Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production

Abstract

Background As methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry. Results Through three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24–410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with PrpmB-cadA and PDnaA-cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply. Conclusions The tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory. Graphical Abstract

Optimization of electroporation method and promoter evaluation for type-1 methanotroph, Methylotuvimicrobium alcaliphilum

“…A recent report showcased the strength of constitutive promoters such as P DnaA (2.81-fold), P Integrase (3.45-fold), P rpmB (22.09-fold), P (2Fe–2S)-binding protein (41.54-fold) using GFP as reporter protein in Methylomonas sp. DH-1 ( Lee et al, 2021 ). P sps earlier studied by our group ( Awasthi et al, 2022 ) is shown to efficiently perform 2.5-fold increase in gene expression, in this work.…”

Transcription regulation strategies in methylotrophs: progress and challenges

“…So far, diversified promoter libraries have been built to evaluate promoter candidates. The original method of building a library is to amplify the promoters of all genes and detect the interaction with proteins by 2D-PAGE, then further screen through fusing fluorescent proteins in vivo (Lee et al 2021 ). However, the tedious process greatly increases the time cost in this way.…”

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum

“…Rational regulation of gene expression by appropriate promoters is crucial for the efficient production of target products in industrial microorganisms. [ 4 ] Various methods, such as error‐prone PCR, [ 5 ] transcriptome data analysis, [ 6 ] promoter rational design, [ 7 ] have been employed to establish promoter libraries in different bacteria and fungi, which include Methylomonas , [ 8 ] Rhodobacter sphaeroides , [ 9 ] Myxobacteria , [ 10 ] Saccharopolyspora erythraea , [ 11 ] Burkholderiales , [ 12 ] Saccharomyces cerevisiae , [ 13 ] Aspergillus nidulans , [ 14 ] Aspergillus oryzae , [ 15 ] and Trichoderma reesei . [ 16 ] The availability of diverse promoters broadens the potential applications of microbial genetic modification, [ 15 ] such as expressing and screening a single gene to obtain optimal expression intensity and corresponding high‐yield strain.…”