Fossil resources (oil, natural gas and coal) are used to generate fuels, electricity and chemicals. Released CO2 contributes towards global warming with significant effects on the climate. One hour of solar energy reaching the surface of the planet is enough to replace all fossil resources and mined uranium combined.
Life on earth is based on solar energy. Evolution has developed photosynthesis as a biological process to convert light energy into chemical energy. Light is captured and used to make the universal energy-storage and reducing-power transport molecules ATP and NADPH. In nature, these two molecules produced in the light reaction are used in the Calvin–Benson–Bassham cycle to fix and convert CO2 into organic compounds, biomass and thereby growth. This is the biological base for the present bioeconomy that includes renewable biofuels, and biomass for district heating, generation of electricity and biogas.
Solar energy can generate renewable CO2-free electricity using commercially available solar panels. However, to generate sustainable carbon-neutral solar fuels and chemical products directly from solar energy and CO2 is more challenging. One option is electro (e)-fuels and chemicals, processes that convert CO2 into products using renewable electricity. A second option is based on the most efficient photosynthetic organisms on earth, globally widespread cyanobacteria, naturally occurring in most environments. These microscopic cells have the highest capacity for growth and biomass formation from solar energy and CO2.
The rapid progress in synthetic biology and metabolic engineering have made it possible to design and engineer microorganisms, including cyanobacteria, for production of chemicals. To date, cyanobacteria have been engineered, as a proof of concept, to synthesise numerous non-native products from solar energy and CO2. Each cyanobacteria cell is modified into a green cell factory for the production of the selected solar chemical/fuel in a direct process – the energy in the product coming from the sun and the carbon from CO2.
The CRISPR-Cas technologies, which have revolutionised modern genetic engineering since the year 2012, have had an impact on not only genome editing of mammalian cells, but also cyanobacteria. Due to the simplicity and versatility of the CRISPR-Cas systems, many successful studies modulating gene expression gave confidence to the cyanobacteria community to build smarter and more powerful cyanobacteria cell factories – to produce value-added chemicals from CO2. In addition to CRISPR-guided genome editing, recent base-editing and prime-editing technologies have enabled precise genome editing upon a synthetic biology-inspired design.
Nowadays, new bioengineering approaches using machine learning and robotic automation platforms (so-called BioFoundry) are emerging in synthetic biology and the metabolic engineering field to reduce human labour and to increase the research productivities, compared with the current research and development platforms. This is another opportunity for the community to further explore the engineering of cyanobacteria. Currently, one of the limitations of the engineered cyanobacteria for feasible CO2 mitigation is low-yield production. Thus, novel approaches such as advanced CRISPR-Cas technologies, machine learning and robotic platforms will accelerate the development of green cell factories.
Cyanobacteria as green cell factories for production of butanol from CO2
Since the available biotechnologies differ in biosynthetic pathways and products, the fuel butanol (isobutanol and 1-butanol) is used here as an example of current work in our laboratory for, and the present status of, sustainable CO2-neutral chemical/fuel production in cyanobacteria. As cyanobacteria lack the butanol biosynthetic pathways and relevant genes, the isobutanol and 1-butanol biosynthetic pathways were first required to be introduced into cyanobacteria. Selected butanol-forming genes were expressed/overexpressed and evaluated, followed by optimising the expression levels of corresponding enzymes and protein engineering of specific enzymes. Deletion of competing pathways, enhancement of native substrates supporting introduced pathways, and rewriting central carbon metabolism were then performed in order to redirect more carbon flow towards butanol synthesis. Finally, a comprehensive combination of the above approaches with optimisation of the cultivation system resulted in the highest photosynthetic production of isobutanol and 1-butanol at 0.9 g/L in 46 days and 4.8 g/L in 28 days, respectively. The maximal production rate observed was 600 mg of photosynthetic 1-butanol/L/day, with a carbon partitioning efficiency of 60%, i.e. 60% of the carbon taken up by the cells was used to generate 1-butanol at the expense of growth/biomass formation.
Besides the approaches discussed above, there are a growing number of engineering advances to custom-design cyanobacteria for specific purposes, such as modelling metabolic networks, enhancing photosynthesis, applying stress conditions, improving tolerance of products, modulating growth rate, performing photo-bioreaction and removing excreted products – to name a few. Applying these more comprehensive strategies in cyanobacteria to further improve the generation of products requires advanced technologies, such as more efficient genetic tools (e.g. CRISPR-Cas) and the high-throughput manipulation platforms discussed above.
The way forward
Concomitantly with the development of green cell factories for sustainable CO2-neutral production of chemicals (e.g. acetone, organic acids, isoprenoids, butanol) and fuels (e.g. ethanol and butanol), public and societal acceptance of this technology is of fundamental importance for successful introduction. A recent pioneering survey-based study examined the opinion of European experts and stakeholders on modified photosynthetic microorganisms for biofuel production. The results indicated that a majority believe that biofuels produced by modified photosynthetic microorganisms can provide strong benefits compared with other fuels.
Initial life-cycle assessments of cyanobacteria-based production of sustainable CO2-neutral chemicals have identified a need for improvements in, for example, light utilisation and a carbon-partitioning efficiency to above 90%. These improvements are needed together with stable high-yield cultivation systems and product extraction technologies at scale, for successful implementation of this technology.
In order to follow the Paris Climate Agreement from 2015 to substantially reduce global greenhouse gas emissions (to limit global warming to well below 2°C), countries and sectors are making commitments to reduce the release of CO2 into the atmosphere. At present, technologies are being developed, and numerous larger-scale industrial projects are being initiated, to separate and capture the CO2 from flue gases before being released into the atmosphere. Obtained CO2 is compressed into liquid form, which can be transported for storage in selected sites deep underground. An alternative scenario is to use separated CO2 as a substrate for sustainable production of CO2-neutral chemicals, including fuels, by green cell factories, as discussed above. Modified photosynthetic cyanobacteria may be our future sustainable CO2-neutral producers of selected products that are currently made from fossil resources.
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