An Artificial Pathway for N-Hydroxy-Pipecolic Acid Production From L-Lysine in Escherichia coli.

Zhou Luo,Hanmei Li,Jiamin Zhang,Lixiu Yan,Bangxu Wang,Yao Lu,Zhiping Zhao,Jie Cheng,Wei Wang,Zhen Wang,Ting Bai

doi:10.3389/fmicb.2022.842804

Abstract

N-hydroxy-pipecolic acid (NHP) is a hydroxylated product of pipecolic acid and an important systemic acquired resistance signal molecule. However, the biosynthesis of NHP does not have a natural metabolic pathway in microorganisms. Here, we designed and constructed a promising artificial pathway in Escherichia coli for the first time to produce NHP from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs six enzymes to sequentially convert lysine into NHP. This artificial route involves six functional enzyme coexpression: lysine α-oxidase from Scomber japonicus (RaiP), glucose dehydrogenase from Bacillus subtilis (GDH), Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida (DpkA), lysine permease from E. coli (LysP), flavin-dependent monooxygenase (FMO1), and catalase from E. coli (KatE). Moreover, different FMO1s are used to evaluate the performance of the produce NHP. A titer of 111.06 mg/L of NHP was yielded in shake flasks with minimal medium containing 4 g/L of lysine. By this approach, NHP has so far been produced at final titers reaching 326.42 mg/L by 48 h in a 5-L bioreactor. To the best of our knowledge, this is the first NHP process using E. coli and the first process to directly synthesize NHP by microorganisms. This study lays the foundation for the development and utilization of renewable resources to produce NHP in microorganisms.

Highlights

Plant metabolites play an important role in plant defense, since they can directly harm attacking pathogens, preventing pathogens from entering plant tissues (Chezem et al, 2017)
The nucleotide sequences of lysine α-oxidase gene raip from Scomber japonicus, piperidine-2-carboxylic acid (Pip2C) reductase gene dpkA from Pseudomonas putida, glucose dehydrogenase gene gdh from Bacillus subtilis, and lysine permease gene lysP from E. coli are available in the GenBank with accession numbers MG423617, MG423618, MG425967, and WP_000253273.1, respectively
N-hydroxy-pipecolic acid (NHP) can be produced in Arabidopsis, tomato (Holmes et al, 2019), and cucumber (Schnake et al, 2020), it has not been reported in microorganisms

Summary

Introduction

Plant metabolites play an important role in plant defense, since they can directly harm attacking pathogens, preventing pathogens from entering plant tissues (Chezem et al, 2017). With the great development of metabolic engineering and synthetic biology, more and more high-value chemicals can be produced from renewable raw materials by natural or artificial pathways in microorganisms (Cheng et al, 2021a). There are many effective strategies that have been developed and used to improve the production of target chemicals, such as enzyme engineering (Ting et al, 2021), cofactor engineering (You et al, 2021), transcription factor engineering (Kang et al, 2021), promoter engineering (Gao et al, 2020), modularity engineering (Cen et al, 2021; Osire et al, 2021), ribosome binding site engineering (Wang et al, 2018), pathway engineering (Wang et al, 2021), fine-tuning gene expression (Wang et al, 2015), biosensor technology (Li et al, 2019), high-through screening (Zeng et al, 2020), and so on

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Conclusion