Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway

Abstract

Engineering Escherichia coli for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1 g/L yeast extract as co-substrate. 13C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid methanol assimilation pathway with Mdh and Das.

Advanced applications of smart electrospun nanofibers in cancer therapy: With insight into material capabilities and electrospinning parameters

“…Currently, the majority of research employs conventional electrospinning techniques to fabricate smart nanofibers for cancer treatment. However, emerging methods such as Janus, trilayer core-shell, and hybrid structures combining core-shell and Janus nanostructures, as well as the integration of electrospraying with electrospinning ( Chen et al, 2024 ; Sun et al, 2023 ; Zhou et al, 2024b ), appear to enhance the efficiency of these systems significantly. Although there have been successful studies on cancer treatment using smart nanofibers, none have successfully transitioned into clinical applications.…”

Host evolution improves genetic circuit function in complex growth environments

“…This technique has been used in both bacteria and yeast for the generation of desired phenotypes such as osmotic, acid, and temperature tolerance, and increased alternate substrate metabolism for bioproduction ( 18 ). Recently, ALE was used to optimize growth performance of a genome-reduced E. coli strain ( 19 ), co-evolve mutually auxotrophic strains of yeast and lactic acid bacteria for improved production of the auxotrophic compound ( 20 ), and improve methanol utilization in E. coli engineered with a hybrid methanol assimilation pathway ( 21 ). In combination with omics technologies, researchers can better understand mutational pathways underlying evolution and create strains that are tolerant to unique and stressful environmental conditions ( 22 ).…”

Effect of adaptive laboratory evolution of engineered Escherichia coli in acetate on the biosynthesis of succinic acid from glucose in two-stage cultivation

“…Adaptive laboratory evolution (ALE) is a technique for selecting strains with a better phenotype by long-term culture under a specific selection pressure or growth environment (Wang et al 2022 ). With the development in recent decades, ALE can be used at the laboratory level to improve many abilities of microbes, such as promoting product yield (Kim et al 2022 ; Godara and Kao 2021 ), increasing substrate utilization (Sun et al 2023 ), improving growth rate (Barten et al 2022 ), and enhancing tolerance (Wang et al 2021 ). In addition to strain improvement, it has also been used to expand intracellular regulatory networks by revealing potential mechanisms that regulate cellular metabolism (SuRin and Pil 2020 ).…”