Abstract
3-Dehydroshikimate (3-DHS) is a key intermediate for the synthesis of various compounds, including the antiviral drug oseltamivir. The Gluconobacter oxydans strain NBRC3244 intrinsically oxidizes quinate to produce 3-dehydroquinate (3-DHQ) in the periplasmic space. Even though a considerable activity is detected in the recombinant G. oxydans homologously overexpressing type II dehydroquinate dehydratase (DHQase) encoded in the aroQ gene at a pH where it grows, an alkaline shift of the culture medium is required for 3-DHS production in the middle of cultivation. Here, we attempted to adopt type I DHQase encoded in the aroD gene of Gluconacetobacter diazotrophicus strain PAL5 because the type I DHQase works optimally at weak acid, which is preferable for growth conditions of G. oxydans. In addition, we anticipated that subcellular localization of DHQase is the cytoplasm, and therefore, transports of 3-DHQ and 3-DHS across the cytoplasmic membrane are rate-limiting steps in the biotransformation. The Sec- and TAT-dependent signal sequences for secretion were attached to the N terminus of AroD to change the subcellular localization. G. oxydans that expresses the TAT-AroD derivative achieved 3-DHS production at a tenfold higher rate than the reference strain that expresses wild-type AroD even devoid of alkaline shift. Enzyme activity with the intact cell suspension and signal sequence cleavage supported the relocation of AroD to the periplasmic space. The present study suggests that the relocation of DHQase improves 3-DHS production in G. oxydans and represents a proof of concept for the potential of enzyme relocation in metabolic engineering.
Key points
• Type-I dehydroquinate dehydratase (DHQase) was expressed in Gluconobacter oxydans.
• Cytoplasmic DHQase was relocated to the periplasmic space in G. oxydans.
• Relocation of DHQase in G. oxydans improved 3-dehydroshikimate production.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank Yoshinori Akiyama (Kyoto University) for invaluable suggestions on signal peptide cleavage. We are grateful to Hirohide Toyama (University of Ryukyus) who continuously encouraged us throughout this study. We thank Oriental Yeast (Tokyo, Japan) and Toyobo (Osaka, Japan) for kindly gifting us with yeast extract and genetic engineering kits, respectively.
Funding
This work was supported by MEXT KAKENHI grant number 20H02902 (to TY). Part of this work was funded by the Core to Core Program, which was supported by the Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT).
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KNAK and TY designed the study. KNAK performed most experiments, analyzed the data, prepared the figures, and wrote a draft manuscript. KNAG assisted experiments. MM performed bioinformatics. OA, NK, YA, GT, KM, and TY supervised. NK, KM, and TY edited the manuscript. All authors read and approved the manuscript.
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Nakamura, K., Nagaki, K., Matsutani, M. et al. Relocation of dehydroquinate dehydratase to the periplasmic space improves dehydroshikimate production with Gluconobacter oxydans strain NBRC3244. Appl Microbiol Biotechnol 105, 5883–5894 (2021). https://doi.org/10.1007/s00253-021-11476-8
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DOI: https://doi.org/10.1007/s00253-021-11476-8