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Leaf-inspired nanocellulose frameworks for next generation photosynthetic cell factories

Periodic Reporting for period 1 - FuturoLEAF (Leaf-inspired nanocellulose frameworks for next generation photosynthetic cell factories)

Reporting period: 2020-09-01 to 2021-08-31

FuturoLEAF envisions to exploit the combined know-how of ligno-cellulosic materials and cell biology to revolutionize the field of industrial algal biotechnology with a conceptually renewed tailored solid-state cell factrories. FuturoLEAF introduces algal-based biocatalysts with a functional architecture formulated from nanocellulose building blocks designed on the principles of plant leaf anatomy and function. We will integrate knowledge on bio-based materials science and photosynthesis with achievements on synthetic biology and biomolecular engineering to conceive the new technology that efficiently captures CO2 and produces solar-driven biofuels and chemicals. The system will maximise light utilization and CO2 capturing by providing controllable transport of moisture, gases, nutrients, products and substrates to living cells, leading to next generation photosynthetic cell factories with high catalytic turn-over time. In addition, the solid-state systems are much less water intensive by nature in contrast to current suspension cultures. The grand target is to enable a sustainable production of a wide spectrum of chemicals, varying from active pharmaceutical ingredients (APIs) and special chemicals to commodity chemicals and biofuels.
FuturoLEAF will contribute significantly to society by developing the biotechnology field with direct impact to existing biotechnological companies utilizing photosynthetic cells. Additionally, it is envisioned that FuturoLEAF will enable the birth of new biotechnological companies, where solid-state cell factories will be utilized with one or multiple cell types, creating new business openings in the EU. The novel technology created within FuturoLEAF will also give EU a marketing advantage over other market areas related to bioproduction. Moreover, FuturoLEAF will contribute to environmental safety and the development of green technologies. Implementation of the knowledge created in FuturoLEAF for green technology will lead to sustainable use of resources by conversion of CO2 and light into chemicals.
We have convincingly demonstrated that our nanocellulose-based materials leading to the solid-state cell factory concept provides long-term mechanical stability and improved cell viability when compared to the existing alginate-based reference materials. We achieved over three times higher gel strength coupled with higher ethylene yield with our nanocellulose-based solid-state cell factory design. Unlike the alginate matrix, the self-standing nanocellulose hydrogel matrix is able to withstand the challenging production conditions, and the colloidal network fully prevents the cell leakage and disintegration of matrix during the photoproduction stage.
Within the FuturoLEAF project, we have engineered recombinant green alga, C. reinhardtii, holding foreign cyclohexanone monooxygenase (CHMO) for converting cyclohexanone to the polymer precursor ε-caprolactone and optimized cultivation conditions for the highest conversion efficiency. We also showed an improved production of sucrose by sucrose-producing cyanobacterium Synechocystis sp. PCC 6803 entrapped in alginate beads.
In the production of monomers for biodegradable polymers, we have shown so far that the cells can be successfully immobilized in different materials and show excellent reaction rates. By molecular engineering, the photosynthetic chassis for monooxygenase reactions was optimized, leading to significantly elevated reaction rates. With the successful proof of concept both for the molecular engineering and implementation of biobased materials, we will focus on the performance of the system in 3D architectures, and the physiological characterization to match molecular cell engineering and material design in an iterative process.
In regard to the production of APIs, or more concretely the production of modified microcystins as novel payload class of antibody-drug conjugates (ADCs) for targeted cancer therapies, we could successfully demonstrate the proof of concept for the encapsulation of microcystin-producing cyanobacteria in both alginate and in nanocellulose.
The efforts towards the leaf-inspired hierarchical cell factory architecture with active immobilization strategy have been initiated. We have developed a FuturoLEAF -architecture allowing efficient light-capturing under low light conditions by creating a gradient of cells with truncated photosynthetic antennae across the immobilization matrix. This architecture can be further applied for improving production yields of commodity chemicals and biofuels, such as H2 and ethylene, as well as for improving the efficiency of biotransformation approaches.
In addition, molecularly imprinted polymers (MIPs) as synthetic receptors exhibiting molecular recognition of the substructures (peptide epitopes) of bacterial surface proteins have been developed, which allow specific binding to bacterial cells (cyanobacteria and E. coli). In addition, the interfacing of these MIPs with nanocellulose matrices has been studied. These combined findings and the resulting nanocomposite materials will allow the construction of self-assembling artificial leaf structures.
We have shown that novel bio-based nanomaterials assemblies based on nanocellulose and other plant-sourced carbohydrates are feasible for cell immobilization matrixes and the developed solid-state immobilisation systems display prolonged cell viability. In the beginning of the FuturoLEAF project, we expected that the engineered multi-layer architecture will convert light energy to the targeted product with maximum efficiency of around 4-6 % under low light conditions. We achieved 4% values of light to H2 conversion already in the first year of the project by developing the films with double-layer structure with truncated photosynthetic antennae strain placed a top of layer with wild-type cells.
In regard to the production of active pharmaceutical ingredients (APIs), our preliminary results indicate that the production rates of modified microcystins in the reference system (alginate beads and in alginate films) are increased by almost two times compared to conventional suspension cultures.
The research related to active immobilization strategy involving MIPs and other approaches to reach the plant leaf-inspired architectures is expected to yield a generic approach to reversible and specific recognition and sequestering of target bacteria by stimuli-responsive nanocomposite materials. This will be an excellent example of biomimicry at the molecular level, based on the principles of bioinspiration and bioresources.
FuturoLEAF is expected to demonstrate a functional solid-state cell factory concept producing molecules for pharmaceuticals, commodity chemicals and fuels. FuturoLEAF will enable a significant step away from dependency of fossil sources, towards sustainable energy and chemicals production while contributing to the green production and climate change mitigation.
FuturoLEAF nanocellulose-based solid-state cell factory concept