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

Abstract

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 that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix‐loop‐helix transcription factor (NsbHLH2). These transformants showed an 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 the 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 a high dilution rate (0.5 day −1 ), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to an increased fatty acid methyl ester production by 83.6 mg l −1 day −1 . To elucidate genetic mechanisms, we identified the genes containing E‐boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in the growth and lipid metabolism, and revealed their positive contribution to the phenotypes via quantitative real‐time polymerase chain reaction. These results provide proof‐of‐concept that NsbHLH2 can be used to produce biomass and lipids.

Improved lipid productivity in Nannochloropsis gaditana in nitrogen-replete conditions by selection of pale green mutants

“…In line with this finding e8 mutant did not present any mutations on light harvesting subunits. Unaltered photosynthetic efficiency in pale green mutant has been previously reported in several microalgae species [22,23,31,[47][48][49], chlorophyll content per cell being not necessarily influencing the functionality of the photosynthetic apparatus, but rather being possibly linked to chloroplast biogenesis. Consistently e8 mutant was characterized by a strong reduction in C20:5 fatty acid accumulation, this lipid being the main constituent of thylakoid glycerolipids MGDG, DGDG and SQDG in N. gaditana [19].…”

“…However, this approach strongly reduces cells growth rate, affecting overall biomass and lipid productivities. Some positive results were obtained by overexpression or downregulation of transcriptional factor increasing the lipid production with moderate or even positive effects for the growth [30,31]. However, the possibility to use genetic modified organisms (GMOs) at industrial scale is still limited by the different acceptance and legislation in the different countries, hampering the application of the promising results obtained.…”

Macronutrient effect on biomass of Microalgae in biofuel production: A review

“…For instance, the recombinant strain of Scenedesmus obliquus CPC2-G1 showed successfully increases in biomass and lipid productivity, at 16.3% and 84.9% higher than the wild-type strain (114). In Nannochloropsis salina, overexpressing a bHLH transcription factor led to increase the biomass production with a simultaneous increase of fatty acid methyl esters in lipid (115).…”

The NanDeSyn database for Nannochloropsis systems and synthetic biology

“…These extensive metabolic and genetic engineering efforts have shown Nannochloropsis to be an excellent model system for industrial applications in aquaculture and nutraceutical fields as well as biofuel industries (Poliner et al, 2018a). In fact, such efforts have been employed to enhance triacylglycerol (TAG) biosynthesis (Li et al, 2014;Wang et al, 2014a;Ajjawi et al, 2017;Xin et al, 2017Xin et al, , 2019, to dissect or engineer CO 2 fixation machineries (Gee et al, 2017;Wei et al, 2017aWei et al, , 2019a, and to characterize carbon partitioning (Jia et al, 2015;Poliner et al, 2015), sterol metabolism (Lu et al, 2014b), synthesis of photosynthetic pigments (Koh et al, 2019), phytohormone function (Lu et al, 2014a;Lu and Xu, 2015) and transcriptional-factor regulatory networks (Hu et al, 2014;Kang et al, 2015Kang et al, , 2017Kang et al, , 2019.…”