Identification and functional characterization of two Δ12-fatty acid desaturases associated with essential linoleic acid biosynthesis in Physcomitrella patens.

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.

Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization.

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ(5)- and Δ(6)-elongases of Marchantia polymorpha as well as the Δ(6)-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ(9)-, Δ(6)-C18, and Δ(5)-C20 LC-PUFAs, whereas only Δ(9)- and Δ(6)-C18 LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C20 fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C20 substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.

Production of biomass and extracellular 5-aminolevulinic acid by Rhodopseudomonas palustris KG31 under light and dark conditions using volatile fatty acid.

Kinetic parameters for growth and extracellular 5-aminolevulinic acid (ALA) production of Rhodopseudomonas palustris KG31 under light and dark conditions in a medium containing volatile fatty acids (VFA) as the carbon sources were estimated using a Gompertz model. The lag phase for growth and the maximum specific growth rate under microaerobic-light cultivations were 7.29-12.49 h and 0.038-0.094 h(-1), respectively, whereas under aerobic-dark cultivations, they were 2.03-14.25 h and 0.016-0.022 h(-1), respectively. The lag phase for extracellular ALA production and the maximum specific extracellular ALA production rate under microaerobic-light cultivations (15.72-24.74 h and 0.222-0.299 h(-1), respectively) were better than those obtained under aerobic-dark cultivations (24.57-44.84 h and 0.103-0.215 h(-1), respectively). The biomass and the extracellular ALA yields of 39.66-56.25 gDCW/l/mol C, and 148.47-245.75 µM/mol C, respectively, under microaerobic-light cultivations were higher than of those obtained under aerobic-dark conditions. An enhancement of extracellular ALA production under aerobic-dark conditions revealed that the ALA yield was markedly increased 8-fold (48.36 µM) by the addition of 10mM succinate, 4.5mM glycine, and 15 mM levulinic acid (LA). By controlling dissolved oxygen (DO) and pH values, a maximum extracellular ALA yield of 66.38 µM was found. The degradation rate of ALA in the culture broth was closely related to the pH value.

High level production of adrenic acid in Physcomitrella patens using the algae Pavlova sp. Delta(5)-elongase gene.

Adrenic acid (ADA), an omega-6 polyunsaturated fatty acid (PUFA), has attracted much interest due to its pharmaceutical potential. Exploiting the wealth of information currently available on in planta oil biosynthesis, and coupling this information with the tool of genetic engineering, it is now feasible to deliberately alter fatty acid biosynthetic pathways to generate unique oils in commodity crops. In this study, a Delta(5)-elongase gene from the algae Pavlova sp. related to the biosynthesis of C(22) PUFAs was targeted to enable production of ADA in the moss Physcomitrella patens. Heterologous expression of this gene was under the control of a tandemly duplicate 35S promoter. It was established that ADA (0.42mg/l) was synthesized in P. patens from endogenous arachidonic acid (ARA) via the expressed Pavlova sp. Delta(5)-elongase in the moss. In an attempt to maximize ADA production, medium optimization was effected by the response surface methodology (RSM), resulting in a significant elevation of ADA (4.51mg/l) production under optimum conditions. To the best of our knowledge, this is the first study describing the expression of a PUFA synthesizing enzyme in non-seed lower plant without supplying the exogenous fatty acid.