Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol‐dependent growth of Corynebacterium glutamicum

Abstract

Methanol is a promising feedstock for biomanufacturing of fuels and chemicals. Although efforts have been made to engineer platform microorganisms for methanol bioconversion, the substrate uptake and cell growth rates on methanol are still unsatisfactory, suggesting certain limiting factors remain unsolved. Herein, we analysed the global metabolic regulation changes between an evolved methanol-dependent Corynebacterium glutamicum mutant and its ancestral strain by transcriptome analysis. Many genes involved in central metabolism including glycolysis, amino acid biosynthesis and energy generation were regulated, implying the adaptive laboratory evolution reprogrammed the cellular metabolism for methanol utilization. We then demonstrated that nitrate could serve as a complementary electron acceptor for aerobic methanol metabolism, and the biosynthesis of several amino acids limited methylotrophic growth. Finally, the sedoheptulose bisphosphatase pathway for generating methanol assimilation acceptor was found effective in C. glutamicum. This study identifies limiting factors of methanol metabolism and provides engineering targets for developing superior synthetic methylotrophs.

Methylomarinovum tepidoasis sp. nov., a moderately thermophilic methanotroph of the family Methylothermaceae isolated from a deep-sea hydrothermal field

“…In addition to the canonical reactions with fructose phosphates, these two enzymes have been shown to catalyse the above-mentioned reactions with sedoheptulose phosphates (regardless of the direction of the reactions) in different bacteria, including methanotrophs [ 43 49 ]. Furthermore, it has been reported that the SBP pathway is feasible or likely to have evolved as part of the RuMP or pentose phosphate pathways in several bacteria, including native [ 50 ] and synthetic [ 51 52 ] methylotrophs and bacteria assimilating pentose sugars without the transaldolase gene [ 43 44 46 ].…”

“…In addition to the canonical reactions with fructose phosphates, these two enzymes have been shown to catalyse the abovementioned reactions with sedoheptulose phosphates (regardless of the direction of the reactions) in different bacteria, including methanotrophs [43][44][45][46][47][48][49]. Furthermore, it has been reported that the SBP pathway is feasible or likely to have evolved as part of the RuMP or pentose phosphate pathways in several bacteria, including native [50] and synthetic [51,52] methylotrophs and bacteria assimilating pentose sugars without the transaldolase gene [43,44,46]. Regarding nitrogen metabolism, IN45 T carries genes for ammonium assimilation (glnA and GDH2), nitrate assimilation (nasA and nirBD), partial denitrification of nitrate to N 2 O (narGHJI, nirK, and norBC) and hydroxylamine dehydrogenase (hao).…”

Adaptive Laboratory Evolution of Microorganisms: Methodology and Application for Bioproduction

“…Many methylotrophic microorganisms are present in nature, but information on the metabolism and genetic engineering tools for these methylotrophs is limited. Therefore, the creation of synthetic methylotrophs from E. coli and Corynebacterium glutamicum has been attempted by many researchers [ 59 , 60 , 61 , 62 ]. Synthetic methylotrophs are constructed by introducing metabolic reactions from methylotrophs for methanol oxidation [methanol dehydrogenase (Mdh)] and fixation of formaldehyde to ribulose-5-phosphate [3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi)], which is known as the formaldehyde fixation pathway and is called ribulose monophosphate (RuMP) cycle.…”

From a Hetero- to a Methylotrophic Lifestyle: Flash Back on the Engineering Strategies to Create Synthetic Methanol-User Strains

“…h −1 ( Tuyishime et al, 2018 ). Based on transcriptome analysis, further work was done on this strain to explore the metabolic regulation that operated during methylotrophic conditions ( Fan et al, 2021 ). Results demonstrated that the evolved C. glutamicum used the SBPase variant to recycle Ru5P as fba and glpX were upregulated while tal was downregulated.…”