Cyanobacteria are photoautotrophic microorganisms which can be engineered to directly convert CO 2 and water into biofuels and chemicals via photosynthesis using sunlight as energy. However, the product titers and rates are the main challenges that need to be overcome for industrial applications.Here we present systematic modular engineering of the cyanobacterium Synechocystis PCC 6803, enabling efficient biosynthesis of 1-butanol, an attractive commodity chemical and gasoline substitute.Through introducing and re-casting the 1-butanol biosynthetic pathway at the gene and enzyme levels, optimizing the 5 0 -regions of expression units for tuning transcription and translation, rewiring the carbon flux and rewriting the photosynthetic central carbon metabolism to enhance the precursor supply, and performing process development, we were able to reach a cumulative 1-butanol titer of 4.8 g L À1 with a maximal rate of 302 mg L À1 day À1 from the engineered Synechocystis. This represents the highest 1-butanol production from CO 2 reported so far. Our multi-level modular strategy for high-level production of chemicals and advanced biofuels represents a blue-print for future systematic engineering in photosynthetic microorganisms.
Broader contextIn order to reduce the impact of human activities on climate change, we must address our dependence on fossil resources. Biological systems are able to make molecules identical to petroleum-derived compounds used in fuels and in the chemical industry. This has led to increasing attention on the field of metabolic engineering, where microorganisms are genetically engineered to produce compounds of industrial interest. Most such microbial systems employ heterotrophic organisms fed substrates generated from plant biomass. However, photosynthetic microorganisms could be used to perform direct production of fuels and chemicals from photosynthesis, enabling a more efficient conversion of solar energy and carbon dioxide to desirable products, thus leading to more sustainable processes. Engineered strains of photosynthetic cyanobacteria can make many different compounds on a proof-of-concept level, but few products so far show titers approaching those achieved in heterotrophic organisms. We have systematically engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce 1-butanol. The resulting titers are among the highest for any heterologous product from cyanobacteria, and we show that long-term productivity is feasible. The employed strategy can be used for other products when engineering photosynthetic microorganisms for direct production of solar chemicals and biofuels.