Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l(-1) hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne's to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species.

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Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.

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Sedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl β-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel-nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.