Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype–phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.

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...

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The actin-associated protein Sla1p, through its SHD1 domain, acts as an adaptor for the NPFX (1,2) D endocytic targeting signal in yeast. Here we report that Wsc1p, a cell wall stress sensor, depends on this signal-adaptor pair for endocytosis. Mutation of NPFDD in Wsc1p or expression of Sla1p lacking SHD1 blocked Wsc1p internalization. By live cell imaging, endocytically defective Wsc1p was not concentrated at sites of endocytosis. Polarized distribution of Wsc1p to regions of cell growth was lost in the absence of endocytosis. Mutations in genes necessary for endosome to Golgi traffic caused redistribution of Wsc1p from the cell surface to internal compartments, indicative of recycling. Inhibition of Wsc1p endocytosis caused defects in polarized deposition of the cell wall and increased sensitivity to perturbation of cell wall synthesis. Our results reveal that the NPFX (1,2) D-Sla1p system is responsible for directing Wsc1p into an endocytosis and recycling pathway necessary to maintain yeast cell wall polarity. The dynamic localization of Wsc1p, a sensor of the extracellular wall in yeast, resembles polarized distribution of certain extracellular matrix-sensing integrins through endocytic recycling. INTRODUCTIONEndocytosis plays a fundamental role in regulating the dynamic organization of the plasma membrane in eukaryotic cells (Conner and Schmid, 2003). A well-characterized endocytic entry route involves formation of clathrin-coated vesicles (CCV). Proteins destined for uptake via CCV generally harbor endocytic targeting signals that direct incorporation into emergent vesicles (Traub, 2005). Such signals are recognized by adaptors that link the cargo to core components of the clathrin coat. Identifying endocytic targeting signals and their partner adaptors, and defining the roles of signal/adaptor pairs in cell physiology are key issues in understanding the endocytic process.Several types of endocytic targeting signals for CCV have been identified in mammalian cells including YXX⌽, FXNPXY (where X is any amino acid and ⌽ is a bulky hydrophobic amino acid), and ubiquitin (Hicke and Dunn, 2003;Traub, 2005). The YXX⌽ signal, present in many endocytic cargo proteins, is recognized by the AP-2 adaptor complex, a core structural component of CCV that plays important roles in clathrin coat assembly. In contrast, the less common FXNPXY and ubiquitin signals are recognized by alternative adaptors known as CLASPs (clathrin-associated sorting proteins), that appear to act more specifically in cargo collection (Traub, 2005).In the yeast Saccharomyces cerevisiae, two distinct classes of endocytic targeting signals have been defined: ubiquitin, which is added post-translationally to lysine residues in endocytic cargo and the peptide signal NPFX (1,2) D (Hicke and Riezman, 1996;Tan et al., 1996). Monoubiquitylation is sufficient to direct internalization and this signal is likely recognized by components of the endocytic machinery such as the epsins Ent1p and Ent2p and the Eps15 homologue Ede1p, all of which contain ub...

Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.