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Project Topic
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The MCM4SB project aims to replace sugar-based feedstocks for bioprocesses with one carbon (C1) compounds, namely methanol (CH3OH), carbon dioxide (CO2) and methylamine (CH3NH2) in order to establish a novel, sustainable production within a low-fossil-fuel bio-economy. Renewable methanol currently is produced e.g. by catalytic reduction of CO2 at the 700,000 tons per annum scale. Methylamine is present in fish wastes and is available from CO2 by direct reduction in the presence of ammonia or indirectly via green methanol. This project aims to combine utilization of methanol with CO2 and/or methylamine for production of the bulk chemical L-malate and the specialty chemical N-methyl-L-glutamate. In order to achieve that goal, two different methylotrophic organisms differing in their central methanol assimilation metabolism as well as their environmental adaptation to different growth temperatures will be used. The application of diverse hosts has a double impact on the establishment of novel biotechnological processes – on the metabolic engineering level, different genetic targets will be selected based on the flux balance analysis (FBA) and on the technological level, different cultivation conditions such as medium components and temperature. Bacillus methanolicus is a thermophilic methylotroph that utilizes ribulose monophosphate cycle to assimilate methanol and is not able to use methylamine as carbon source; on the other hand, Methylobacterium extorquens utilizes methylamine via the N-methyl-Lglutamate pathway. Those different metabolisms will be appropriately adjusted, in order to expand the methylotrophy towards additional C1 compounds. In case of B. methanolicus, a two-faceted approach will be undertaken, namely the metabolic flux will be redirected towards the precursor metabolite, oxaloacetate through anaplerotic reactions in order to increase CO2 assimilation. In parallel some of decarboxylation reactions will be silenced to decrease CO2 release. This will lead to creation of a platform strain that can be used for production of L-malate. Furthermore, the genes encoding γ-glutamylmethylamide synthetase and N-methyl-L-glutamate synthase will be heterologously overexpressed to enable use of methylamine for production of N-methyl-L-glutamate. In the case of M. extorquens, methylamine assimilation into N-methyl-L-glutamate will be achieved through interruption of the methylamine catabolic pathway and L-malate formation through overexpression of malate dehydrogenase. The planned goals will be achieved by combining systems and synthetic biology approaches. Specifically, target identification for strain development will be guided by the genome-scale metabolic models, which will be iteratively fine-tuned based on experimental test results, and the newly developed strains will be characterized on the transcriptome level in order to detect the bottlenecks in the metabolism, which will subsequently be relieved to steer the next round of strain development. The techno-economic assessment will help to steer the bioprocess design by assessing the economic feasibility, bottlenecks, and operation targets for process improvement and identify possible trade-offs during early stages of design and development. Furthermore, Responsible Research and Innovation will be a cross-cutting and integrated research activity in MCM4SB with a focus on sustainable bioeconomy based on renewable feedstocks.
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