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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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Schematic representation of changes in lipid titer in the wild type and transformed Colletotrichum fungus after adopting different strategies.
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pone-0111253-g004: Schematic representation of changes in lipid titer in the wild type and transformed Colletotrichum fungus after adopting different strategies.

Mentions: We have found that the wild type fungus can produce about 7.8±0.6 g/l of total lipid under nutrient (either nitrogen or phosphorus) stress condition, which is ∼1.6-fold more than the lipid titer (4.8±0.1 g/l) at standard growth condition. However, after model-based optimization of culture conditions, the wild type strain produces 9.1±0.8 g/l of total lipid i.e., ∼1.9-fold higher lipid titer without imposition of any nutrient stress (Figure 4). On the contrary, while the transformants at standard growth condition yield ∼1.7-fold more total lipid compared to the wild type (4.8±0.1 g/l increases to 8.1±0.2 g/l), optimization of culture conditions further increases the lipid titer upto 14.1±0.5 g/l or ∼1.7-fold higher in transformants (Figure 4). Taken together, ∼2.9-fold greater lipid titer over the wild type has been achieved in transformed Colletotrichum fungus following model-based optimization of culture condition and without any nutrient stress (Figure 4). Therefore, it is evident from the present study that not only biochemical engineering (nutrient stress) but the metabolic engineering of fungus using suitable gene together with bioprocess optimization could be an effective combined strategy to increase the storage lipid productivity. Critical analysis of our experimental data (Table 3) reveals that the lipid content in the transformed Colletotrichum is ∼1.9-fold higher under standard growth condition, and ∼2.2-fold more after model-based optimization of culture conditions as compared to the wild type strain grown at standard condition. In a previous report, the fungal genes coding for mailc enzyme from Mucor circinelloides (malEMt) and Mortierella alpine (malEMc) were overexpressed in M. circinelloides, which resulted 2.5- and 2.4-fold higher lipid content in the transformed malEMt and malEMc strains, respectively [22]. A recent report has documented 4-fold increase in lipid content compared to wild type through overexpression of DGA1 gene (encoding for a type-1 DGAT) in Yerrowia lipolytica oleaginous yeast [43]. In the same organism, increased lipid content of 2-fold over control was observed after overexpression of acetyl-CoA carboxylase (ACC1) gene. Moreover, when both the genes (ACC1+DGA1) were overexpressed a lipid content of 4.9-fold was recorded in the same study. This ‘push-and-pull’ strategy can achieve large flux towards lipid accumulation with less feedback regulation as it maintain a balance between upstream and downstream metabolite formation pathway.


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)

Schematic representation of changes in lipid titer in the wild type and transformed Colletotrichum fungus after adopting different strategies.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111253-g004: Schematic representation of changes in lipid titer in the wild type and transformed Colletotrichum fungus after adopting different strategies.
Mentions: We have found that the wild type fungus can produce about 7.8±0.6 g/l of total lipid under nutrient (either nitrogen or phosphorus) stress condition, which is ∼1.6-fold more than the lipid titer (4.8±0.1 g/l) at standard growth condition. However, after model-based optimization of culture conditions, the wild type strain produces 9.1±0.8 g/l of total lipid i.e., ∼1.9-fold higher lipid titer without imposition of any nutrient stress (Figure 4). On the contrary, while the transformants at standard growth condition yield ∼1.7-fold more total lipid compared to the wild type (4.8±0.1 g/l increases to 8.1±0.2 g/l), optimization of culture conditions further increases the lipid titer upto 14.1±0.5 g/l or ∼1.7-fold higher in transformants (Figure 4). Taken together, ∼2.9-fold greater lipid titer over the wild type has been achieved in transformed Colletotrichum fungus following model-based optimization of culture condition and without any nutrient stress (Figure 4). Therefore, it is evident from the present study that not only biochemical engineering (nutrient stress) but the metabolic engineering of fungus using suitable gene together with bioprocess optimization could be an effective combined strategy to increase the storage lipid productivity. Critical analysis of our experimental data (Table 3) reveals that the lipid content in the transformed Colletotrichum is ∼1.9-fold higher under standard growth condition, and ∼2.2-fold more after model-based optimization of culture conditions as compared to the wild type strain grown at standard condition. In a previous report, the fungal genes coding for mailc enzyme from Mucor circinelloides (malEMt) and Mortierella alpine (malEMc) were overexpressed in M. circinelloides, which resulted 2.5- and 2.4-fold higher lipid content in the transformed malEMt and malEMc strains, respectively [22]. A recent report has documented 4-fold increase in lipid content compared to wild type through overexpression of DGA1 gene (encoding for a type-1 DGAT) in Yerrowia lipolytica oleaginous yeast [43]. In the same organism, increased lipid content of 2-fold over control was observed after overexpression of acetyl-CoA carboxylase (ACC1) gene. Moreover, when both the genes (ACC1+DGA1) were overexpressed a lipid content of 4.9-fold was recorded in the same study. This ‘push-and-pull’ strategy can achieve large flux towards lipid accumulation with less feedback regulation as it maintain a balance between upstream and downstream metabolite formation pathway.

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