Two Δ(12)-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from Physcomitrella patens and their functions identified. The open reading frames (ORFs) of PpFAD2-1 and PpFAD2-2 consisted of 1,128 bp and code for 375 amino acids. Their deduced polypeptides showed 62-64 % identity to microsomal Δ(12)-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. Yeast cells transformed with plasmid constructs containing PpFAD2-1 or PpFAD2-2 produced an appreciable amount of hexadecadienoic (16:2 Δ(9,12)) and linoleic acids (18:2 Δ(9,12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ(12)-desaturase enzymes. In addition, reduction of the growth temperature from 30 to 15 °C resulted in increased accumulation of unsaturated fatty acid products.

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Identification of the parameters that had significant effects on polyunsaturated fatty acids (PUFAs) and biomass production by the moss Physcomitrella patens was performed using nine culture variables (temperature, agitation speed, pH, sucrose, di-ammonium tartrate, CaCl 2 Á2H 2 O, MgSO 4 Á7H 2 O, KH 2 PO 4 and KNO 3 ) with the statistical design technique of Plackett-Burman. Statistical analysis revealed that two physical variables (pH and temperature) had significant effects on the production of both biomass and PUFAs (linoleic acid, LA; c-linolenic acid, GLA; a-linolenic acid, ALA; eicosadienoic acid, EDA; di-homo-c-linolenic acid, DHGLA; arachidonic acid, ARA; eicosapentaenoic acid, EPA). Three nutritional variables (sucrose, CaCl 2 and MgSO 4 ) had an influence only on the production of some of the PUFAs. Of the two levels used in this study, higher concentrations of sucrose had a positive effect on LA, ARA and EPA production, whereas higher concentrations of metal ions (CaCl 2 and MgSO 4 ) had a negative effect only on ARA and EPA production. After adjustment by multiple linear regression, it can be concluded that pH, temperature, sucrose, CaCl 2 and MgSO 4 were the most statistically significant parameters for the growth of P. patens and for PUFA production by this moss.

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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.