The Involvement of hybrid cluster protein 4, HCP4, in Anaerobic Metabolism in Chlamydomonas reinhardtii.

Adam C Olson,Clay J Carter,Andrew Webber

doi:10.1371/journal.pone.0149816

Adam C Olson, Clay J Carter + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0149816

Copy DOI

Abstract

The unicellular green algae Chlamydomonas reinhardtii has long been studied for its unique fermentation pathways and has been evaluated as a candidate organism for biofuel production. Fermentation in C. reinhardtii is facilitated by a network of three predominant pathways producing four major byproducts: formate, ethanol, acetate and hydrogen. Previous microarray studies identified many genes as being highly up-regulated during anaerobiosis. For example, hybrid cluster protein 4 (HCP4) was found to be one of the most highly up-regulated genes under anoxic conditions. Hybrid cluster proteins have long been studied for their unique spectroscopic properties, yet their biological functions remain largely unclear. To probe its role during anaerobiosis, HCP4 was silenced using artificial microRNAs (ami-hcp4) followed by extensive phenotypic analyses of cells grown under anoxic conditions. Both the expression of key fermentative enzymes and their respective metabolites were significantly altered in ami-hcp4, with nitrogen uptake from the media also being significantly different than wild-type cells. The results strongly suggest a role for HCP4 in regulating key fermentative and nitrogen utilization pathways.

Highlights

Chlamydomonas reinhardtii is a predominantly soil dwelling microalgae found globally [1] that has long been used as a model system for studying photosynthesis, nutrient deprivation, flagellar function, and H2 production [2]
Interest in C. reinhardtii as model organism for biofuel production has been renewed in recent years due to: 1) the discovery of its ability to perform anaerobiosis in the light, 2) its rapid growth rates compared to terrestrial plants, and 3) development of ‘omics’ based approaches to elucidating metabolic pathways, including the development of genetic manipulation techniques, which can be used for the optimization of metabolic processes [3,4]
Wild-type cc425 and ami-hcp4 cells were grown in Tris Acetate Phosphate (TAP) media, pelleted, washed, and resuspended in 28mM HEPES buffer and subjected to five hours of anoxic conditions through growth in the dark while being bubbled with a constant stream of N2 gas

Summary

Introduction

Chlamydomonas reinhardtii is a predominantly soil dwelling microalgae found globally [1] that has long been used as a model system for studying photosynthesis, nutrient deprivation, flagellar function, and H2 production [2]. Interest in C. reinhardtii as model organism for biofuel production has been renewed in recent years due to: 1) the discovery of its ability to perform anaerobiosis in the light, 2) its rapid growth rates compared to terrestrial plants, and 3) development of ‘omics’ based approaches to elucidating metabolic pathways, including the development of genetic manipulation techniques, which can be used for the optimization of metabolic processes [3,4]. The generation of stable mutants in C. reinhardtii has traditionally been achieved by random genomic integration [4]. This approach is cumbersome and requires the screening of thousands of mutants using suitable phenotypic criteria or extensive DNA analysis. Tools have been developed that enable targeted gene disruption through the use of artificial microRNAs (amiRNAs) [5,6] and CRISPR/Cas technologies [7].

Objectives

Methods

Results

Discussion

Conclusion