Ectoine hydroxylase displays selective trans-3-hydroxylation activity towards l-proline

Ryotaro Hara, Takeyuki Nishikawa, Takuya Okuhara, Kento Koketsu, Kuniki Kino*


研究成果: Article査読

9 被引用数 (Scopus)


l-Hydroxyproline (Hyp) is a valuable intermediate for the synthesis of pharmaceuticals; consequently, a practical process for its production has been in high demand. To date, industrial processes have been developed by using l-Pro hydroxylases. However, a process for the synthesis of trans-3-Hyp has not yet been established, because of the lack of highly selective enzymes that can convert l-Pro to trans-3-Hyp. The present study was designed to develop a biocatalytic trans-3-Hyp production process. We speculated that ectoine hydroxylase (EctD), which is involved in the hydroxylation of the known compatible solute ectoine, may possess the ability to hydroxylate l-Pro, since the structures of ectoine and 5-hydroxyectoine resemble those of l-Pro and trans-3-Hyp, respectively. Consequently, we discovered that ectoine hydroxylases from Halomonas elongata, as well as some actinobacteria, catalyzed l-Pro hydroxylation to form trans-3-Hyp. Of these, ectoine hydroxylase from Streptomyces cattleya also utilized 3,4-dehydro-l-Pro, 2-methyl-l-Pro, and l-pipecolic acid as substrates. In the whole-cell bioconversion of l-Pro into trans-3-Hyp using Escherichia coli expressing the ectD gene from S. cattleya, only 12.4 mM trans-3-Hyp was produced from 30 mM l-Pro, suggesting a rapid depletion of 2-oxoglutarate, an essential component of enzyme activity as a cosubstrate, in the host. Therefore, the endogenous 2-oxoglutarate dehydrogenase gene was deleted. Using this deletion mutant as the host, trans-3-Hyp production was enhanced up to 26.8 mM from 30 mM l-Pro, with minimal loss of 2-oxoglutarate. This finding is not only beneficial for trans-3-Hyp production, but also for other E. coli bioconversion processes involving 2-oxoglutarate-utilizing enzymes.

ジャーナルApplied Microbiology and Biotechnology
出版ステータスPublished - 2019 7月 20

ASJC Scopus subject areas

  • バイオテクノロジー
  • 応用微生物学とバイオテクノロジー


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