The diversity and biotechnological application of marine microbes producing omega-3 fatty acids

PhD ThesisOmega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) play a role in the modulation and prevention of human diseases, in particular cardiovascular diseases. The omega-3 family is found mainly in fish, of which wild stocks are becoming limited. Therefore production of omega-3 PUFAs by marine microbes may provide an alternative source of such componds. The diversity of marine microbes was studied using 16S/18S rRNA gene sequencing of different marine biota with 1500 bacterial strains and 50 microalgae were isolated. The diversity of culturalbe microorganisms inhabiting Mid-Atlantic Ridge (MAR) non-vent sediments was examined for the first time in this area with findings of high diversily of Gram-positive strains, good production of squalene by an unusual strain Bacillus sp. MAR089 and the highest yield of EPA ever recovered from strain Shewanella sp. MAR441. North Sea sponge associated Vibrio sp. strain NSP560 produced considerable levels of EPA, whereas no PUFAs producers were found from tropic Caribbean marine sponge associated bacteria. Photobacterium sp. strain MA665, isolated from the coast of North Sea, was described for the first time of this genus and could be cultured easily under atmospheric conditions with appreciable levels of EPA -1 with up to 25 % of total fatty acids (TFA) (or 10.6 mg g in dried cell). Strain MAR441 was identified as a new species, designated as Shewanella dovemarina sp. T nov. (Type strain MAR441 ). The level of EPA production of strain MAR441 has been optimized by varying fermentation conditions, and 15-25 % EPA of TFA (or 17-30 mg -1 g in dried cell) could be achieved with 40 % improvement. In order to understand the PUFAs biosynthesis pathways and better predict the maximum EPA production, EPA gene clusters (pfaA, pfaB, pfaC, pfaD and pfaE) were cloned and sequenced from the following three species Shewanella, Vibrio and Photobacterium. Great potential was found in marine algae Phaeodactylum tricornutum strain M7 with lipid content of 10 % in dry wt biomass and 22-30 % EPA of TFA when it was cultured outdoors under local weather conditions in UK. Under anaerobic conditions, strain MAR441 contained less -2 amount of EPA and produced electricity of ~100 mW m . Enhanced electricity production using artificial consortia of estuarine bacteria grown as biofilms was -2 observed with power generation of ~200 mW m . In conclusion, bacteria taxonomic resolution based on complete cell fatty acid composition is possible and marine microbes with considerable production of EPA could be potential candidates for industrial production of PUFAs

Gradient type optimization methods for electronic structure calculations

The density functional theory (DFT) in electronic structure calculations can be formulated as either a nonlinear eigenvalue or direct minimization problem. The most widely used approach for solving the former is the so-called self-consistent field (SCF) iteration. A common observation is that the convergence of SCF is not clear theoretically while approaches with convergence guarantee for solving the latter are often not competitive to SCF numerically. In this paper, we study gradient type methods for solving the direct minimization problem by constructing new iterations along the gradient on the Stiefel manifold. Global convergence (i.e., convergence to a stationary point from any initial solution) as well as local convergence rate follows from the standard theory for optimization on manifold directly. A major computational advantage is that the computation of linear eigenvalue problems is no longer needed. The main costs of our approaches arise from the assembling of the total energy functional and its gradient and the projection onto the manifold. These tasks are cheaper than eigenvalue computation and they are often more suitable for parallelization as long as the evaluation of the total energy functional and its gradient is efficient. Numerical results show that they can outperform SCF consistently on many practically large systems.Comment: 24 pages, 11 figures, 59 references, and 1 acknowledgement

Dramatic Improvement of Crystals of Large RNAs by Cation Replacement and Dehydration

SummaryCompared to globular proteins, RNAs with complex 3D folds are characterized by poorly differentiated molecular surfaces dominated by backbone phosphates, sparse tertiary contacts stabilizing global architecture, and conformational flexibility. The resulting generally poor order of crystals of large RNAs and their complexes frequently hampers crystallographic structure determination. We describe and rationalize a postcrystallization treatment strategy that exploits the importance of solvation and counterions for RNA folding. Replacement of Li+ and Mg2+ needed for growth of crystals of a tRNA-riboswitch-protein complex with Sr2+, coupled with dehydration, dramatically improved the resolution limit (8.5–3.2 Å) and data quality, enabling structure determination. The soft Sr2+ ion forms numerous stabilizing intermolecular contacts. Comparison of pre- and posttreatment structures reveals how RNA assemblies redistribute as quasi-rigid bodies to yield improved crystal packing. Cation exchange complements previously reported postcrystallization dehydration of protein crystals and represents a potentially general strategy for improving crystals of large RNAs