Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline.
A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized. The proline 4-hydroxylase activity was l600-fold higher than that in Dactylosporangium sp. RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.
Shibasaki
T
T
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, Machida, Japan.
Mori
H
H
Ozaki
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002264:Carboxylic Acids; D015870:Gene Expression; D005822:Genetic Vectors; D006900:Hydroxylation; D006909:Hydroxyproline / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D013237:Stereoisomerism
D002264:Carboxylic Acids; D007656:Ketoglutaric Acids; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline