Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09

Abstract

Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C−H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co‐synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non‐heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.

Direct Methane Oxidation by Copper- and Iron-Dependent Methane Monooxygenases

“…Coexpression of the Methylomonas methanica MC09 sMMO operon with E. coli GroEL and GroES led to assembly of the MMOH dimer as detected by native PAGE. 351 This MMOH exhibited nitrobenzene oxidation activity at about half the level of M. trichosporium OB3b sMMO and an EPR signal consistent with the presence of a mixed valent Fe(II)Fe(III) center. While the sMMO operon encodes a GroEL homolog, MmoG (Figure 5a), it is unclear whether it interacts with a GroES homolog and MmoG alone is not sufficient to yield soluble MMOH.…”

“…More recently, attempts to express sMMO have focused on coexpression with the E. coli chaperone proteins GroES and GroEL. Coexpression of the Methylomonas methanica MC09 sMMO operon with E. coli GroEL and GroES led to assembly of the MMOH dimer as detected by native PAGE . This MMOH exhibited nitrobenzene oxidation activity at about half the level of M. trichosporium OB3b sMMO and an EPR signal consistent with the presence of a mixed valent Fe­(II)­Fe­(III) center.…”

A rationally designed miniature of soluble methane monooxygenase enables rapid and high-yield methanol production in Escherichia coli

“…sMMO can oxidize a wide range of carbon feedstocks (C1 to C8) directly using intracellular NADH (a native reductant) and thus is a promising enzyme in developing green routes for industrial-scale manufacturing of chemicals; however, the high-throughput biosynthesis of active recombinant sMMO in E. coli-which rapidly grows to a high-cell density and has been used for producing many recombinant proteins at industrial scale-has long been exceptionally difficult because of the structural and functional complexity of sMMO, comprised of three components-MMOH, MMOR, and MMOB. Recently, the co-expression of recombinant sMMO and E. coli chaperonins was reported 50 ; however, the effectiveness of the expression system was not fully evidenced, although catalytic oxidation of p-nitrobenzene was verified with the EPR-based characterization of iron center of hydroxylase. To circumvent this issue, in this work, we attempted a molecular editing of sMMO (from M. capsulatus (Bath)), which is based on an optimal reassembly of the minimal sub-structures (NH 2 -R FAD -ΔHα-COOH) and modified version (retroB) of the three components on the catalytically inert and stable huHF scaffold.…”

“…Compared to the traditional methanol production by various methanotrophs (pure culture of Methylocystis 51 , co-culture of Methylosinus sporium and Methylocella tundrae 52 , etc. ), other recombinant expression systems 50,53,54 , or MMO-mimetic iron-embedded zeolite catalysts [55][56][57] , the recoverable methanol yield and productivity were improved up to 2.9 g/L and 0.11 g/L/h (22 to 55-fold higher than methanotrophic production), respectively, through the in situ methane oxidation in the high-cell-density culture of mini-sMMOexpressing recombinant E. coli. This seems partly due to the fact that, unlike methanotrophs that utilize the methanol produced by MMO as a carbon and energy source, E. coli does not have such a methanol oxidation pathway 58 , indicating that this approach is a superior way of achieving the highest rate of methanol production from methane in microbial cultures.…”