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.
Highlights
Lipid is an important raw material for the production of different essential compounds related to food and non-food industrial applications
Four stable transformants were investigated for the expression of CtDGAT2b transgene, qualitative assay and quantitative estimation of total lipid as well as neutral lipid
One of the stable transformants having single copy CtDGAT2b transgene in the fungal genome was further examined for the fatty acid profile of the total lipid and subjected to modelbased bioprocess optimization for enhancement of lipid titer
Summary
Lipid is an important raw material for the production of different essential compounds related to food and non-food industrial applications. Storage lipid has been considered since many years as source of important fatty acids polyunsaturated fatty acids (PUFAs) and other nutraceuticals as dietary supplements for fishes, poultry birds, domestic animals and human [1,2]. Among non-food industrial applications, biodiesel production from lipid feedstock, from plant seed oil, has massively increased recently. Oil-rich microorganisms, microalgae have been demonstrated to be a promising alternative source of lipids for biodiesel production [3,4,5]. The important criteria for a microorganism to be acceptable for production of lipid feedstock are its total amount of lipid and the types of fatty acids inherently present in it
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