Shibasaki T, Mori H, Ozaki A
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, Machida, Japan.
Biosci Biotechnol Biochem. 2000 Apr;64(4):746-50. doi: 10.1271/bbb.64.746.
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.
从指孢囊菌属菌株RH1中克隆出的脯氨酸4-羟化酶基因，在对该基因5'端的密码子使用进行优化后，在色氨酸串联启动子控制下的质粒上于大肠杆菌W1485中过表达。脯氨酸4-羟化酶活性比指孢囊菌属菌株RH1中的活性高1600倍。当重组大肠杆菌在含有L-脯氨酸和葡萄糖的培养基中培养时，反式-4-羟基-L-脯氨酸（Hyp）在100小时内产生并积累至41 g/L（L-脯氨酸的产率为87%）。脯氨酸4-羟化酶将L-脯氨酸羟基化所必需的2-氧代戊二酸显然是通过细胞代谢途径从葡萄糖中提供的。W1485的putA突变体不能降解L-脯氨酸，这使得L-脯氨酸能够定量转化为Hyp。未观察到羟脯氨酸其他异构体的形成。在大规模培养中，Hyp的生产率几乎相同。建立了从L-脯氨酸生产Hyp的方法。
