• Energy Research

Depletion of the fossil fuel reserves of the Earth has prompted research into sources of renewable and sustainable energy, and feedstock for the chemical and pharmaceutical industries to support the transition towards a bio-based society. Photosynthesis efficiently captures solar energy, but its subsequent conversion into chemical energy in the form of biomass is limited to a final output in the 1-4% range. Re-routing of photosynthetic electron transport and reducing power directly into desired biosynthetic pathways offers a new avenue for sustainable production of high-value products.

Depletion of the fossil fuel reserves of the Earth has prompted research into sources of renewable and sustainable energy, and feedstock for the chemical and pharmaceutical industries to support the transition towards a bio-based society. Photosynthesis efficiently captures solar energy, but its subsequent conversion into chemical energy in the form of biomass is limited to a final output in the 1-4% range. Re-routing of photosynthetic electron transport and reducing power directly into desired biosynthetic pathways offers a new avenue for sustainable production of high-value products.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.

AbstractA Danish strain of the green microalgae Scenedesmus sp. was isolated, identified and characterized with respect to productivity under outdoor cultivation conditions at northern latitudes. The algae were cultivated outdoors in Denmark in closed tubular photobioreactors using only sunlight, simple inorganic nutrients and under ambient temperatures. The biomass composition was evaluated in terms of protein content and quality. The average volumetric and areal biomass productivity obtained for the Scenedesmus sp. isolate during outdoor cultivation was 0.083 g dry matter L−1 and 6.40 g dm m−2 day−1, respectively. Thus, productivities are comparable to data reported in the literature under similar conditions. A strain‐specific nitrogen to protein conversion factor of 5.5 was determined for the Scenedesmus sp. strain enabling more accurate protein estimations from simple nitrogen determination methods like Kjeldahl analysis in the future. The protein content was determined to be 52.4% of dried biomass for this Scenedesmus strain. The sum of essential amino acids was 42% which is high compared to other microalgae. The results are compared and discussed in comparison to other microalgae and soybean as a common plant protein source.

AbstractA Danish strain of the green microalgae Scenedesmus sp. was isolated, identified and characterized with respect to productivity under outdoor cultivation conditions at northern latitudes. The algae were cultivated outdoors in Denmark in closed tubular photobioreactors using only sunlight, simple inorganic nutrients and under ambient temperatures. The biomass composition was evaluated in terms of protein content and quality. The average volumetric and areal biomass productivity obtained for the Scenedesmus sp. isolate during outdoor cultivation was 0.083 g dry matter L−1 and 6.40 g dm m−2 day−1, respectively. Thus, productivities are comparable to data reported in the literature under similar conditions. A strain‐specific nitrogen to protein conversion factor of 5.5 was determined for the Scenedesmus sp. strain enabling more accurate protein estimations from simple nitrogen determination methods like Kjeldahl analysis in the future. The protein content was determined to be 52.4% of dried biomass for this Scenedesmus strain. The sum of essential amino acids was 42% which is high compared to other microalgae. The results are compared and discussed in comparison to other microalgae and soybean as a common plant protein source.

Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO2, light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca.Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L-1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.