Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in Chlamydomonas reinhardtii.

Natarajan Velmurugan,Minji Sung,Ji Won Yang,Min S Park,Ki Jun Jeong,Sung Sun Yim

doi:10.1186/s13068-014-0117-7

Abstract

BackgroundThe concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions.ResultsThe successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution.ConclusionsAdaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0117-7) contains supplementary material, which is available to authorized users.

Highlights

The concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions
To obtain a healthy population, the seed cultures of wild-type CC124 and mutant sta6-1 were obtained from the late-log phase in nitrogen-supplemented Tris Acetate Phosphate liquid Medium (TAP) medium
C. reinhardtii strains CC124 and sta6-1 have been subjected to adaptive evolution to understand the molecular mechanisms of lipid accumulation under nitrogen-depleted conditions

Summary

Introduction

The concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. A flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions. The intracellular lipid body formation in C. reinhardtii depends on several factors including stress conditions such as nutrient starvation, temperature, salinity, and light intensity [4]. In the past few years, researchers have investigated the intracellular lipid accumulation in microalgae under different stress conditions [1,5]. Detailed studies on the molecular mechanism of lipid accumulation in microalgae under stress conditions should facilitate improvements in the lipid productivity, cultivation processes, and strain development for biofuels production [4]. Under nitrogen-depleted conditions, neutral lipids and starch will accumulate to high levels to serve as a primary form of energy storage [4]

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