Engineering of nannochloropsis salina for enhanced lipid biosynthesis by high-throughput screening and genome editing

Abstract

Because of the depletion of energy resources and the global warming caused by the use of fossil fuels, biofuels, which are sustainable and environmentally friendly energy resources, are attracting attention as alternative energy sources. Among the various biomass feedstock, microalgae for biofuels are promising biomass with the advantage that relatively narrow cultivation land and short cultivation time are required, and do not compete with human food. Recently, the necessity for carrying out biofuel production research is being highlighted in accordance with introduction Renewable Fuel Standard (RFS) and increase in the planned biofuel supplement. In this respect, we carried out studies for the high efficiency biosynthesis of lipids to reduce in oleaginous microalgae Nannochoropsis salina due to reduce the biomass raw material cost for economical biofuel production. In particular, the present study focused on exploring gene information useful for the development of N. salina. Herein, isolation of gene locations were performed for the purpose of finding novel target genes related to lipid biosynthesis and establishment of high-level gene expression system. First, a study for identification of genes related to increased lipid biosynthesis in the event of gene disruption was performed. N. salina library including mutants having single gene disruption in random was constructed using the insertional mutagenesis technique and screened by using FACS. As a result, a mutant was isolated and exhibited 53% faster growth rate and 34% increase in fatty acid methyl ester (FAME) contents after incubation for eight days, resulting in 75% increase in FAME productivity compared to wild type. And we identified the disrupted gene in isolated mutant, gene of trehalose-6-phosphate phosphatase domain (TPP domain) included in trehalose-6-phosphate synthase (TPS). By performing metabolite analyses, the intracellular concentration of trehalose was significantly decreased and trehalose-6-phosphate was slightly increase than wild type. With these results, a novel hypothesis for correlation among TPS gene disruption, cell growth, and lipid biosynthesis was suggested. In addition, isolation of transcriptional hotspot was conducted to construct a gene overexpression system for efficient genetic engineering of N. salina. The gene of superfolder green fluorescent protein (sfGFP) was introduced at a random chromosomal location to construct library for integration site screening. By performing fluorescence-activated cell sorting (FACS) of constructed library, mutants having transcriptional hotspot for high-level expression were successfully isolated. Also, several fatty acid desaturases (FADs) for high-level biosynthesis of polyunsaturated fatty acids (PUFAs) were overexpressed by using the isolated transcriptional hotspot and CRISPR/Cas9 technique. As a result, PUFAs were successfully produced in FADs overexpressing N. salina and finally we demonstrated the use of transcriptional hotspot as a novel gene overexpression system successfully. In summary, the two studies mentioned above revealed the novel genes related to gene overexpression and lipid biosynthesis, and the genetic information is expected to be widely used for genetic and metabolic engineering studies for microalgae as well as N. salina through the use of CRISPR/Cas9 genome editing. Also, it is expected that these studies can be contribute to metabolic engineering of high-lipid biosynthesizing microalgae, followed by reducing raw material and the commercialization of microalgal biofuels.