Abstract
BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesisResultsNeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years.ConclusionsOur results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.
Highlights
Microalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels
All four canonical motifs attributed to Lysophosphatidic acid acyltransferase (LPAAT) proteins [23]: motif I, NH(X4)D; motif II, GVIFIDR; motif III, EGTR and motif IV, IVPIVM were observed in NeoLPAAT1
N-terminus of NeoLPAAT1, as predicted by PredAlgo [25], contained a putative chloroplast transit peptide ( ~ 69 amino acids, highlighted with red in Fig. 1a), suggesting that NeoLPAAT1 could be targeted to the chloroplast of N. oleoabundans
Summary
Microalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Products derived from microalgal TAG are not yet economically feasible; increas‐ ing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engi‐ neered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesis. Microalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural. Increasing TAG content in microalgae could be achieved by targeted genetic engineering of genes in TAG biosynthesis pathway [4, 6, 7]. No LPAAT1 overexpression has been reported so far in oleaginous microalga Neochloris oleoabundans
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