Methylorubrum extorquens AM1 is an alpha-proteobacteria that can utilize C1 compounds (e.g., methanol [1], formate [2], and methylamine [3]) as the sole carbon and energy source. As many technologies have been developed to generate these C1 feedstocks from carbon dioxide (CO2) and sustainable energy [4], the C1-fixing ability of this methylotrophic strain represents significant opportunities to produce biomaterials, such as bioplastic [5], microbial proteins [6], and pharmaceuticals [7], from CO2, potentially replacing sugar-based fermentation [8]. Moreover, this C1-activating pathway and enzymes in M. extorquens AM1 provide valuable insight into the development of the enzymatic CO2 conversion or bio-inspired chemical catalysts due to the remarkable selectivity and reaction rate of biocatalysts [9], [10], [11], [12].

Formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) is an attractive enzyme to apply into biocatalytic CO2 conversion to formate [13], [14]. Although four different types of FDHs were reported in this strain, a previous study demonstrated the responsible activity of MeFDH1 for CO2 reduction through gene knockout and whole-cell biocatalytic reaction [15]. Despite many reports suggesting the feasibility of MeFDH1, the enzyme has been obtained from the wild-type strain through multi-step and time-consuming purification [16], [17]. Consequently, the lack of recombinant expression system hinders the application of this CO2-converting enzyme in industrial field. In addition, MeFDH1 requires a homologous expression system in M. extorquens AM1 to express efficiently due to the presence of several unique cofactors required for its maturation [18]. This enzyme is composed of alpha and beta subunits as a heterodimer containing many unique cofactors such as tungsten-bis-(molybdenum guanine dinucleotide) (W-bis-MGD) at the active center, iron-sulfur (Fe-S) cluster, and flavin mononucleotide (FMN) [17].

Despite these interests and challenges in C1-converting microbe, the recombinant expression system for microbial engineering is premature due to a lack of synthetic biology tools and its information (e.g., plasmid stability, untranslated region (UTR), terminator efficiency) [19], while several types of homologous (PmxaF) and hybrid (PR/cmtO and PL/O4) promoters were developed for recombinant expression [20], [21], [22].

Here, we presented the construction of recombinant expression system to increase the expression level of MeFDH1 for efficient CO2 conversion activity in whole-cell biocatalysis. The type of terminator played a significant role in determining the levels of both mRNA and enzyme production. Subsequently, screening of 5’-UTR sequences was carried out with candidates from homologous sequences which were referred to proteomics data of protein abundance [23] and UTR designer (Web server: http://sbi.postech.ac.kr/utr_designer) [24]. This improved expression system resulted in enhanced productivity of formate by the engineered whole-cell biocatalyst.