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Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina.

Kang NK, Jeon S, Kwon S, Koh HG, Shin SE, Lee B, Choi GG, Yang JW, Jeong BR, Chang YK - Biotechnol Biofuels (2015)

Bottom Line: These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.

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.

Conclusion: 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.

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Related in: MedlinePlus

Expression of NsbHLH2 in transformants under different culture conditions. a qRT-PCR of transgenic NsbHLH2 mRNA by using qbH1 and qbH2 primers, where qbH1 is located in the vector. b qRT-PCR of transgenic and endogenous NsbHLH2 mRNA by using qbH3 and qbH4 primers. c Western blotting of FLAG-tagged NsbHLH2. The expected size of FLAG-tagged NsbHLH2 was 65 kD. AtpB [expected sizes of 72.6 kD (F-type H-ATPase ß subunit) and 53.13 kD (CF1ß subunit of ATP synthase)] was used as a loading control. Accession number of CF1ß subunit of ATP synthase from N. salina CCMP537 is YP_008519835; accession number of F-type H-ATPase ß subunit from N. gaditana B-31 is EWM25142. Homologs of these proteins were present in N. salina CCMP1776, and appeared to be good loading controls with constant expression level under different culture conditions. WT wild type; N normal conditions; NL nitrogen limitation; O osmotic stress. The data points represent the average of samples and error bars indicate standard error (n = 3). Significant differences, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)
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Fig2: Expression of NsbHLH2 in transformants under different culture conditions. a qRT-PCR of transgenic NsbHLH2 mRNA by using qbH1 and qbH2 primers, where qbH1 is located in the vector. b qRT-PCR of transgenic and endogenous NsbHLH2 mRNA by using qbH3 and qbH4 primers. c Western blotting of FLAG-tagged NsbHLH2. The expected size of FLAG-tagged NsbHLH2 was 65 kD. AtpB [expected sizes of 72.6 kD (F-type H-ATPase ß subunit) and 53.13 kD (CF1ß subunit of ATP synthase)] was used as a loading control. Accession number of CF1ß subunit of ATP synthase from N. salina CCMP537 is YP_008519835; accession number of F-type H-ATPase ß subunit from N. gaditana B-31 is EWM25142. Homologs of these proteins were present in N. salina CCMP1776, and appeared to be good loading controls with constant expression level under different culture conditions. WT wild type; N normal conditions; NL nitrogen limitation; O osmotic stress. The data points represent the average of samples and error bars indicate standard error (n = 3). Significant differences, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)

Mentions: We also measured the expression of endogenous and transgenic NsbHLH2 under different culture conditions using qRT-PCR and Western blotting (Fig. 2). For these experiments, samples were collected at 0 h under normal conditions and at 24 h after the change in culture conditions (normal, N limitation, and osmotic stress condition). First, we examined the transcription of transgenic NsbHLH2 using the forward primer in the untranslated region of the plasmid (qbH1) in combination with the reverse primer located in the coding sequence of NsbHLH2 (qbH2) (Additional file 5: Table S1). These primers amplified correct products only in the transformants, and qRT-PCR revealed variable expression of the transgenic NsbHLH2 (Fig. 2a). Although the constitutive TUB promoter was used to express NsbHLH2, expression depended on the culture conditions. In particular, there was significantly reduced expression of NsbHLH2 under osmotic stress. We also used Western blot to show expression of FLAG-tagged NsbHLH2 under the three different culture conditions. Consistent with the qRT-PCR results, the FLAG-tagged proteins were found only in the transformants (Fig. 2c). However, expression pattern was not consistent with that of RNAs. Expression of the NsbHLH2 protein was rather constant and high in transformant 3–6, while that of 3–11 was low except for the 24 h sample under the normal condition. These discrepancies suggest that accumulation of transgenic NsbHLH2 protein mainly depends on the stability of the protein rather than on the amount of RNAs.Fig. 2


Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina.

Kang NK, Jeon S, Kwon S, Koh HG, Shin SE, Lee B, Choi GG, Yang JW, Jeong BR, Chang YK - Biotechnol Biofuels (2015)

Expression of NsbHLH2 in transformants under different culture conditions. a qRT-PCR of transgenic NsbHLH2 mRNA by using qbH1 and qbH2 primers, where qbH1 is located in the vector. b qRT-PCR of transgenic and endogenous NsbHLH2 mRNA by using qbH3 and qbH4 primers. c Western blotting of FLAG-tagged NsbHLH2. The expected size of FLAG-tagged NsbHLH2 was 65 kD. AtpB [expected sizes of 72.6 kD (F-type H-ATPase ß subunit) and 53.13 kD (CF1ß subunit of ATP synthase)] was used as a loading control. Accession number of CF1ß subunit of ATP synthase from N. salina CCMP537 is YP_008519835; accession number of F-type H-ATPase ß subunit from N. gaditana B-31 is EWM25142. Homologs of these proteins were present in N. salina CCMP1776, and appeared to be good loading controls with constant expression level under different culture conditions. WT wild type; N normal conditions; NL nitrogen limitation; O osmotic stress. The data points represent the average of samples and error bars indicate standard error (n = 3). Significant differences, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4666162&req=5

Fig2: Expression of NsbHLH2 in transformants under different culture conditions. a qRT-PCR of transgenic NsbHLH2 mRNA by using qbH1 and qbH2 primers, where qbH1 is located in the vector. b qRT-PCR of transgenic and endogenous NsbHLH2 mRNA by using qbH3 and qbH4 primers. c Western blotting of FLAG-tagged NsbHLH2. The expected size of FLAG-tagged NsbHLH2 was 65 kD. AtpB [expected sizes of 72.6 kD (F-type H-ATPase ß subunit) and 53.13 kD (CF1ß subunit of ATP synthase)] was used as a loading control. Accession number of CF1ß subunit of ATP synthase from N. salina CCMP537 is YP_008519835; accession number of F-type H-ATPase ß subunit from N. gaditana B-31 is EWM25142. Homologs of these proteins were present in N. salina CCMP1776, and appeared to be good loading controls with constant expression level under different culture conditions. WT wild type; N normal conditions; NL nitrogen limitation; O osmotic stress. The data points represent the average of samples and error bars indicate standard error (n = 3). Significant differences, as determined by Student’s t test, are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)
Mentions: We also measured the expression of endogenous and transgenic NsbHLH2 under different culture conditions using qRT-PCR and Western blotting (Fig. 2). For these experiments, samples were collected at 0 h under normal conditions and at 24 h after the change in culture conditions (normal, N limitation, and osmotic stress condition). First, we examined the transcription of transgenic NsbHLH2 using the forward primer in the untranslated region of the plasmid (qbH1) in combination with the reverse primer located in the coding sequence of NsbHLH2 (qbH2) (Additional file 5: Table S1). These primers amplified correct products only in the transformants, and qRT-PCR revealed variable expression of the transgenic NsbHLH2 (Fig. 2a). Although the constitutive TUB promoter was used to express NsbHLH2, expression depended on the culture conditions. In particular, there was significantly reduced expression of NsbHLH2 under osmotic stress. We also used Western blot to show expression of FLAG-tagged NsbHLH2 under the three different culture conditions. Consistent with the qRT-PCR results, the FLAG-tagged proteins were found only in the transformants (Fig. 2c). However, expression pattern was not consistent with that of RNAs. Expression of the NsbHLH2 protein was rather constant and high in transformant 3–6, while that of 3–11 was low except for the 24 h sample under the normal condition. These discrepancies suggest that accumulation of transgenic NsbHLH2 protein mainly depends on the stability of the protein rather than on the amount of RNAs.Fig. 2

Bottom Line: These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 Republic of Korea.

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus