Overexpression of fructose-1,6-bisphosphate aldolase 1 enhances accumulation of fatty acids in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtti is a promising source of bioenergy and understanding its carbon metabolism is important. In this organism, carbon accumulation can be manipulated with nitrogen supply. Chlamydomonas reinhardtii is a mixotroph that can act as an autotroph or heterotroph, depending on conditions. Fructose-1,6-bisphosphate aldolases (FBAs) are important enzymes in energy pathways of algae; they convert fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Expression of both CrFBA1 and CrFBA2 was less under nitrogen-deplete conditions than under nitrogen-replete conditions in Chlamydomonas cells. To explore CrFBA1 function, CrFBA1-overexpressing Chlamydomonas lines were generated. Compared to wild-type, transformed cells grew more slowly in photoautotrophic conditions, but at the same rate in mixotrophic condition. We suggest that overexpression of CrFBA1 affected photosynthetic carbon fixation in the Calvin-Benson cycle. The CrFBA1-overexpressing lines had less starch than the wild-type under mixotrophic, low-nitrogen conditions, but more fatty acids. In the transgenic cells, expression of granule bound starch synthase 1 (GBSS1) determined by quantitative reverse transcription-polymerase chain reaction was down-regulated, but acetyl-CoA synthetases (ACS1 and ACS2) and phosphoglucomutase (PGM) were up-regulated compared to wildtype. We propose three possible explanations for the observed low starch and high fatty acid content of the transgenic cells. Firstly, abnormal carbon flux by CrFBA1 overexpression leads to GBSS1 down-regulation in a feed-forward regulation, which resulted in the low starch accumulation. Secondly, increase of exogenous acetate assimilation by ACS1 and ACS2 up-regulation results in high accumulation of total fatty acid in transformants, perhaps due to compensation for reduced-carbon fixation. Lastly, activation of glycolytic pathway by CrFBA1 overexpression enhances conversion of starch to fatty acid. We conclude that overexpression of CrFBA1 deregulates carbon metabolism in Chlamydomonas reinhardtii.