Chemical Mutating And High Throughput Screening Of Lipid-rich Nannochloropsis Oceanica

As fossil fuels are being run out of, microalgae-based biodiesel has currently been viewed as a perfect substitute for petroleum. Nannochloropsis have received a lot of attention for biodiesel production. To meet the requirements of the commercial production, outstanding microalgal species have been hotspot of research. Integrating random mutation with a new high-throughput screening method, we attempted to achieve desirable Nannochloropsis species with higher performance in both lipid content and biomass. Initially we compare the efficiciency of ethyl methane sulfonate (EMS) with nitrosoguanidine (NTG) in mutagenesis. Three mutants (LAMB001, LAMB002 and LAMB003) of Nannochloropsis oceanica with higher lipid were bred by NTG mutating and high-throughput screening in this study. The high-throughput screening included BODIPY 505/515 staining and fluorescence microplate reading. The optimal concentration (0.240?g/mL) of BODIPY 505/515 was determined for staining wild N. oceanica. NTG was proved to be more effective than EMS for producing lipid-rich mutants. In comparison with wild N. oceanica, the lipid content of LAMB001, LAMB002 and LAMB003 increased by 17.4%,23.7% and 29.40%, respectively. Although the growth of mutant LAMB003 was obviously slow, it accumulated the richest lipid (31.23%). In order to explain the mechanism of mutagenesis, LAMB003 was further analyzed. The lipid increase of LAMB003 results in the decrease of 16.09% and 18.24% in protein and carbohydrate content. Triacylglycerol (TAG) composition of LAMB003 and wild N.oceanica was measured by lipidomics and there ws a distinction between them. Wild N. oceanica contains two more kinds--TAG (14:0/16:0/20:4) and TAG (16:0/16:0/20:5), both of which contain a PUFA chain, while none of TAG in LAMB003 is comprised of PUFA chain, which is in accordance with the fatty acid profile. PUFA content of LAMB003 decreased significantly and only accounted for 29.46% of total fatty acid, while wild N.oceanica 34.54%. We measured the transcriptional level of some key genes along the lipid biosynthesis path way, especially the fatty acid synthesis, by real-time quantitative PCR. The results indicate that the transcriptional level of phosphoenolpyruvate carboxylase (PEPC), which competes phosphoenolpyruvate (PEP) with lipid synthesis, A6 desaturase (D6), elongase (NE) and acyl-CoA syntheta (ACS) decreased dramatically, while that of Acetyl-CoA carboxylase (ACCase) increases significantly. No difference is found in ?12 desaturase (D6). Given all the results, we can draw a conclusion that the decreased transcriptional level of PEPC and increased transcriptional level of ACCase cause less PEP in tricarboxylic acid cycle and simultaneously the decreased transcriptional level of D6 and NE also reduce the synthesis of PUFA. Transcriptional level of ACS, as a key gene in lipid synthesis, also declined, since the ACS may be responsible for lipid degradation rather than synthesis. The successful isolation of the three outstanding mutants proved the viability of our high-throughput screening method.