Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Abstract

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ؎ 0.3 mM/h (mean ؎ standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13 C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m ؉ 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum ⌬ald ⌬adhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO 2 , the m ؉ 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

“…Methanol utilization and 13 C-methanol-derived labeling of intracellular metabolites were first demonstrated in Corynebacterium glutamicum by expressing the mdh gene from B. methanolicus and the hps and phi genes from Bacillus subtilis (16,17). A similar approach was also demonstrated in E. coli, where Mdh2 from B. methanolicus PB1 was coexpressed with Hps and Phi from B. methanolicus MGA3 to increase the production of 13 C-labeled intracellular metabolites compared with the control strain expressing Mdh3 alone (15).…”

“…Recent work has demonstrated the ability to transfer methylotrophic genes to other organisms to enable nonnative methanol oxidation (15)(16)(17). Methanol utilization and 13 C-methanol-derived labeling of intracellular metabolites were first demonstrated in Corynebacterium glutamicum by expressing the mdh gene from B. methanolicus and the hps and phi genes from Bacillus subtilis (16,17).…”

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate

“…Subsequently, formate is oxidized to CO 2 by formate dehydrogenase FdhF (cg0618), involving a currently unknown electron acceptor and the gene products encoded by cg0616 and cg0617 (Witthoff et al 2012). Recently, assimilation of methanol into central metabolites by engineered bacteria has been described for Pseudomonas putida (Koopman et al 2009), Escherichia coli (Müller et al 2015b), and also C. glutamicum (Witthoff et al 2015). Based on this engineering strategy, the study described here aimed one step further by engineering conversion of methanol into the industrially relevant non-native product cadaverine while retaining (some) carbon from methanol in the secreted product.…”

Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway

“…On the contrary, the linear formaldehyde assimilation pathway used in this study only requires one enzyme FLS and directly produces C3 intermediate DHA, which could be a great advantage for pathway engineering. Previous and the present studies revealed that constructing synthetic methylotrophs was far more complicated than complementing metabolic pathways where several crucial factors need to be considered, such as how to keep the intracellular formaldehyde concentration below the toxicity threshold (Witthoff et al 2015) and how to balance the reducing equivalent generated by methanol oxidation (Price et al 2016). In this case, combining metabolic engineering and adaptive evolution could be an easy strategy to prepare a desirable mutant that assimilates methanol efficiently.…”

“…Recently, synthetic methylotrophs were constructed by introducing native methanol assimilation pathways into non-native methylotrophs such as Escherichia coli (Dai et al 2017;Leßmeier et al 2015;Müller et al 2015;Rohlhill et al 2017;Whitaker et al 2017;Witthoff et al 2015). To date, ribulose monophosphate (RuMP) cycle that utilizes ribulose-5-phosphate (Ru5P) as a formaldehyde acceptor is the only pathway used for synthetic methylotrophs.…”