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Enhancement of lipid productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast CtDGAT2b gene under model-optimized growth condition.

Dey P, Mall N, Chattopadhyay A, Chakraborty M, Maiti MK - PLoS ONE (2014)

Bottom Line: Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress.Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively.Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.

View Article: PubMed Central - PubMed

Affiliation: Adv. Lab. for Plant Genetic Engineering, Advanced Technology Development Center, Indian Institute of Technology Kharagpur, Kharagpur, India.

ABSTRACT
Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.

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Development of Colletotrichum transformants with the CtDGAT2b gene expression construct, pCAM-GpdA-CtDGAT2b-TrpC through Agrobacterium-mediated transformation.(A) Schematic diagram of the genetic construct prepared in pCAMBIA 1300 binary vector. (B) Ethidium bromide-stained 1.2% agarose gel showing characteristic restriction enzyme digestion profile of the genetic construct with EcoRI+HindIII (lane1), EcoRI+BamHI (lane2), BamHI (lane3), SalI+BglII (lane 4), BamHI+HindIII (lane 5) and XhoHI (lane 7) along with the pUC18 DNA digested with Hinf1 as molecular weight marker (lane 6). (C) PCR-based screening for the presence of 550 bp CtDGAT2b transgene-specific amplicon in four (Td#2, Td#9, Td#18 and Td#23) chosen transformed lines. Lane M =  HinfI digested pUC18 DNA as molecular weight marker. (D) Confirming genomic integration of the CtDGAT2b transgene through Southern hybridization. Genomic DNA samples of four transformed lines were digested with EcoR1, and hybridized with the CtDGAT2b gene-specific probe. Lambda (λ) DNA digested with EcoRI+HindIII was used as molecular weight marker. In case of (C) and (D), the wild type Colletotrichum sp. DM06 was taken as control (lane C). (E) Transcriptional expression of the CtDGAT2b transgene in four fungal transformants as revealed by RT-PCR for 26 cycles. (F) Transcriptional expression of the CtDGAT2b in one of the fungal transformants, Td#18 (T) as revealed by RT-PCR for 26 cycles, showing upregulation of CtDGAT2b transcript upto 3rd day of growth and declined at 4th day onward. In case of (E) and (F), the wild type Colletotrichum sp. DM06 was taken as control (C), and the relative transcript level was measured based upon the densitometric scanning of the RT-PCR amplicons (550 bp for CtDGAT2b and 400 bp for endogenous β-actin as internal control) as shown in lower panel.
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pone-0111253-g002: Development of Colletotrichum transformants with the CtDGAT2b gene expression construct, pCAM-GpdA-CtDGAT2b-TrpC through Agrobacterium-mediated transformation.(A) Schematic diagram of the genetic construct prepared in pCAMBIA 1300 binary vector. (B) Ethidium bromide-stained 1.2% agarose gel showing characteristic restriction enzyme digestion profile of the genetic construct with EcoRI+HindIII (lane1), EcoRI+BamHI (lane2), BamHI (lane3), SalI+BglII (lane 4), BamHI+HindIII (lane 5) and XhoHI (lane 7) along with the pUC18 DNA digested with Hinf1 as molecular weight marker (lane 6). (C) PCR-based screening for the presence of 550 bp CtDGAT2b transgene-specific amplicon in four (Td#2, Td#9, Td#18 and Td#23) chosen transformed lines. Lane M =  HinfI digested pUC18 DNA as molecular weight marker. (D) Confirming genomic integration of the CtDGAT2b transgene through Southern hybridization. Genomic DNA samples of four transformed lines were digested with EcoR1, and hybridized with the CtDGAT2b gene-specific probe. Lambda (λ) DNA digested with EcoRI+HindIII was used as molecular weight marker. In case of (C) and (D), the wild type Colletotrichum sp. DM06 was taken as control (lane C). (E) Transcriptional expression of the CtDGAT2b transgene in four fungal transformants as revealed by RT-PCR for 26 cycles. (F) Transcriptional expression of the CtDGAT2b in one of the fungal transformants, Td#18 (T) as revealed by RT-PCR for 26 cycles, showing upregulation of CtDGAT2b transcript upto 3rd day of growth and declined at 4th day onward. In case of (E) and (F), the wild type Colletotrichum sp. DM06 was taken as control (C), and the relative transcript level was measured based upon the densitometric scanning of the RT-PCR amplicons (550 bp for CtDGAT2b and 400 bp for endogenous β-actin as internal control) as shown in lower panel.

Mentions: The pCAM-GpdA-GusA-TrpC genetic construct was prepared using the backbone of pCAMBIA 1300 binary vector. Initially the pNOM102 vector carrying the intron-containing gusA (uidA) gene placed under the regulation of gpdA promoter of Aspergillus nidulans and trpC terminator of A. nidulans in pUC18 vector (kindly provided by Prof. Francisco JL Aragão, Embrapa Recursos Genéticos e Biotecnologia, Brazil) was doubly digested with EcoRI and HindIII to obtain the DNA fragment GpdA-GusA-TrpC, which was subcloned into the pCAMBIA 1300 using the same set of restriction enzymes. The resulting recombinant plasmid was named as pCAM-GpdA-GusA-TrpC (Figure 1A). Cloning, sequencing and characterization of the CtDGAT2b gene (GenBank accession number KJ437598) from Candida tropicalis SY005 have already been described previously [30]. The CtDGAT2b gene was obtained after digesting with BamHI from pYES2/CtDGAT2b recombinant plasmid, which was used for the study of transgene expression in Saccharomyces cerevisiae[30]. The promoter GpdA was PCR amplified from pCAM-GpdA-GusA-TrpC recombinant plasmid (using Expand High Fidelity PCR mix, Roche) with a set of specific primers- GpdAFp and GpdARp (Table S1) to incorporate the restriction sites- EcoRI at 5' end and BamHI at 3' end of the promoter. The pCAM-GpdA-GusA-TrpC construct was digested with EcoRI and BamHI to remove the GpdA promoter and gusA gene. Thereafter, a tripartaite ligation was carried out using the BamHI digested CtDGAT2b gene, EcoRI and BamHI digested PCR amplified GpdA promoter and the above-mentioned EcoRI and BamHI digested pCAM-GpdA-GusA-TrpC to generate the pCAM-GpdA-CtDGAT2b-TrpC recombinant plasmid (Figure 2A).


Enhancement of lipid productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast CtDGAT2b gene under model-optimized growth condition.

Dey P, Mall N, Chattopadhyay A, Chakraborty M, Maiti MK - PLoS ONE (2014)

Development of Colletotrichum transformants with the CtDGAT2b gene expression construct, pCAM-GpdA-CtDGAT2b-TrpC through Agrobacterium-mediated transformation.(A) Schematic diagram of the genetic construct prepared in pCAMBIA 1300 binary vector. (B) Ethidium bromide-stained 1.2% agarose gel showing characteristic restriction enzyme digestion profile of the genetic construct with EcoRI+HindIII (lane1), EcoRI+BamHI (lane2), BamHI (lane3), SalI+BglII (lane 4), BamHI+HindIII (lane 5) and XhoHI (lane 7) along with the pUC18 DNA digested with Hinf1 as molecular weight marker (lane 6). (C) PCR-based screening for the presence of 550 bp CtDGAT2b transgene-specific amplicon in four (Td#2, Td#9, Td#18 and Td#23) chosen transformed lines. Lane M =  HinfI digested pUC18 DNA as molecular weight marker. (D) Confirming genomic integration of the CtDGAT2b transgene through Southern hybridization. Genomic DNA samples of four transformed lines were digested with EcoR1, and hybridized with the CtDGAT2b gene-specific probe. Lambda (λ) DNA digested with EcoRI+HindIII was used as molecular weight marker. In case of (C) and (D), the wild type Colletotrichum sp. DM06 was taken as control (lane C). (E) Transcriptional expression of the CtDGAT2b transgene in four fungal transformants as revealed by RT-PCR for 26 cycles. (F) Transcriptional expression of the CtDGAT2b in one of the fungal transformants, Td#18 (T) as revealed by RT-PCR for 26 cycles, showing upregulation of CtDGAT2b transcript upto 3rd day of growth and declined at 4th day onward. In case of (E) and (F), the wild type Colletotrichum sp. DM06 was taken as control (C), and the relative transcript level was measured based upon the densitometric scanning of the RT-PCR amplicons (550 bp for CtDGAT2b and 400 bp for endogenous β-actin as internal control) as shown in lower panel.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222912&req=5

pone-0111253-g002: Development of Colletotrichum transformants with the CtDGAT2b gene expression construct, pCAM-GpdA-CtDGAT2b-TrpC through Agrobacterium-mediated transformation.(A) Schematic diagram of the genetic construct prepared in pCAMBIA 1300 binary vector. (B) Ethidium bromide-stained 1.2% agarose gel showing characteristic restriction enzyme digestion profile of the genetic construct with EcoRI+HindIII (lane1), EcoRI+BamHI (lane2), BamHI (lane3), SalI+BglII (lane 4), BamHI+HindIII (lane 5) and XhoHI (lane 7) along with the pUC18 DNA digested with Hinf1 as molecular weight marker (lane 6). (C) PCR-based screening for the presence of 550 bp CtDGAT2b transgene-specific amplicon in four (Td#2, Td#9, Td#18 and Td#23) chosen transformed lines. Lane M =  HinfI digested pUC18 DNA as molecular weight marker. (D) Confirming genomic integration of the CtDGAT2b transgene through Southern hybridization. Genomic DNA samples of four transformed lines were digested with EcoR1, and hybridized with the CtDGAT2b gene-specific probe. Lambda (λ) DNA digested with EcoRI+HindIII was used as molecular weight marker. In case of (C) and (D), the wild type Colletotrichum sp. DM06 was taken as control (lane C). (E) Transcriptional expression of the CtDGAT2b transgene in four fungal transformants as revealed by RT-PCR for 26 cycles. (F) Transcriptional expression of the CtDGAT2b in one of the fungal transformants, Td#18 (T) as revealed by RT-PCR for 26 cycles, showing upregulation of CtDGAT2b transcript upto 3rd day of growth and declined at 4th day onward. In case of (E) and (F), the wild type Colletotrichum sp. DM06 was taken as control (C), and the relative transcript level was measured based upon the densitometric scanning of the RT-PCR amplicons (550 bp for CtDGAT2b and 400 bp for endogenous β-actin as internal control) as shown in lower panel.
Mentions: The pCAM-GpdA-GusA-TrpC genetic construct was prepared using the backbone of pCAMBIA 1300 binary vector. Initially the pNOM102 vector carrying the intron-containing gusA (uidA) gene placed under the regulation of gpdA promoter of Aspergillus nidulans and trpC terminator of A. nidulans in pUC18 vector (kindly provided by Prof. Francisco JL Aragão, Embrapa Recursos Genéticos e Biotecnologia, Brazil) was doubly digested with EcoRI and HindIII to obtain the DNA fragment GpdA-GusA-TrpC, which was subcloned into the pCAMBIA 1300 using the same set of restriction enzymes. The resulting recombinant plasmid was named as pCAM-GpdA-GusA-TrpC (Figure 1A). Cloning, sequencing and characterization of the CtDGAT2b gene (GenBank accession number KJ437598) from Candida tropicalis SY005 have already been described previously [30]. The CtDGAT2b gene was obtained after digesting with BamHI from pYES2/CtDGAT2b recombinant plasmid, which was used for the study of transgene expression in Saccharomyces cerevisiae[30]. The promoter GpdA was PCR amplified from pCAM-GpdA-GusA-TrpC recombinant plasmid (using Expand High Fidelity PCR mix, Roche) with a set of specific primers- GpdAFp and GpdARp (Table S1) to incorporate the restriction sites- EcoRI at 5' end and BamHI at 3' end of the promoter. The pCAM-GpdA-GusA-TrpC construct was digested with EcoRI and BamHI to remove the GpdA promoter and gusA gene. Thereafter, a tripartaite ligation was carried out using the BamHI digested CtDGAT2b gene, EcoRI and BamHI digested PCR amplified GpdA promoter and the above-mentioned EcoRI and BamHI digested pCAM-GpdA-GusA-TrpC to generate the pCAM-GpdA-CtDGAT2b-TrpC recombinant plasmid (Figure 2A).

Bottom Line: Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress.Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively.Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.

View Article: PubMed Central - PubMed

Affiliation: Adv. Lab. for Plant Genetic Engineering, Advanced Technology Development Center, Indian Institute of Technology Kharagpur, Kharagpur, India.

ABSTRACT
Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.

Show MeSH
Related in: MedlinePlus