ERA-NET: Microbial conversion of C1 to value-added products by integrated systems and synthetic biology

In the reporting period since November 2020 the focus has been on concluding experimental work in all five research WPs and writing of joint scientific publications. The interdisciplinary aspects of C1Pro project, including genome scale modelling, genetic tools and strain engineering, fermentation technology and downstream processing, was very much in play during this period requiring efficient communication including all project partners. C1Pro had an ambition to demonstrate microbial production of four different value-added products from methanol and this goal was achieved although the production level of some products is still low. In parallel with this, novel genetic tools were developed for the model host organism Bacillus methanolicus, including CRISPRi for gene knockdown, as well as riboswitches for regulatory circuits. These tools and techniques have high impact for research and strain development of this bacterium and other bacilli beyond this project. Two physical projects meetings were planned for this period and due to COVID-19 restrictions they were converted into 4 shorter web-based project meetings. We should also mention that three of the C1Pro partners, NTNU, UNIBI and Acies Bio, together with one new partner Marmara University from Turkey, in this reporting period was granted a new ERA CoBioTech project - MCM4SB - which takes advantage of competence, knowledge, tools and technology developed in C1Pro to further explore bacterial methylotrophy in industrial biotechnology perspective. The MCM4SB project had kick-off in April 2021 and is already well up and running.

C1Pro promoted the transition to new technologies for bioproduction by establishment of a sustainable platform for methanol-based biosynthesis. Our work paved the way for future development of 5AVA production. Our methods for CRISPRi-based gene repression and a riboswitch-mediated regulatory system constitute refined engineering strategies to develop new strains of B. methanolicus and other bacilli, as we proved that the CRISPRi method is transferable to Paenibacillus sonchi. Our research was disseminated both in peer-reviewed papers and during scientific conferences and is available for scientific public access. The successful collaboration forged throughout the C1Pro activities led to the formation of a new consortium, as NTNU together with Acies Bio and UNIBI collaborate in the newly funded project MCM4SB, in which they will continue their efforts towards a methanol-based bioeconomy.

Society urgently needs a sustainable production processes for key platform chemicals with various applications in several industries, in light of resource scarcity. The C1Pro project aims to establish a sustainable platform for methanol-based production of value-added products (GABA, 5AVA, L-Pro and L-PA) with proven industrial applications. Methanol is an attractive and alternative raw material for biotechnological processes because of its chemical properties, relatively low price and availability from both fossil and renewable sources. The Gram-positive, methylotrophic and thermophilic bacterium Bacillus methanolicus was chosen as model organism in this project for several reasons: it utilizes methanol as raw material for growth and energy, it grows at elevated temperatures (50 – 55 °C), it naturally overproduces L-glutamate, and its classical mutants have demonstrated a high potential to overproduce L-lysine. The omega-amino acids GABA and 5AVA can be cyclized by lactamization, while L-Pro and L-PA are cyclic amino acids. The targeted products serve as building blocks of polymers or precursors of pharmaceuticals and other biologically active substances. Systems and synthetic biology approaches are key to the proposed strain and process development, which is facilitated by common biosynthesis pathways. Novel genetic tools will simplify regulated gene expression on different levels via CRISPR/Cas9 for genome modifications and riboswitches for regulatory circuits. Pathway design will be guided by the genome-scale metabolic model which will be iteratively fine-tuned based on experimental test results. All generated data will be collected in an LCA-compliant way and used to select the most promising strain(s) for up-scaling where methanol-based production at 20-150 L scale will be performed in a clean bioprocess. Finally, GABA isolation to at least 80% purity will be demonstrated.