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Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

  • Applied genetics and molecular biotechnology
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Abstract

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.

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Acknowledgments

We greatly appreciate Dr. Akira Nishimura and Ryo Nasuno (Nara Institute of Science and Technology, Japan) for their helpful assistance and discussion on this work. We thank Dr. Goh Matsuo (Institute for Advanced Biosciences, Keio University) and Dr. Hirotada Mori (Nara Institute of Science and Technology, Japan) providing N-acetyl CHOP, E. coli strains, and plasmids for this works. This work was supported by a Grant-in-Aid for Scientific Research (B) (22380061) from Japan Society for the Promotion of Science to H.T. and Global COE Program in NAIST from the Ministry of Education, Science, Culture, Sports and Technology of Japan.

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Authors and Affiliations

  1. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

    Thi Mai Hoa Bach, Iwao Ohtsu, Nobuyuki Yoshida & Hiroshi Takagi

  2. Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo, 169-8555, Japan

    Ryotaro Hara & Kuniki Kino

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  1. Thi Mai Hoa Bach
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  2. Ryotaro Hara
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  3. Kuniki Kino
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  5. Nobuyuki Yoshida
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Correspondence to Hiroshi Takagi.

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Bach, T.M.H., Hara, R., Kino, K. et al. Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1. Appl Microbiol Biotechnol 97, 247–257 (2013). https://doi.org/10.1007/s00253-012-4204-z

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