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

Abstract

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 wild-type. 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.