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

Abstract

Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.

Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery

“…). The resulting recombinant C. glutamicum strain was able to produce 1.5 g L −1 cadaverine when methanol was supplied with glucose as co‐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).…”

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

“…L-lysine can be converted to other value-added compounds such as the diamine cadaverine (Mimitsuka et al 2007;Tateno et al 2009;Kind et al 2014;Oh et al 2015;Shimizu 2013;Leßmeier et al 2015), 5-aminovalerate (5AVA) (Park et al 2013) and L-PA (Fujii et al 2002;Gatto et al 2006;Neshich et al 2013). C. glutamicum has been engineered for cadaverine production but not for production of L-PA.…”