ERA-NET: Replacing food-competing feedstocks with Methanol, CO2 and Methylamine for a Sustainable Bioeconomy

During the reporting period we have included previous NTNU postdoc Marta Irla and her new employer University of Aarhus in Denmark as a formalized associate partner in the MCM4SB consortium; this was done in agreement with ERACoBioTech and the respective MCM4SB partners. Marta was engaged in the NCM4SB as NTNU postdoc and has been actively involved in the project after moving to Denmark; contribution is most valuable for the scientific progress of the project. Our research focus in this reporting period has been further strain optimization, testing and analysis in the methylotrophic model bacterium Bacillus methanolicus. We have published one important publication on overproduction of riboflavin from methanol (Klein et al 2023) which has been a key activity in the project from NTNU side. We are also now using the genome-scale model (GSM) of B. methanolicus for further metabolic engineering activities including overproduction of the key chemical L-malate. The B. methanolicus GSM is also used as basis for a techno-economic analysis to predict economic feasibility, bottlenecks, and operation targets for process improvement. We have constructed recombinant B. methanolicus strains overexpressing genes encoding two key CO2 incorporating enzymes PEP carboxylase and pyruvate carboxylase from different genetic backgrounds. We have established mutant strains overproducing the malate precursor molecule oxaloacetate through biochemical reactions that increase CO2 assimilation. The genes encoding citrate synthase and aspartate aminotransferase have been knocked down by using our recently established CRISPR technology in B. methanolicus; this downregulation caused higher levels of the L-malate precursor molecule oxaloacetate. Among the strains tested the most promising results were obtained with strain overexpressing the PEP carboxylase gene, and logically this strain was selected for further strain improvements. We have also made huge progress on metabolic engineering of Bacillus methanolicus for the production of 3-hydroxypropionic acid from methanol and the results of this was also presented as a mini-symposium on the Bacell conference in Kobe Japan in November 2023. A publication is under preparation. In also good association with this project, we have continued our research on fundamental knowledge on bacterial methylotrophy, and a manuscript entitled “Identification and characterization of a novel formaldehyde dehydrogenase in Bacillus subtilis" was submitted for publication in December 2023. We have arranged two physical project meetings, one in Ljubljana (hosted by industry partner Acies Bio) and one in Aarhus (hosted by new associate partner Marta Irla, Aarhus University); several joint publications were discussed and are now in the pipeline.

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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. We will 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, by using two different methylotrophic organisms Bacillus methanolicus and Methylobacterium extorquens. The application of two 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 and on the technological level, different cultivation conditions such as medium components and temperature. B. methanolicus utilizes ribulose monophosphate cycle to assimilate methanol and is not able to use methylamine as carbon source while M. extorquens utilizes methylamine via the N-methyl-L glutamate pathway. This project combines 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. 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.