A process for the recovery of poly(hydroxyalkanoates) from Pseudomonads Part 1: Solubilization

Poly(hydroxyalkanoates) (PHAs) are bacterial storage polyesters, currently receiving much attention because of their potential as biodegradable and renewable plastics. Well known are poly(hydroxybutyrate) and its copolymers with hydroxyvalerate, which have been commercialised under the trademark Biopol. In addition to these rigid materials, the elastomeric medium-chain-length PHAs (mcl-PHAs) from fluorescent Pseudomonads are now emerging. Here we describe the development of a non-solvent based process for the recovery of mcl-PHAs from the biomass. This first paper addresses a procedure to solubilize the biomass, so that it can be separated from the particulate mcl-PHA. The solubilization procedure, involving heat, protease and detergent, leaves the peptidoglycan intact, which facilitates the separation. The purity of the resulting mcl-PHA exceeds 95%. In a subsequent paper, we utilize the solubilization procedure in a downstream process and we discuss the economics of the corresponding mcl-PHA production.     

High-pressure small-angle neutron scattering (SANS) study of 1,2-dielaidoyl-sn-glycero-3-phosphocholine bilayers

Small-angle neutron scattering of the trans-unsaturated DEPC has been investigated as a function of pressure at 12, 18.6 and 35 degrees C. A pressure-induced structural phase transition from a liquid-crystalline state to a gel state is observed at the temperatures studied. The critical pressure of this transition increases with increasing temperature with a delta P/delta T value of 51 bar/C degrees. The small-angle neutron scattering results indicate that the effect of the trans double bonds in DEPC is to enhance the conformational disorder in the hydrocarbon chains. In DEPC bilayers, a pressure-induced conformational ordering process is observed not only in the liquid-crystalline phase but also in the gel phase, which indicates that conformational disorder exists in the liquid-crystalline phase as well as in the gel phase.     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C6 to C14 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and 13C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5-87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C8 and C10 monomers as longer and smaller monomers were incorporated less efficiently.     

Synthesis and characterization of poly(3-hydroxyalkanoates) from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.     

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.     

Studies of thermoluminescence properties of liquid crystalline N-phenyl substituted phenyl polysiloxane hydroxamic acids

The investigation of thermoluminescence (TL) glow curves in liquid crystalline side chain N-phenyl-substituted phenyl polysiloxane hydroxamic acids (PHAs) has yielded significant insights. These polymers demonstrated TL behavior when exposed to β-radiation between 0 and 220°C, indicating inherent luminescent properties when irradiated. Notably, a dose-dependent relationship was observed in reported derivatized polymers; this study elucidates the diverse TL characteristics exhibited by various liquid crystalline side chain N-phenyl-substituted phenyl PHAs when exposed to β-radiation. Understanding these dose-dependent and dose-independent behaviors enhances the knowledge of their luminescent properties and potential applications in radiation detection.     

Isolation of polyhydroxyalkanoates-producing bacteria using a combination of phenotypic and genotypic approach

AIMS: To develop an efficient approach using a combination of phenotypic and genotypic methods for isolation of environmental bacteria that produce mid-chain-length polyhydroxyalkanoates (mcl-PHAs). METHODS AND RESULTS: A viable-colony staining method using Nile red was used to screen for PHA-producing bacteria followed by a polymerase chain reaction (PCR) screen using primers to amplify the partial nucleic acid sequence of the phaC1 synthase gene for confirmation. Microbes containing lipophilic storage compounds isolated from environmental samples could readily be detected by the colony staining method. They were further examined by Sudan Black staining to highlight the inclusions inside the cells. These isolates were subsequently subjected to PCR analysis. As a result, more than a hundred strains were identified as PHA-positive isolates from this screening approach. CONCLUSIONS: These results conclusively demonstrate that environmental bacterial strains able to accumulate the PHAs could readily be obtained by this screening method. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose a polyphasic approach using a combination of phenotypic and genotypic screening method to rapidly screen and identify bacteria able to produce significant amounts of mcl-PHAs from environment. This approach can be adopted as a rapid screen for micro-organisms able to accumulate PHAs to be used for potential manufacture and other industrial applications.     

Polyunsaturated hydrocarbon chains: computer study of the characteristics of intramolecular ordering of chains

The conformational properties of isolated unbranched hydrocarbon polyunsaturated molecules of cis-C18:4 and cis-C18:5 under theta-conditions (T = 298 K) were studied using Monte Carlo simulations. The conformations were generated by a computer (the continuum model was used; the energy of nonbonded interactions and torsion and electrostatic terms were taken into account). A molecule-fixed coordinate system with the axes along inertia tensor eigenvectors of each molecule conformation (principal axes of inertia) were used for the calculations. C-H and C-C bond orientation distribution functions rho and ordering parameters S with respect to the maximum molecule span axis were calculated. It was shown that the presence of five methylene-interrupted cis double bonds in C18 chain has a maximum effect on the intramolecular ordering properties of the molecule. The widths of function rho CH for pentaenes differed significantly from those of other C18-chains: the widths of function rho for all CH2-groups were nearly twice as large as that for C-H-bonds flanking the double bonds C=C, and roughly constant along the chain sequence. The mean magnitudes of magnitudes of SCH in the molecule decreased when unsaturation increased.     

Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.

The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.     

Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.     

Average structural and motional properties of a diunsaturated acyl chain in a lipid bilayer: effects of two cis-unsaturated double bonds

Isolinoleic acid (18:2 delta 6,9) deuterated at 10 different positions was esterified to form 1-palmitoyl-2-isolinoleoyl-sn-glycero-3-phosphocholine (PiLPC), and the average structural and motional properties of the diunsaturated chain, in aqueous dispersions of PiLPC, were examined by 2H NMR spectroscopy. For each sample, 2H spectra were acquired over a temperature range of 1-40 degrees C and the quadrupolar splittings interpreted in terms of carbon-deuterium bond order parameters, SCD. Furthermore, definition of the average orientation of the C8 methylene unit with respect to the bilayer normal [Baenziger, J. E., Smith, I. C. P., Hill, R. J., & Jarrell, H. C. (1988) J. Am. Chem. Soc. 110, 8229-8231] provided sufficient information to calculate both the average orientations and the molecular order parameters, Smol (which reflects the amplitudes of motion), for the C6-C7 and the C9-C10 double bonds. The results indicate that both the motional freedom (reflected in the order profile) and the average structure (reflected in the orientation of carbon segments with respect to the bilayer normal) are strongly affected by the presence of two cis-unsaturated double bonds. The data were interpreted in terms of two possible models whereby, in each case, the chain adopts a conformation consistent with the low-energy conformation of 1,4-pentadiene [Applegate, K. R., & Glomset, J. A. (1986) J. Lipid Res. 27, 658-680] but undergoes a two-site jump between the conformations. The jump motion arises mainly from rotations about the C7-C8 and the C8-C9 single bonds that disorder the C8 and the C9-C10 segments (Smol = 0.15 and 0.08, respectively) but leave the C6-C7 double bond relatively immobile (Smol = 0.55; all at 40 degrees C). It is suggested that acyl chains containing three or more double bonds could not undergo a similar jump motion and therefore would be highly ordered and not "fluid" as is generally thought.     

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

Contribution of hydrogen bonding to the conformational stability of ribonuclease T1.

For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1). When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed. Twelve mutants of RNase T1 [Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)] have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A. On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond. This estimate is in good agreement with results from several related systems. Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect. Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides.     

Autoxidation of medium chain length polyhydroxyalkanoate

Polyhydroxyalkanoates (PHAs) are a class of biopolymers that are currently the subject of intensive research for various applications (packaging, consumer products, medical applications, etc.). It is known from synthetic polymers that all plastic materials show more or less pronounced autoxidation (aging induced by UV radiation, temperature, heavy metal ions, etc.). There is less knowledge as yet regarding the autoxidation behavior of biopolymers. The autoxidative behavior of medium chain length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) was therefore investigated. mcl-PHA (co)polymers with amounts of 0, 10, 50, and 75 mol % of olefinic side chains with terminal double bonds were tempered at 60 degrees C in air for 3 months. After 1, 2, 4, 8, and 12 weeks, samples were removed and analyzed for changes in chemical and physical properties by sol-gel analysis (Soxhlet extraction), size exclusion chromatography (SEC), infrared analysis (IR), and gas chromatography/flame ionization detection (GC/FID). It became apparent that the content of double bonds greatly influences the autoxidation of mcl-PHA. A low amount of unsaturated moiety (0 and 10 mol %) resulted in chain scission, whereas samples with 50 and 75 mol % olefinic side chains showed cross-linking and became insoluble after a few weeks. Kinetic data of oxidation behavior were investigated by performing isothermal DSC experiments at elevated temperatures. The kinetic data combined with the experiment enabled the gelation time to be predicted and the shelf-life of mcl-PHA to be estimated. Because of the detected sensitivity of mcl-PHA regarding autoxidation, it is recommended that these biopolymers should be stored cold (at least -5 degrees C) and in an inert gas atmosphere or stabilized by suitable additives (antioxidants).     

Proton nuclear magnetic resonance as a probe of differences in structure between the C102T and F82S,C102T variants of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae.

Differences in chemical shifts and in nuclear Overhauser effects between the C102T and F82S,C102T variants of Saccharomyces cerevisiae iso-1-cytochrome c in both the reduced and oxidized forms are reported and analyzed. There is evidence for small conformational differences in both oxidation states of the double variant near position 82. Differences in structure are more evident in the oxidized forms of the variants. These differences extend to distant parts of the protein. It is concluded that the oxidized double variant has undergone a small rearrangement of several regions of the protein that are linked by a hydrogen-bond network. It is shown that the rearrangement involves hydrogen bonds associated with the two heme propionates and associated water molecules. The deductions from nuclear magnetic resonance data are compared with the differences in the crystal structures of the reduced forms of wild-type protein and the F82S variant [Louie, G. V., Pielak, G. J., Smith, M., & Brayer, G. D. (1988) Biochemistry 27, 7870-7876].     

Conformational characterization of ceramides by nuclear magnetic resonance spectroscopy

Ceramide (Cer) has been identified as an active lipid second messenger in the regulation of cell growth, differentiation, and apoptosis. Its analog, dihydroceramide, without the 4 to 5 trans double bond in the sphingoid backbone lacks these biological effects. To establish the conformational features that distinguish ceramide from its analogs, nuclear magnetic resonance spectral data were acquired for diluted samples of ceramides (C2- and C18-Cer), dihydroceramide (C16-DHCer), and deoxydihydroceramide (C18-DODHCer). Our results suggest that in both C2- and C18-Cer, an H-bond network is formed in which the amide proton NH is donated to the OH groups on carbons C1 and C3 of the sphingosine backbone. Two tightly bound water molecules appear to stabilize this network by participating in flip-flop interactions with the hydroxyl groups. In DHCer, the lack of the trans double bond leads to a conformational distortion of this H-bonding motif. Without the critical double bond, the degree with which water molecules stabilize the H bonds between the two OH groups of the sphingolipid is reduced. This structural alteration might preclude the participation of DHCer in signaling-related interactions with cellular targets.     

Formation of Polyesters by Pseudomonas oleovorans: Effect of Substrates on Formation and Composition of Poly-(R)-3-Hydroxyalkanoates and Poly-(R)-3-Hydroxyalkenoates

Pseudomonas oleovorans grows on C₆ to C₁₂n-alkanes and 1-alkenes. These substrates are oxidized to the corresponding fatty acids, which are oxidized further via the β-oxidation pathway, yielding shorter fatty acids which have lost one or more C₂ units. P. oleovorans normally utilizes β-oxidation pathway intermediates for growth, but in this paper we show that the intermediate 3-hydroxy fatty acids can also be polymerized to intracellular poly-(R)-3-hydroxyalkanoates (PHAs) when the medium contains limiting amounts of essential elements, such as nitrogen. The monomer composition of these polyesters is a reflection of the substrates used for growth of P. oleovorans. The largest monomer found in PHAs always contained as many C atoms as did the n-alkane used as a substrate. Monomers which were shorter by one or more C₂ units were also observed. Thus, for C-even substrates, only C-even monomers were found, the smallest being (R)-3-hydroxyhexanoate. For C-odd substrates, only C-odd monomers were found, with (R)-3-hydroxyheptanoate as the smallest monomer. 1-Alkenes were also incorporated into PHAs, albeit less efficiently and with lower yields than n-alkanes. These PHAs contained both saturated and unsaturated monomers, apparently because the 1-alkene substrates could be oxidized to carboxylic acids at either the saturated or the unsaturated ends. Up to 55% of the PHA monomers contained terminal double bonds when P. oleovorans was grown on 1-alkenes. The degree of unsaturation of PHAs could be modulated by varying the ratio of alkenes to alkanes in the growth medium. Since 1-alkenes were also shortened before being polymerized, as was the case for n-alkanes, copolymers which varied with respect to both monomer chain length and the percentage of terminal double bonds were formed during nitrogen-limited growth of P. oleovorans on 1-alkenes. Such polymers are expected to be useful for future chemical modifications.     

Polyunsaturated fatty acid-cholesterol interactions: Domain formation in membranes

Polyunsaturated fatty acids (PUFA) constitute an influential group of molecules that promote health by an as yet unknown mechanism. They are structurally distinguished from less unsaturated fatty acids by the presence of a repeating CH-CH₂-CH unit that produces an extremely flexible chain rapidly reorienting through conformational states. The most highly unsaturated case in point is docosahexaenoic acid (DHA) with 6 double bonds. This review will summarize how the high disorder of DHA affects the properties of the membrane phospholipids into which the PUFA incorporates, focusing upon the profound impact on the interaction with cholesterol. Results obtained with model membranes using an array of biophysical techniques will be presented. They demonstrate DHA and the sterol possesses a mutual aversion that drives the lateral segregation of DHA-containing phospholipids into highly disordered domains away from cholesterol. These domains are compositionally and organizationally the opposite of lipid rafts, the ordered domain enriched in predominantly saturated sphingolipids “glued” together by cholesterol that is believed to serve as the platform for signaling proteins. We hypothesize that DHA-rich domains also form in the plasma membrane and are responsible, in part, for the diverse range of health benefits associated with DHA.