Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor.

Microalgae are promising feedstocks for sustainable and eco-friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix-loop-helix transcription factor (NsbHLH2). These transformants showed improved growth and lipid production particularly during the early phase of culture under batch culture. However, they had faster uptake of nutrients, resulting in earlier starvation and reduced growth during the later stages. We attempted to optimize growth and lipid production by growing one of the transformants in continuous culture with variable dilution rate and feed nitrogen concentration. Relative to wild type, NsbHLH2 transformant consumed more nitrate at high dilution rate (0.5 day-1 ), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to increased FAME production by 83.6 mg L-1 d-1 . To elucidate genetic mechanisms, we identified genes containing E-boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in growth and lipid metabolism, and revealed their positive contribution to the phenotypes via qRT-PCR. These results provide proof-of-concept that NsbHLH2 can be employed to produce biomass and lipids. This article is protected by copyright. All rights reserved.