Division of Agricultural Science, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan.
Joint Degree Program of Kasetsart University and Yamaguchi University, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan.
Appl Microbiol Biotechnol. 2021 Aug;105(14-15):5883-5894. doi: 10.1007/s00253-021-11476-8. Epub 2021 Aug 14.
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.
3-去氢莽草酸（3-DHS）是合成各种化合物的关键中间体，包括抗病毒药物奥司他韦。氧化葡萄糖酸杆菌 NBRC3244 株内在地将奎宁酸氧化为 3-去氢奎宁酸（3-DHQ），产生于周质空间。尽管在重组 G. oxydans 中检测到相当大的活性，该重组菌过表达了编码在 aroQ 基因中的 II 型脱水酶（DHQase），在其生长的 pH 值下，但在培养中期生产 3-DHS 需要将培养基的 pH 值碱化。在这里，我们试图采用来自 Gluconacetobacter diazotrophicus 菌株 PAL5 的编码 I 型 DHQase 的 aroD 基因，因为 I 型 DHQase 在弱酸下工作最佳，这有利于 G. oxydans 的生长条件。此外，我们预计 DHQase 的亚细胞定位是细胞质，因此，3-DHQ 和 3-DHS 穿过细胞质膜的运输是生物转化中的限速步骤。将用于分泌的 Sec 和 TAT 依赖性信号序列附加到 AroD 的 N 末端，以改变亚细胞定位。表达 TAT-AroD 衍生物的 G. oxydans 实现了比表达野生型 AroD 的参考菌株高十倍的 3-DHS 生产速率，甚至无需碱性转移。具有完整细胞悬浮液的酶活性和信号序列切割支持 AroD 向周质空间的重新定位。本研究表明，DHQase 的重新定位提高了 G. oxydans 中 3-DHS 的产量，并为酶重新定位在代谢工程中的潜力提供了概念验证。要点：• 在氧化葡萄糖酸杆菌中表达了 I 型脱水酶（DHQase）。• 在 G. oxydans 中，DHQase 从细胞质重新定位到周质空间。• G. oxydans 中 DHQase 的重新定位提高了 3-去氢莽草酸的产量。
