Methanol-Based Production of Value-Added Products like 1,5-Diaminopentane

Authors: 
Wendisch, V. F., Bielefeld University


Methanol, a one-carbon compound, is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. Methanol can be utilized by a variety of bacteria and other organisms as carbon and energy source. Two strategies were followed to utilize methanol for cadaverine production, namely using non-methylotrophic Corynebacterium glutamicum and the methylotrophic Bacillus methanolicus.

A strain of non-methylotrophic C. glutamicum was engineered to produce the polyamide building block 1,5-diaminopentane (cadaverine) as non-native product. Moreover, this strain was engineered further for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph B. 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.

B. methanolicus, a thermophilic methylotrophic bacterium, was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg/l during shake flask conditions. A volume-corrected concentration of 11.3 g/l of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and C. glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol.