title: Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in Chlamydomonas reinhardtii.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Adaptive 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea.
authors.forename: Natarajan
authors.initials: N
authors.lastname: Velmurugan
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea.
authors.forename: Minji
authors.initials: M
authors.lastname: Sung
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea.
authors.forename: Sung Sun
authors.initials: SS
authors.lastname: Yim
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea ; Bioscience Division, Los Alamos National Laboratory, Bikini Atoll Road, Los Alamos, NM 87545 USA.
authors.forename: Min S
authors.initials: MS
authors.lastname: Park
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea.
authors.forename: Ji Won
authors.initials: JW
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea ; KI for the Biocentury, KAIST, 291 Daehak-ro, Yuseong-gu Daejeon, 305-701 Republic of Korea.
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
keywords: Adaptive evolution; Chlamydomonas reinhardtii; Flow cytometry; Proteomics