New Synthetic Operon Vectors for Expressing Multiple Proteins in the Chlamydomonas reinhardtii Chloroplast.

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.

Outdoor cultivation of Dunaliella salina KU 11 using brine and saline lake water with raceway ponds in northeastern Thailand.

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 × 106 cells mL-1 ). Increasing KNO3 and NaHCO3 from 0.5 and 0.043 g L-1 to 1 and 2.1 g L-1 , respectively, significantly improved the yields of biomass (0.33 g L-1 ) and beta-carotene (19 mg L-1 ). 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).

Transposon mutagenesis of Planctomyces limnophilus and analysis of a pckA mutant.

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.