High-throughput Raman-activated cell sorting of microalgal genome-wide edited library revealed a regulatory pathway for carotenoid synthesis.

Abstract

Functional genomics have been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated cell sorting (RACS). The CRISPR/Cas-generated library consisted of 3567 microalgal mutants derived from 2397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unraveled mutations in the violaxanthin de-epoxidase (noVDE) or in the proteasome assembly chaperone 4 (noPAC4) genes. Knocking out all five known noVDEs revealed that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidence suggested two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this forward-genetics approach can greatly accelerate the discovery of genes and pathways.

Plant phenotyping relevance

微細藻類の単細胞カロテノイド含量という生理形質を、Raman-activated cell sortingでラベルフリーかつハイスループットに取得・選別するプラットフォームが研究の中心であり、単なる生物学的測定ではない。

Code and data availability

植物フェノタイピング解析を再現する公開資産であることを、入力本文と直接リンクから確認できなかったため保留しました。

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