Metabolic Engineering and Oil Supplementation of Physcomitrella patens for Activation of C₂₂ Polyunsaturated Fatty Acid Production

Abstract

Long chain (C C 20) polyunsaturated fatty acids (LC-PUFAs) represent important components of the human diet. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but high production costs and diminishing feedstock, limit their supply and usage. A more regular sustainable source of these compounds is urgently required and therefore research is being conducted to develop a sustainable, land-based production system. This work describes the metabolic engineering of an artificial pathway that activates the production of C 22 -PUFAs, docosatetraenoic acid or adrenic acid (ADA) and n-3 docosapentaenoic acid (DPA) in Physcomitrella patens using a gene from a marine algae Pavlova sp. encoding D 5 -elongase and vegetable oil supplementation. The accumulation of ADA and x-3 DPA were dramatically increased to 24.3 and 11.7 mg L -1 and accounted for 2.3 and 1.1% of total fatty acids, respectively. This is the first report on producing n-3 DPA, DHA precursor, in P. patens. The obtained results prove that this enzyme appears to be more active when fused to a green fluorescence protein reporter gene. These finding reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation and nutritional supplementation, to produce specific PUFAs in a non-seed lower plant, is a promising technique.

High contents of very long-chain polyunsaturated fatty acids in different moss species

“…The high content of vlPUFAs in mosses highlights their potential for biotechnological application. Especially ω-3 PUFAs such as eicosapentaenoic acid (EPA, 20:5 Δ5,8,11,14,17 ) and docosahexaenoic acid (DHA, 22:6 Δ4,7,10,13,16,19 ) are of importance for human nutrition and need to be produced in larger amounts, as limited natural sources basically comprise algae and marine fish (Chodok et al 2012 ; Xue et al 2013 ). Artificial production of EPA is already achieved with metabolic engineering of the yeast Yarrowia lipolytica (Xue et al 2013 ).…”

“…However, well-directed modifications of metabolic pathways are also possible in P. patens due to its well-annotated genome sequence (Zimmer et al 2013 ) and the high rate of homologous recombination in mitotic cells that facilitates the generation of genetically modified strains. This technique enables the production of vlPUFAs of interest via metabolic engineering (Kaewsuwan et al 2010 ; Chodok et al 2012 ). On the other hand, transgenic engineering of crops, e.g., oil seed crops using moss genes, as recently reviewed regarding genes from microalgae or fish (Jiao and Zhang 2013 ) is also a promising research area.…”

“…On the other hand, mosses themselves provide the potential for the discovery of yet uncharacterized metabolites (Cove et al 2006 ; Asakawa 2007 ; Xie and Lou 2009 ; Erxleben et al 2012 ), but also for the production of metabolites in the moss bioreactor that was established for cultivation of P. patens (Decker and Reski 2008 , 2012 ). Due to the high rate of homologous recombination, i.e., the ability to integrate homologous nucleotide sequences into the genome, metabolic engineering, but also the production of recombinant proteins, has already been realized in P. patens (Büttner-Mainik et al 2011 ; Chodok et al 2012 ; Parsons et al 2012 ). The high rate of homologous recombination in P. patens is unique among land plants at the current state of knowledge, being comparable with the gene targeting efficiency in yeast and several times higher than in vascular plants (Strepp et al 1998 ; Schaefer 2001 ; Hohe et al 2004 ; Kamisugi et al 2006 ).…”

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“…ω -6 metabolic pathway or ω-3 metabolic pathway) [53]. Therefore, proteins or enzymes encoded by many genes can improve the level of ultra long chain polyunsaturated fatty acids such as FAD3, D5 prolongase gene, ω-3 fatty acid desaturase gene, Δ 8-desaturase gene and Δ 5-desaturase gene [54]; [55]; [56]; [57]. FAD3 protein can catalyze the synthesis of α-linolenic acid in rice grains, and then it can be used to increase the content of α-linolenic acid in rice.…”

Influence of Supplementation of Vegetable Oil Blends on Omega-3 Fatty Acid Production inMortierella alpinaCFR-GV15

“…All the previous studies of this fungus were concentrated on variation of cultural parameters for enhanced production of omega-6 fatty acids [ 13 – 15 ]. Vegetable oils containing different fatty acids served as precursors for biosynthesis of long-chain fatty acids (LC-FA) and supplementation with vegetable oil at 1% level in Mortierella alpina ATCC 32222 and microalgae Physcomitrella patens has been reported to enhance biomass with PUFAs [ 16 , 17 ]. LSO and GCO have been reported to be rich sources of ALA and may serve as natural precursors for production of very long chain PUFAs.…”