Ethanol synthesis by genetic engineering in cyanobacteria

  • Deng M
  • Coleman J
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Abstract

Cyanobacteria are autotrophic prokaryotes which carry out oxygenic photosynthesis and accumulate glyco-gen as the major form of stored carbon. In this research, we introduced new genes into a cyanobacterium in order to create a novel pathway for fixed carbon utilization which results in the synthesis of ethanol. The coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium Synechococcus sp. strain PCC 7942. Under control of the promoter from the rbcLS operon encoding the cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase, the pdc and adh genes were expressed at high levels, as demonstrated by Western blotting and enzyme activity analyses. The transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium. As cyanobacteria have simple growth require-ments and use light, CO 2 , and inorganic elements efficiently, production of ethanol by cyanobacteria is a potential system for bioconversion of solar energy and CO 2 into a valuable resource. Cyanobacteria, also known as blue-green algae, are autotro-phic prokaryotes which exhibit diversity in metabolism, struc-ture, morphology, and habitat. However, all of these organisms perform oxygenic photosynthesis, and this photosynthesis is similar to that performed by higher plants (29, 32). As the cyanobacteria have simple growth requirements, grow to high densities, and use light, carbon dioxide, and other inorganic nutrients efficiently, they could be attractive hosts for produc-tion of valuable organic products. In fact, many cyanobacteria can be used directly as food and fodder since they are non-pathogenic and have high nutrient value (27). Some cyanobac-teria also synthesize secondary metabolites which have been reported to have significant therapeutic effects (4). In addition, mass cultivation for commercial production of some cyanobac-teria can be performed efficiently. Synechococcus sp. strain PCC 7942 (previously referred to as Anacystis nidulans R2), a unicellular cyanobacterium that lives in freshwater, is one of the few cyanobacterial strains which have been relatively well-characterized in terms of physiology, biochemistry, and genetics. This organism is able to take up foreign DNA and can be transformed either by using shuttle vectors capable of replicating in both Escherichia coli and the cyanobacterium or by integrating foreign DNA into the chro-mosome through homologous recombination at targeted sites (14, 36). In recent years, workers have achieved limited success in expressing foreign genes in this cyanobacterium, as well as other transformable strains. For example, the human carbonic anhydrase gene caII used to investigate CO 2 -concentrating mechanisms (26), E. coli and human superoxide dismutase genes used to investigate oxidative stress (15, 34), E. coli pet genes used to increase salt stress resistance (25), and Bacillus thuringiensis larvicidal genes used to develop bioinsecticidal hosts (33, 35) have all been expressed in Synechococcus sp. at sufficiently high levels to generate discernible phenotypes. In this paper, we describe our attempts to transform Synechococ-cus sp. strain PCC 7942 with bacterial genes in order to create a novel pathway for ethanol production in cyanobacteria.

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Authors

  • Ming De Deng

  • John R. Coleman

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