A synthetic biology platform for sustainable, climate-friendly conversion of CO2 to products using cyanobacteria

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Cyanobacteria are the simplest and most genetically-tractable organisms capable of oxygenic photosynthesis, using CO2 and sunlight as sole carbon and energy sources. Their photosynthetic yield and growth rate are similar to the fastest-growing microalgae, and greater than terrestrial plants. Consequently, cyanobacteria have great potential as whole-cell biocatalysts in light-driven, carbon-negative bioprocesses converting atmospheric or waste CO2 to products of interest, while avoiding competition with the food chain. Cyanobacteria have been genetically modified to synthesise a wide range of non-native compounds from commodity chemicals and biofuels to high-value products.CHALLENGE:Production using cyanobacteria is not yet commercially competitive with production by conventional approaches due to the current low economic cost of using fossil carbon and emitting CO2. It is also difficult to rationally design DNA encoding an optimal metabolic pathway. If expression is too high, too low, or poorly 'balanced', then low productivity and/or genetic instability results.SOLUTION:Modern DNA assembly allows construction of large libraries of many pathway-encoding construct variants, varying the expression of each enzyme combinatorially. High-performance variants can be identified by screening. This approach has been well proven, but in only a few model organisms. We recently developed a platform for combinatorial construction and optimisation of metabolic pathway-encoding constructs in cyanobacteria, applied it to production of lycopene from CO2 in Synechocystis sp. PCC 6803, and successfully obtained strains with pathways that were both productive and genetically stable, overcoming this key problem.AIM:The new combinatorial metabolic pathway construction platform will be applied and developed to generate photoautotrophic cell factories with highly-productive, genetically stable engineered metabolic pathways, using key strains.APPROACH:Using DNA synthesis, coding sequences for foreign pathway enzymes and native 'bottleneck' enzymes, as well as host-appropriate expression control parts (promoters, ribosome-binding sites, terminators, etc) will be formatted for Start-Stop Assembly, our recently-published DNA assembly system optimised for metabolic engineering. Hierarchical multi-part assembly with controlled part mixtures will generate combinatorial pathway libraries for insertion into host cells and screening using analytical methods e.g. GC/LC/MS/NMR.A) HOSTS:The platform is already validated for the standard strain Synechocystis sp. PCC 6803, allowing work to start quickly and focus entirely on pathways, without need for platform development. The newly-described, fast-growing, high-producing, but little-studied strain Synechococcus sp. PCC 11901 has great potential, and will first require the development of expression control parts. Similarly, the platform will be transferred to Spirulina, a safe, food-grade strain suitable for nutraceutical and medical products.B) PRODUCTS:A wide range of natural and non-natural products could be biosynthesised including alcohols, organic acids, fatty acids, fatty alcohols, alkanes, terpenoids, alkaloids, peptides, sugars, flavours, fragrances, specialised metabolites, pharmaceutical precursors, nutraceuticals, drugs and vaccines. The high-throughput assembly allows multiple different products to be targeted in parallel.C) INTEGRATION WITH OTHER APPROACHES & TECHNIQUES:Combinatorial pathway optimisation will be combined with classical metabolic engineering, knocking out competing pathways, guided by physiological understanding and/or metabolic modelling, subject to the student's interests. Finally, our new, patented, high-throughput enzyme evolution system can be applied to problematic enzymes in important pathways, such as alkane biosynthesis.