title: Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina.
abstract: BACKGROUND
  Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loop-helix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake.
  
  
  RESULTS
  We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3-6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes.
  
  
  CONCLUSIONS
  Overexpression of NsbHLH2 led to enhanced growth rate and nutrient uptake during the early growth phase, and increased biomass and FAME productivity, especially in the later period under normal and stressed conditions. Based on these results, we postulate that NsbHLH2 can be employed for the industrial production of biodiesel from N. salina.
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Nam Kyu
authors.initials: NK
authors.lastname: Kang
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Seungjib
authors.initials: S
authors.lastname: Jeon
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Sohee
authors.initials: S
authors.lastname: Kwon
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Hyun Gi
authors.initials: HG
authors.lastname: Koh
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Sung-Eun
authors.initials: SE
authors.lastname: Shin
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Bongsoo
authors.initials: B
authors.lastname: Lee
authors.affiliation: Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Gang-Guk
authors.initials: GG
authors.lastname: Choi
authors.affiliation: Department of Chemical and Biomolecular Engineering, 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, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Byeong-Ryool
authors.identifier: 0000-0003-0642-1650
authors.initials: BR
authors.lastname: Jeong
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea ; Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
authors.forename: Yong Keun
authors.initials: YK
authors.lastname: Chang
keywords: Basic helix-loop-helix; Fatty acid methyl ester; Microalgae; Nannochloropsis salina; Specific growth rate; Transcription factor