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

I rapporteringsperioden siden november 2020 har det vært fokus på å avslutte eksperimentelt arbeid i alle fem arbeidspakkene (WP1 - W5) og skrive felles vitenskapelige publikasjoner. De tverrfaglige aspektene ved C1Pro-prosjektet, inkludert genom-skala modellering, utvikling av genetiske verktøy og bruk av disse for stamme-utvikling, fermenteringsteknologi og nedstrøms prosessering, har vært sentrale i denne perioden og påkrevd tett og effektiv kommunikasjon mellom samtlige prosjektpartnere. C1Pro hadde en ambisjon om å demonstrere mikrobiell produksjon av fire ulike kommersielle produkter fra råmaterialet metanol og dette målet ble nådd, selv om produksjonsnivået for enkelte produkter fortsatt er lavt. Parallelt med dette ble det utviklet nye genetiske verktøy for vertsorganismen Bacillus methanolicus, inkludert CRISPRi for å nedregulere gener (knockdown), samt såkalte riboswitcher for å kontrollere regulatoriske kretser. Disse verktøyene og teknikkene har stor betydelighet på forskning og stammeutvikling av denne bakterien og andre beslektede bacilli bakterier utover dette prosjektet. To fysiske prosjektmøter var planlagt for denne perioden og på grunn av Covid-restriksjoner ble de omgjort til 4 kortere nettbaserte prosjektmøter. Vi bør også her nevne at tre av C1Pro-partnerne, NTNU, UNIBI og Acies Bio, sammen med en ny partner Marmara University fra Tyrkia, i denne rapporteringsperioden ble innvilget et nytt ERA CoBioTech-prosjekt - MCM4SB - som utnytter kompetanse, kunnskap, verktøy og teknologi utviklet i C1Pro for å utforske bakteriell metylotrofi ytterligere i et industriell bioteknologi perspektiv. MCM4SB-prosjektet hadde kick-off i april 2021 og er allerede godt i gang.

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.