Genetic engineering and multi-omics analysis of chlamydomonas reinhardtii for improved lipid production

Abstract

Microalgae are considered as an excellent biofuel feedstocks due to their renewable and sustainable nature, and genetic engineering of microalgae are in progress to enhance production of biomass and lipids. In an effort to improve microalgae, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was overexpressed in Chlamydomonas reinhardtii. One transformant, designated PNG, showed increased growth under nitrogen starvation. Interestingly, PNG accumulated more lipids under the same condition compared to wild type. To investigate underlying metabolic mechanisms for the concomitant increase in growth and lipid accumulation, transcriptomic and metabolomic analyses were performed. The multi-omics data consistently pointed to enhanced lipid synthetic pathway and Calvin cycle, while deactivation of starch metabolism in PNG. To increase more lipid productivity of PNG, we applied a genome editing technique the clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) system. Due to the toxicity of Cas9 and off-target effect, we transiently delivered the Cas9 protein and sgRNAs as the Cas9 RNP. Testing two loci including the MAA7 and CpSRP43 genes, we isolated small indel mutants and NHEJ-mediated knock-in mutants, respectively. Based on the developed CRISPR/Cas9 system in C. reinhardtii, PNG transformant was transformed with additional Cas9 RNP to knockout the ADP-glucose pyrophosphorylase small subunit (AGPase) gene that is the key enzyme for starch biosynthesis. For better genome editing efficiency, various trials including delivery methods, cell penetrating peptides (CPPs), antisense RNAs of Ku70/80, and high amount of Cas9. In this study, an improved microalgal strain was isolated by overexpression of GAPDH, which showed concomitant increase in biomass and lipid accumulation. Its metabolic mechanisms were revealed by multi-omics analyses of its metabolome and transcriptome. In addition, successful genome editing technique (CRISPR/Cas9) was demonstrated in the model alga C. reinhardtii, opening exciting possibility for other industrial microalgae for production of biofuels and value-added biomaterials.