Production of dicarboxylates from ω-amino acids using a cofactor- and co-substrate-free in vitro biosynthetic system

Abstract

Dicarboxylates are valuable platform compounds with a broad range of applications. The in vitro biosynthetic system used to produce dicarboxylates from ω-amino acids via the natural pathway requires costly cofactors and co-substrates, which restricts its economic feasibility of use. In this study, we designed a cofactor- and co-substrate-free artificial pathway for the production of dicarboxylates from ω-amino acids. Only three enzymes (viz., amine oxidase from Kluyveromyces marxianus DMKU3-1042, xanthine oxidase from bovine milk, and catalase from Aspergillus niger) were required for dicarboxylate production. Succinate (0.79 g g^-1), glutarate (0.83 g g^-1), and adipate (0.77 g g^-1) were produced in high yields from the corresponding ω-amino acids through the in vitro biosynthetic system with the artificial pathway. Glutarate could also be produced from l-lysine by further introducing l-lysine monooxygenase and 5-aminovaleramide amidohydrolase from Pseudomonas putida KT2440 into the in vitro biosynthetic system, with the cofactor- and co-substrate-free system achieving a product yield of 0.63 g g^-1. Considering its desirable characteristics, this artificial pathway-based in vitro biosynthetic system may be a promising alternative for dicarboxylate production from biotechnologically produced ω-amino acids.

Keywords

Dicarboxylate / ω-amino acid / Cofactor / Artificial pathway / In vitro biosynthetic system

Data Availability Statement

The data supporting the findings of this study are included in this published article as well as in the Supplementary Materials available online.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Jinxin Yan: Writing - original draft, Data curation. Hui Zhang: Writing - review & editing, Data curation. Hongxu Zhang: Writing - review & editing, Data curation. Hairong Yu: Investigation. Wenjia Tian: Investigation. Mingyuan Liu: Investigation. Weikang Sun: Investigation. Leilei Guo: Investigation. Xiaoxu Tan: Investigation. Kaiyu Gao: Investigation. Tianyi Jiang: Funding acquisition. Chuanjuan Lü: Writing - review & editing, Validation, Supervision, Funding acquisition, Conceptualization. Qianjin Kang: Validation, Investigation. Wensi Meng: Funding acquisition. Cuiqing Ma: Validation, Investigation. Chao Gao: Writing - review & editing, Validation, Funding acquisition, Conceptualization. Ping Xu: Validation, Investigation.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (32300045, 32300029), Youth Program of Natural Science Foundation of Shandong Province (ZR2022QC092, ZR2023QC237), China Postdoctoral Science Foundation (2023M742085), Youth Program of Natural Science Foundation of Qingdao City (23-2-1-31-zyyd-jch), Postdoctoral Innovation Program of Shandong Province (SDCX-ZG-202400150), Qingdao Postdoctoral Research Project (QDBSH20230201011), State Key Laboratory of Microbial Technology Open Projects Fund (M2023-02), Open Funding Project of State Key Laboratory of Microbial Metabolism (MMLKF24-07), China Postdoctoral Science Foundation (2024M751800) and State Key Laboratory of Microbial Technology (SKLMTFCP-2023-03). We also thank Dr. Jing Zhu, Dr. Jingyao Qu, Dr. Zhifeng Li, and Dr. Guannan Lin from State Key Laboratory of Microbial Technology for assistance in mass spectrographic analysis.

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