Research

Microbial cell factories like specialized bacteria, yeast and fungi, are used to produce relevant compounds and engineered biomolecules such as commodities, fine chemicals, food ingredients and biopharmaceuticals. Tailor-made robust microorganisms displaying novel biological behaviors produce these products in a non-chemical way utilizing nature’s toolset, in general using renewable inputs such as glucose or industrial side streams. C1 feedstocks, such as methane, methanol, formate, CO2 and CO, have important advantages over traditional organic carbon sources like glucose. They are cheap, can be obtained from CO2 in a renewable way, do not compete as food or animal feed and do not require extensive pre-processing from complex agricultural side-streams. Implementing C1 substrates in microbial cell factories would ensure a circular carbon economy that is inherently sustainable. However, due to the relative novelty of this approach, further work will be required to have abiotic C1 substrates compete with biological feedstocks. The CiTrY project will contribute to this goal by investigating and improving transport mechanisms of the C1 substrates over outer and organelle membranes of the microbial cell factories. Underexplored proteins and proteins families will be investigated, advanced protein engineering strategies and high-throughput screening will be conducted, and a novel organelle membrane targeting approach will be developed.

Methanol is an attractive low-cost substrate for biotechnology that does not need agricultural land for its production and can be produced sustainably from the greenhouse gas CO2. Methylotrophy, i.e. the ability of microorganisms to use methanol as carbon and energy source, has evolved in several groups of bacteria, and in a branch of budding yeasts. In yeasts, the methanol assimilation pathway is encapsulated in peroxisomes which may protect the cytosol from toxic intermediates while in bacteria the processes are cytosolic and the reactions are thoroughly balanced to prevent the accumulation of toxic compounds. Research questions We plan to elucidate the role of compartmentation on the functioning of the methanol metabolism in the naturally methylotrophic yeast Komagataella phaffii, and engineered Escherichia coli, by answering the following research questions: • Why and to which degree is compartmentation essential for methylotrophy in yeast? • Can we establish synthetic methylotrophy in bacteria using an “artificial” methylotrophic organelle? Approach We will re-target the entire assimilation pathway to the cytosol of K. phaffii, and exchange the first enzyme from an O2-dependent oxidase to an NAD+ dependent dehydrogenase to understand if any, or all pathway reactions depend on the peroxisomal localization. Artificial organelles based on bacterial microcompartments will be built to harbor the pathway, and introduced into E. coli to create synthetic E. coli strains with the yeast methanol utilization pathway. Functionality of the pathway variants will be assessed by in vitro and in vivo 13C based metabolomics, and the metabolic network and its interplay will be further balanced by adaptive laboratory evolution.

Emissions reductions necessary to meet the Paris Agreement are unachievable without large-scale capture and use of CO2 emissions. VIVALDI offers an innovative integrated solution for the valorisation of CO2 emissions from different biomass-based industrial sectors (pulp/paper, biomass and ethanol) into added-value organic acids. VIVALDI embraces the whole value chain: 1) CO2 capture using a amine-based absorption combined with immobilised carbonic anhydrases 2) electrocatalytical CO2 reduction into C1 building blocks using novel electrode materials and reactor configurations, 3) fermentation of the C1s into organic acids via the Pichia pastoris microbial chassis and 4) recovery of nutrient/trace elements/energy from their own industrial wastewaters using bioelectroconcentration. VIVALDI will profit from the recent research advances of the partners to develop and validate, in strong collaboraton with industry sector, a feasible and sustainable value chain to valorise CO2 into organic acids with different market demands: lactic (already established), itaconic (recently industry-adopted) and 3-hydroxypropionic (still poorly established at industrial scale). Besides the concept, VIVALDI’s strength is the multidisciplinary and complementary consortium: partners with well-known R&D experience, large CO2-producers from each of the industry sectors, early technology adopters, experts in the market of novel solutions and large end-users of the chemical products.