Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

Abstract

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance

“…The (2 S )-sakuranetin titer decreased with increasing (2 S )-naringenin concentrations, which may due to the low tolerance of strain to (2 S )-naringenin. Ribosomal subunits (RpsQ, RpsQ His31Pro , and RpsL [ 26 ]), molecular chaperones (SecB [ 27 ], Ycdy [ 28 ], Nfua [ 29 ], and Yajl [ 30 ]), sRNAs (RpoS [ 31 ], ProQ [ 32 ], NusB [ 33 ], AcrR [ 34 ], and Asr [ 35 ]), and the global regulatory transcription factor CRP [ 36 ], which can participate in cellular stress response, were expressed to select the most suitable gene to improve strain tolerance to (2 S )-naringenin. (2 S )-Naringenin was supplemented in batches to a final concentration of 400 mg/L, 500 mg/L and 600 mg/L (substrate was added as described in Section 3.4 ).…”

“…Strain NS21, NS23 and NS24 harbor plasmid pET-28a (+)- PfOMT3 - rpsQ His31Pro , pET-28a (+)- PfOMT3 - rpoS and pET-28a (+)- PfOMT3-secB , respectively. Genes rpsQ , rpoS and secB can relieve cell pressure to a certain extent; the gene rpsQ can reduce protein mistranslation [ 37 ] and rpsQ His31Pro has been proven to have better stress tolerance [ 26 ], the gene secB can prevent protein aggregation [ 38 ] and promote normal transport of protein [ 39 , 40 ], and the gene rpoS can increase energy metabolism under stress [ 41 ] and regulate protein expression in normal state [ 42 ]. To evaluate intuitively the tolerance ability of strain containing rpsQ His31Pro , rpoS and secB to (2 S )-naringenin, spot assay was conducted.…”

Methylotrophy of Pichia pastoris: Current Advances, Applications, and Future Perspectives for Methanol-Based Biomanufacturing

“…Based on current knowledge, methanol toxicity is supposed to be one of the primary obstacles limiting methanol utilization . In other methylotrophs, increasing methanol has been confirmed to be important for improved methanol utilization. , As the molecular mechanism of methanol toxicity in P. pastoris is poorly understood, the adaptive laboratory evolution (ALE), a powerful strategy generally applied to improve strain tolerance, would be preferred for developing methanol-tolerant P. pastoris. A previous study has adapted P. pastoris for 250 generations to improve its growth rate on methanol .…”

Strategies to improve the stress resistance of Escherichia coli in industrial biotechnology

“…As a result, it obviously demonstrated a strong outlook for the development of methanol tolerance of non-methylotrophic E. coli strains. 68 In many cases, genetically engineered methanol-dependent strains were unable to utilize methanol efficiently without co-substrates. However, the evolved strain was enabled to grow on threonine without methanol.…”