Genetic engineering of photosynthesis and lipid biosynthesis pathways in nannochloropsis salina for enhanced lipid productivity

Abstract

Nannochloropsis salina, a marine alga with high growth rate and lipid composition has been considered to be one of the most appropriate species for the feed stock of biofuels or value-added products. In this research, for the purpose of increasing the overall lipid production rate of N. salina, genetic engineering was adopted to enhance the photosynthetic efficiency and lipid biosynthesis metabolism within the strain. To improve the photosynthetic efficiency, new kinds of light harvesting pigments were heterogeneously synthesized to broaden the light spectrum that N. salina can utilize. For this, a vector system that can deliver the expressed protein to the complex chloroplast of the heterokonts were developed, which were then used to express chlorophyllide a oxygenase (CAO) gene and chlorophyll f synthase (ChlF) gene from Chlamydomonas reinhardtii and Chlorogloeopsis fritschii respectively, after optimizing the sequence for N. salina. As a result, transformants with chlorophyll b and f synthesis could be achieved for the first time in N. salina. Transformants with Chlb showed an improved photosynthetic efficiency under low to high light conditions. The additional energy synthesized from the photosynthetic improvements were used for growth under moderate light conditions (90 uE) whereas the energy was used for lipid synthesis under high light condition (200 uE). However, transformants with Chlf did not grow well and lost the ChlF functions within 2 months, implying toxic effects of Chlf in N. salina.

Furthermore, in order to increase the portion of carbon flux that enters the lipid synthesis pathway, key metabolic enzymes for lipid bio-synthesis were overexpressed in a wild type N. salina. For this, two genes, each encoding Glycerol-3-phosphate dehydrogenase(GPDH) and Phospholipid: diacylglycerol acyltransferase(PDAT) that are responsible for the reaction of first and last reactions for TAG synthesis were selected. The transformants with overexpressed NsGPDH and NsPDAT enzyme showed a tendency of hampered proliferation but the change was not significant. However, the lipid accumulation in each transformants showed up to 40 % increase compared to the wild type. To assure its possibility for producing biodiesel, FAME contents of each transformants were also analyzed and an increase of FAME productivity by up to 40 % were observed in the transformants.

After confirming the beneficial effects of chlorophyll b and GPDH/PDAT in photosynthesis and lipid biosynthesis pathways, multigene expression system in N. salina was developed to provide a tool for co-expression. For this, four kinds of 2A self-cleaving peptides in N. salina was tested and E2A turned out to be the best performer in the strain. It is expected that the study on expression of CAO, GPDH, and PDAT genes along with the development of multigene expression system in N. salina will be beneficial for industrial uses and further research in this field.