Campus Units

Genetics, Development and Cell Biology, Plant Pathology and Microbiology, Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of

Document Type

Article

Publication Date

4-2014

Journal or Book Title

Plant Biotechnology Journal

Volume

12

Issue

7

First Page

872

Last Page

882

DOI

10.1111/pbi.12190

Abstract

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.

Comments

This article is published as Xie, Bo, Dan Stessman, Jason H. Hart, Haili Dong, Yingjun Wang, David A. Wright, Basil J. Nikolau, Martin H. Spalding, and Larry J. Halverson. "High‐throughput fluorescence‐activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants." Plant biotechnology journal 12, no. 7 (2014): 872-882. 10.1111/pbi.12190. Posted with permission.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Copyright Owner

Society for Experimental Biology

Language

en

File Format

application/pdf

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