A synthetic gene encoding a Streptomyces L-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250L A biotransformation process was developed using cell suspensions of the recombinant E. colt and subsequently scaled-up to 10 L for conversion of L-proline to cis-3-hydroxy-L-proline. A reaction yield of 85 M% and d e. of 99.9% was obtained for cis-3-hydroxy-L-proline. (C) 2009 Elsevier Inc All rights reserved.