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To evaluate the potential of algal biotechnology to replace traditional agriculture in northeastern Thailand, an open raceway cultivation system was developed to produce biomass and beta-carotene. Dunaliella salina KU 11 isolated from local saline soil was cultured in open raceway tanks using brine and saline lake water. Grown in modified Johnson's medium (with 2-3.5 M NaCl), the algae reached a maximum cell density on the fourth day (1.8 × 10 cells mL ). Increasing KNO and NaHCO from 0.5 and 0.043 g L to 1 and 2.1 g L , respectively, significantly improved the yields of biomass (0.33 g L ) and beta-carotene (19 mg L ). Expected profits for algal production were evaluated, and it was found that this strain was suitable for outdoor cultivation and the developing algal industry in northeastern Thailand could produce high economic benefits (at least $64,120 per year per 0.16 ha).

A Planctomyces limnophilus mutant generated using the EZ-Tn5 transposome was found to possess an insertion within pckA, encoding phosphoenolpyruvate carboxykinase. Disruption of pckA expression and elimination of enzymatic activity resulted in poor growth in glucose-free medium, demonstrating a gluconeogenic role for pckA in P. limnophilus. Bacteria belonging to the phylum Planctomycetes possess several distinctive phenotypic and molecular characteristics, including a proteinaceous cell wall that is devoid of peptidoglycan (17), reproduction by yeast-like division, and in many cases, internal cell compartmentalization resembling the eukaryotic nucleus (2, 19). They are found in aquatic and soil environments (3), as well as among microbial communities associated with the intestinal tracts of many organisms, including mammals (11) and fish (15). The Planctomycetes are significant participants in the global carbon and nitrogen cycles and contain a unique group capable of carrying out anaerobic ammonium oxidation (anammox) (5,8). While the availability of genome sequences for several Planctomycetes (cited in reference 6), as well as biochemical, physiological, and ecological studies, have provided some insight into this fascinating group of bacteria, the lack of molecular genetic tools has made it difficult to examine them in greater detail.Recently, Jogler et al. (6) described a genetic approach for studying Planctomyces limnophilus and suggested that this bacterium be used as a model species for the Planctomycetes. P. limnophilus is a chemoheterotroph that has a distinct cell cycle, with sessile cells that form stalks; its ability to form multicellular rosettes, its low salt tolerance, pinkish red pigmentation, high growth rate compared to the growth of other Planctomycetes (4), intracytoplasmic membrane structure, and the availability of a genome sequence (10) make it an ideal subject for molecular genetic studies. We have been examining the use of the Epicentre (Madison, WI) R6K␥ori/Kan-2 transposon kit (EZ-Tn5) (9) for creating P. limnophilus mutants by a procedure similar to that described by Jogler et al. (6). Here, we report the isolation of mutants obtained using this approach and describe the characterization of one mutant that contains an insertion within the gene encoding phosphoenolpyruvate carboxykinase, pckA.Transposon mutagenesis and isolation of mutants. Electrocompetent cells of P. limnophilus (DSM 3776), a gift from Naomi Ward, University of Wyoming, were prepared from a 5-day-old culture grown in 500 ml of modified 621 medium (DSMZ) (peptone and yeast extract concentrations were doubled to 0.05% each) at 28°C and 190 rpm that was washed with 10% glycerol and suspended in 1.5 ml 10% glycerol. Cells prepared in this manner were electrocompetent for several months when stored at Ϫ80°C. Electroporation was carried out by mixing 1 l of EZ-Tn5 transposome solution and 1 l of TypeOne restriction inhibitor (Epicentre) with 70-l cells in a microcentrifuge tube, incubating on ice for 5 min, and then transf...

Microalgae are a promising platform for generating valuable commercial products, including proteins that may not express well in more traditional cell culture systems. In the model green alga Chlamydomonas reinhardtii, transgenic proteins can be expressed from either the nuclear or chloroplast genome. Expression in the chloroplast has several advantages, but technology is not yet well developed for expressing multiple transgenic proteins simultaneously. Here, we developed new synthetic operon vectors to express multiple proteins from a single chloroplast transcription unit. We modified an existing chloroplast expression vector to contain intercistronic elements derived from cyanobacterial and tobacco operons and tested the ability of the resulting operon vectors to express two or three different proteins at a time. All operons containing two of the coding sequences (for C. reinhardtii FBP1 and atpB) expressed the products of those genes, but operons containing the other two coding sequences (C. reinhardtii FBA1 and the synthetic camelid antibody gene VHH) did not. These results expand the repertoire of intercistronic spacers that can function in the C. reinhardtii chloroplast, but they also suggest that some coding sequences do not function well in the context of synthetic operons in this alga.

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