Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate.

Carsten Haupka,Volker F Wendisch,Daniel Wibberg,Luciana F Brito,Tobias Busche

doi:10.3389/fmicb.2021.664598

Carsten Haupka, Volker F Wendisch + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2021.664598

Copy DOI

Abstract

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4–C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants—AVA6 and AVA10—with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

Highlights

The production of bio-based plastics is predicted to increase in the recent future1
To further our physiological understanding of L-lysine metabolism in B. methanolicus and as a basis for application to the methanol-based production of Llysine and L-lysine-derived compounds, we studied the response of this methylotroph to these compounds
B. methanolicus was grown in a medium with methanol as a carbon and energy source in the presence of various concentrations of L-lysine and its potential degradation products (Supplementary Figure 1) as well as some structural analogs differing in carbon chain lengths (Figure 1)

Summary

Introduction

The production of bio-based plastics is predicted to increase in the recent future. Polyamides belong to plastics, and they can be synthesized chemically in two ways: (1) via anionic ring-opening polymerization of lactams derived from ω-amino acids and (2) condensation of diamines with dicarboxylic acids. Microorganisms used in biotechnological industry at large scale, e.g., Escherichia coli and Corynebacterium glutamicum, an industrial producer of about 2.6 million tons of L-lysine in 2018 (Wendisch, 2020), are a suitable choice for the sustainable production of polyamide precursors by fermentation. The fermentative production of C4 and C5 dicarboxylic acids, for example, has been achieved with metabolically engineered E. coli and C. glutamicum strains (Pérez-García et al, 2018; Rohles et al, 2018; Chae et al, 2020). The C4 ω-amino acid γ-aminobutyrate (GABA) can be produced by E. coli and C. glutamicum strains overproducing L-glutamate and expressing a glutamate decarboxylase (Takahashi et al, 2012; Xiong et al, 2017) or by extending the putrescine production pathway by heterologous expression of putrescine transaminase and γ-aminobutyraldehyde dehydrogenase genes (Jorge et al, 2016). C. glutamicum strains overproducing L-lysine were engineered for production of 5-aminovalerate (5AVA), and three alternative biosynthesis pathways were established (Supplementary Figure 1; Shin et al, 2016; Pérez-García et al, 2018; Haupka et al, 2020)

Objectives

Methods

Results

Conclusion