Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens.

Chandran Sathesh-Prabu,Young Shin Ryu,Sung Kuk Lee

doi:10.3389/fbioe.2021.797020

Abstract

Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/P hpdH ) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%–100%) to that of the widely used strong promoters such as P mxaF and P L/O4 . The HpdR/P hpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.

Highlights

Microbial utilization of single-carbon (C1) compounds as a feedstock to produce fuels, chemicals, and other value-added products is an ideal strategy to combat the depletion of fossil fuels and environmental pollution (Humphreys and Minton, 2018)
In our previous study (Sathesh-Prabu et al, 2021), we evaluated the efficiency of these inducible expression systems in Pseudomonas putida KT2440 by constructing promoter candidates in the pPROBE_eGFP+ plasmid (Kim et al, 2019), which served as a template for PCR amplification in the present study
The inducibility of the constructed expression systems was analyzed by measuring the green fluorescent protein (GFP) fluorescence of the strains cultivated in minimal salt medium (MSM) supplemented with glucose (MRHZ01), levulinic acid (LA) or 3-hydroxypropionic acid (3-HP) (MLRL01, MHRH01, and MMRM01), xylose (MXRX01), or IPTG (MPLO4)

Summary

Introduction

Microbial utilization of single-carbon (C1) compounds as a feedstock to produce fuels, chemicals, and other value-added products is an ideal strategy to combat the depletion of fossil fuels and environmental pollution (Humphreys and Minton, 2018). When cultivated on media containing formate or methanol, the metabolic flux largely goes through the tetrahydrofolate, serine, and EMC pathways, generating a stable supply of acetyl-CoA, which could serve as a precursor for the production of value-added chemicals in engineered M. extorquens AM1 strains (Peyraud et al, 2011; Zhang et al, 2019; Hwang et al, 2020). This strain has been engineered to produce a variety of value-added chemicals or biofuels such as PHB (Hwang et al, 2020; Yoon et al, 2021), polyhydroxyalkanoate terpolymers (Orita et al, 2014), 1-butanol (Hu and Lidstrom, 2014), 3-hydroxypropionic acid (3-HP) (Yang et al, 2017), mevalonic acid (Zhu et al, 2016), butadiene precursor (Yang et al, 2018), terpenoids (Sonntag et al, 2015a), mesaconic acids (Sonntag et al, 2015b), crotonic acids (Schada Von Borzyskowski et al, 2018), and itaconic acids (Lim et al, 2019) from formate or methanol

Objectives

Methods

Results

Conclusion