Conversion of CO 2 for the synthesis of chemicals by photosynthetic organisms is an attractive target for establishing independence from fossil reserves. However, synthetic pathway construction in cyanobacteria is still in its infancy compared with model fermentative organisms. Here we systematically developed the 2,3-butanediol (23BD) biosynthetic pathway in Synechococcus elongatus PCC7942 as a model system to establish design methods for efficient exogenous chemical production in cyanobacteria. We identified 23BD as a target chemical with low host toxicity, and designed an oxygen-insensitive, cofactor-matched biosynthetic pathway coupled with irreversible enzymatic steps to create a driving force toward the target. Production of 23BD from CO 2 reached 2.38 g/L, which is a significant increase for chemical production from exogenous pathways in cyanobacteria. This work demonstrates that developing strong design methods can continue to increase chemical production in cyanobacteria.metabolic engineering | synthetic biology | biofuel | renewable energy A mid rising global energy demands and pressing environmental issues, interest is growing in the production of fuels and chemicals from renewable resources. Petroleum consumption reached 37.1 quadrillion BTU in the United States in 2008, of which a large majority (71%) was liquid fuel in the transportation sector. Petroleum and natural gas account for 99% of the feedstocks for chemicals, such as plastics, fertilizers, and pharmaceuticals in the chemical industry (1). Considering rapidly increasing world population and exhaustion of fossil fuels, the development of sustainable processes for energy and carbon capture to produce fuels and chemicals is crucial for human society.Energy and carbon capture by cyanobacteria is also directed toward mitigating increasing atmospheric CO 2 concentrations. According to the US Energy Information Administration (2), world energy-related CO 2 emissions in 2006 were 29 billion metric tons, which is an increase of 35% from 1990. Accelerating accumulation of atmospheric CO 2 is not only a result of increased emissions from world growth and intensifying carbon use, but also from a possible attenuation in the efficiency of the world's natural carbon sinks (3). As a result, atmospheric levels of CO 2 have increased by ∼25% over the past 150 y and it has become increasingly important to develop new technologies to reduce CO 2 emissions. Many creative solutions have been proposed and argued for carbon capture, each with varied environmental side-effects and costs (4). Sequestration by photosynthetic microorganisms in which CO 2 is biologically converted to valuable chemicals is an important addition to the toolbox for overall capture of CO 2 (5-7).Photosynthetic microorganisms, including cyanobacteria, are currently being engineered for platforms to convert solar energy to biochemicals renewably (5-7). These microorganisms possess many advantages over traditional terrestrial plants with regard to biochemical production. For example, the pho...

Cyanobacteria have great potential as a platform for biofuel production because of their fast growth, ability to fix carbon dioxide gas, and their genetic tractability. Furthermore they do not require fermentable sugars or arable land for growth and so competition with cropland would be greatly reduced. In this perspective we discuss the challenges and areas for improvement most pertinent for advancing cyanobacterial fuel production, including: improving genetic parts, carbon fixation, metabolic flux, nutrient requirements on a large scale, and photosynthetic efficiency using natural light.

Summary The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life and death depending on environment. Here are presented “essentializer” and “cryodeath” circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 105 after ten days of growth in the mammalian gut.

No abstract