-proline and N-boc-5-hydroxy-L-proline from L-proline.

D-proline and N-boc-5-hydroxy-L-proline are key chiral intermediates in the production of eletriptan and saxagliptin, respectively. An efficient proline racemase-proline dehydrogenase cascade was developed for the enantioselective production of D-proline. It included the racemization of L-proline to DL-proline and the enantioselective dehydrogenation of L-proline in DL-proline. The racemization of L-proline to DL-proline used an engineered proline racemase (ProR). L-proline up to 1000 g/L could be racemized to DL-proline with 1 g/L of wet Escherichia coli cells expressing ProR within 48 h. The efficient dehydrogenation of L-proline in DL-proline was achieved using whole cells of proline dehydrogenase-producing Pseudomonas pseudoalcaligenes XW-40. Moreover, using a cell-recycling strategy, D-proline was obtained in 45.7% yield with an enantiomeric excess of 99.6%. N-boc-5-hydroxy-L-proline was also synthesized from L-glutamate semialdehyde, a dehydrogenated product of L-proline, in a 16.7% yield. The developed proline racemase-proline dehydrogenase cascade exhibits great potential and economic competitiveness for manufacturing D-proline and N-boc-5-hydroxy-L-proline from L-proline.