Sugar transport for enhanced xylose utilization in Ashbya gossypii.

The co-utilization of mixed (pentose/hexose) sugars constitutes a challenge for microbial fermentations. The fungus Ashbya gossypii, which is currently exploited for the industrial production of riboflavin, has been presented as an efficient biocatalyst for the production of biolipids using xylose-rich substrates. However, the utilization of xylose in A. gossypii is hindered by hexose sugars. Three A. gossypii homologs (AFL204C, AFL205C and AFL207C) of the yeast HXT genes that code for hexose transporters have been identified and characterized by gene-targeting approaches. Significant differences in the expression profile of the HXT homologs were found in response to different concentrations of sugars. More importantly, an amino acid replacement (N355V) in AFL205Cp, introduced by CRISPR/Cas9-mediated genomic edition, notably enhanced the utilization of xylose in the presence of glucose. Hence, the introduction of the afl205c-N355V allele in engineered strains of A. gossypii will further benefit the utilization of mixed sugars in this fungus.

Microbial lipids from industrial wastes using xylose-utilizing Ashbya gossypii strains.

This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main Δ9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.

Importance of hydrophobic interactions in the single-chained cationic surfactant-DNA complexation.

The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency.

Host-guest interactions between cyclodextrins and surfactants with functional groups at the end of the hydrophobic tail.

The aim of this work was to investigate the influence of the incorporation of substituents at the end of the hydrophobic tail on the binding of cationic surfactants to α-, ß-, and γ-cyclodextrins. The equilibrium binding constants of the 1:1 inclusion complexes formed follow the trend K1(α-CD)>K1(ß-CD)≫K1(γ-CD), which can be explained by considering the influence of the CD cavity volume on the host-guest interactions. From the comparison of the K1 values obtained for dodecyltriethylammonium bromide, DTEAB, to those estimated for the surfactants with the substituents, it was found that the incorporation of a phenoxy group at the end of the hydrocarbon tail does not affect K1, and the inclusion of a naphthoxy group has some influence on the association process, slightly diminishing K1. This makes evident the importance of the contribution of hydrophobic interactions to the binding, the length of the hydrophobic chain being the key factor determining K1. However, the presence of the aromatic rings does influence the location of the host and the guest in the inclusion complexes. The observed NOE interactions between the aromatic protons and the CD protons indicate that the aromatic rings are partially inserted within the host cavity, with the cyclodextrin remaining close to the aromatic rings, which could be partially intercalated in the host cavity. To the authors' knowledge this is the first study on the association of cyclodextrins with monomeric surfactants incorporating substituents at the end of the hydrophobic tail.

Binding of 12-s-12 dimeric surfactants to calf thymus DNA: Evaluation of the spacer length influence.

Several cationic dimeric surfactants have shown high affinity towards DNA. Bis-quaternary ammonium salts (m-s-m) have been the most common type of dimeric surfactants investigated and it is generally admitted that those that posses a short spacer (s≤3) show better efficiency to bind or compact DNA. However, experimental results in this work show that 12-s-12 surfactants with long spacers make the surfactant/ctDNA complexation more favorable than those with short spacers. A larger contribution of the hydrophobic interactions, which control the binding Gibbs energy, as well as a higher average charge of the surfactant molecules bound to the nucleic acid, which favors the electrostatic attractions, could explain the experimental observations. Dimeric surfactants with intermediate spacer length seem to be the less efficient for DNA binding.

Reversibility of the interactions between a novel surfactant derived from lysine and biomolecules.

In this work the novel cationic surfactant derived from lysine (S)-5-acetamido-6-(dodecylamino)-N,N,N-trimethyl-6-oxohexan-1-ammonium chloride, LYCl, was prepared and the physicochemical characterization of its aqueous solutions was carried out. The binding of LYCl to bovine serum albumin, BSA, and to double stranded calf thymus DNA, ctDNA, was investigated using several techniques. Results show that LYCl binding to BSA is followed by a decrease in the α-helix content caused by the unfolding of the protein. LYCl association to ctDNA mainly occurs through groove binding and electrostatic interactions. These interactions cause morphological changes in the polynucleotide from an elongated coil structure to a more compact globular structure, resulting in the compaction of ctDNA. Addition of ß-cyclodextrin, ß-CD, to the BSA-LYCl and ctDNA-LYCl complexes is followed by the refolding of BSA and the decompaction of ctDNA. This can be explained by the ability of ß-CD to hinder BSA-LYCl and ctDNA-LYCl interactions due to the stronger and more specific ß-CD-LYCl hydrophobic interactions. The stoichiometry of the ß-CD:LYCl inclusion complex and its formation equilibrium constant were determined in this work. The reported procedure using ß-CD is an efficient way to refold proteins and to decompact DNA, after the morphological changes caused in the biomolecules by their interaction with cationic surfactants.

Self-aggregation of cationic dimeric surfactants in water-ionic liquid binary mixtures.

The micellization of four dimeric cationic surfactants ("gemini surfactants") derived from N-dodecyl-N,N,N-trimethylammonium chloride was studied in pure water and in water-ionic liquid (IL) solutions by a wide range of techniques. The dimeric surfactants are distinguished by their rigid spacer groups separating the two surfactant motifs, which range from C3 to C5 in length. In order to minimize organic ion pairing effects as well as the role of the ionic liquids as potential co-surfactants, ILs with inorganic hydrophilic anions and organic cations of limited hydrophobicity were chosen, namely ethyl, butyl, and hexyl-3-imidazolium chlorides. (1)H NMR two-dimensional, 2D, rotating frame nuclear Overhauser effect spectroscopy measurements, ROESY, supported this premise. The spacer nature hardly affects the micellization process, neither in water nor in water-IL solutions. However, it does influence the tendency of the dimeric surfactants to form elongated micelles when surfactant concentration increases. In order to have a better understanding of the ternary water-IL surfactant systems, the micellization of the surfactants was also studied in aqueous NaCl solutions, in water-ethylene glycol and in water-formamide binary mixtures. The combined results show that the ionic liquids play a double role in the mixed systems, operating simultaneously as background electrolytes and as polar organic solvents. The IL role as organic co-solvent becomes more dominant when its concentration increases, and when the IL alkyl chain length augments.

Colloidal and biological properties of cationic single-chain and dimeric surfactants.

The colloidal and biological properties of the two single-chain surfactants N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium bromide (PH12) and N-cyclohexylmethyl-N,N-dimethyl-N-(1-dodecyl)ammonium bromide (CH12) and their two dimeric counterparts N,N'-(1,3-phenylenebis(methylene))bis(N,N-dimethyl-N-(1-dodecyl)ammonium dibromide (12PH12) and N,N'-(cyclohexane-1,3-diylbis(methylene))bis(N,N-dimethyl-N-(1-dodecyl)ammonium dibromide (12CH12) were investigated. The thermodynamic functions of the self-aggregation process were estimated by using calorimetric measurements and the micellization enthalpy values, ΔHM, were examined considering the different enthalpic contributions to ΔHM. In the investigation of the structure-property relationship, it was found that the surfactant structure does not influence practically the foamability of the surfactants, but it plays a key role in their solubilization capacity, antimicrobial activity and biodegradability.

Binding of cationic single-chain and dimeric surfactants to bovine serum albumin.

The interactions between bovine serum albumin (BSA) and the single-chain surfactants N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium bromide (PH12) and N-cyclohexylmethyl-N,N-dimethyl-N-(1-dodecyl)ammonium bromide (CH12) and their two dimeric counterparts, N,N'-[1,3-phenylenebis(methylene)]bis[N,N-dimethyl-N-(1-dodecyl)]ammonium dibromide (12PH12) and N,N'-[cyclohexane-1,3-diylbis(methylene)]bis[N,N-dimethyl-N-(1-dodecyl)]ammonium dibromide (12CH12), respectively, have been investigated by surface tension, fluorescence, circular dichroism, ζ potential, and atomic force microscopy. The results obtained permit the examination of the way an increase in the number of hydrophobic chains and the substitution of a cyclohexyl ring by a phenyl ring, either in the headgroup of single-chain surfactants or in the spacer of dimeric surfactants, affect BSA-surfactant interactions. The comparison of the fluorescence results with those obtained by ζ potential measurements shows that the sites of binding of the surfactants with aromatic rings to the BSA are somewhat different from those of the surfactants with no aromatic rings.

Binary mixtures with novel monomeric and dimeric surfactants: influence of the head group nature and number of hydrophobic chains on non-ideality.

The micellization and micellar growth in the mixtures of N,N-dimethyl, N-phenyl,N-dodecylammonium bromide, PH12, N,N-dimethyl,N-ciclohexylmethyl,N-dodecylammonium bromide, CH12, and their two dimeric counterparts m-dimethylphenyl-α-ω-bis(dodecyldimethylammonium) bromide, 12PH12, and m-dimethylciclohexyl-α-ω-bis(dodecyldimethylammonium) bromide, 12CH12, with dodecyltrimethylammoniumbromide, DTAB, and with N-decanoyl N-methylglucamide, MEGA10, were investigated at 303 K. Circular dichroism, CD, experiments showed the formation of mixed micelles. Two-dimensional, 2D, rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments indicated that the arrangement of the rings in the pure and mixed micelles is similar, with the rings bent into the micelle interior avoiding contact with water. Application of different theoretical approaches shows that PH12 and CH12 mixtures with DTAB and with MEGA10 behave almost ideally. The binary systems of 12PH12 and 12CH12 with DTAB and with MEGA10 show a non-ideal behavior. An increment in the solution mole fraction of MEGA10 and DTAB diminishes the tendency of the micellar aggregates to grow.

Physicochemical characterization of bromide mono- and dimeric surfactants with phenyl and cyclohexyl rings in the head group.

In this work the two monomeric surfactants N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium bromide (PH12,Br(-)) and N-cyclohexylmethyl-N,N-dimethyl-N-(1-dodecyl)ammonium bromide (CH12,Br(-)) and their two dimeric counterparts N,N'-(1,3-phenylenebis(methylene))bis(N,N-dimethyl-N-(1-dodecyl)ammonium dibromide (12PH12,2Br(-)) and N,N'-(cyclohexane-1,3-diylbis(methylene))bis(N,N-dimethyl-N-(1-dodecyl)ammonium dibromide (12CH12,2Br(-)) were prepared and characterized. The critical micelle concentration, the micellar ionization degree and the average aggregation number were obtained by using different techniques. The results are discussed with the help of (1)H NMR two-dimensional, 2D, rotating frame nuclear Overhauser effect spectroscopy measurements, ROESY, which seem to indicate that the phenyl and cyclohexyl rings present in the head groups of the surfactants are bent towards the micellar interior in order to avoid contact with water. The surface activity of the surfactants was studied by means of surface tension measurements. The occurrence of morphological transitions upon increasing surfactant concentration and the variations in the polarity and in the microviscosity of the interfacial region accompanying this sphere-to-rod transition were investigated. It was shown that the formation of spherocylindrical micelles also causes changes in the (1)H NMR spectra of the surfactant solutions.

Concentration and medium micellar kinetic effects caused by morphological transitions.

The reaction methyl naphthalene-2-sulfonate + Br(-) was investigated in several alkanediyl-α-ω-bis(dodecyldimethylammonium) bromide, 12-s-12,2Br(-) (with s = 2, 3, 4, 5, 6, 8, 10, 12), micellar solutions in the absence and in the presence of various additives. The additives were 1,2-propylene glycol, which remains in the bulk phase, N-decyl N-methylglucamide, MEGA10, which forms mixed micelles with the dimeric surfactants, and 1-butanol, which distributes between the aqueous and micellar phases. Information about the micellar reaction media was obtained by using conductivity and fluorescence measurements. In all cases, with the exception of water-1,2-prop 12-5-12,2Br(-) micellar solutions, with 30% weight percentage of the organic solvent, a sphere-to-rod transition takes place upon increasing surfactant concentration. In order to quantitatively explain the experimental data within the whole surfactant concentration range, a kinetic equation based on the pseudophase kinetic model was considered, together with the decrease in the micellar ionization degree accompanying micellar growth. However, theoretical predictions did not agree with the experimental kinetic data for surfactant concentrations above the morphological transition. An empirical kinetic equation was proposed in order to explain the data. It contains a parameter b which is assumed to account for the medium micellar kinetic effects caused by the morphological transition. The use of this empirical equation permits the quantitative rationalization of the kinetic micellar effects in the whole surfactant concentration range.

Study of the micellization and micellar growth in pure alkanediyl-alpha-omega-bis(dodecyldimethylammonium) bromide and MEGA10 surfactant solutions and their mixtures. Influence of the spacer on the enthalpy change accompanying sphere-to-rod transitions.

The micellization and micellar growth in pure aqueous alkanediyl-alpha-omega-bis(dodecyldimethylammonium) bromide, 12-s-12,2Br(-) (with s = 2,5,6,8,10,12), and N-decanoyl-N-methylglucamide MEGA10 solutions and their mixtures are investigated at 303 K. Application of different theoretical approaches to the binary mixtures shows a nonideal behavior. It also shows that the spacer length does not play an important role in the attractive interactions shown by the mixed systems. The sphere-to-rod morphological transition in the pure dimeric micellar solutions is studied at 303 K. From comparison of these results with those at 298 K the key role played by the spacer in the micellar growth is shown. The spacer length controls not only the surfactant concentration at which the morphological transition happens but also the sign of the enthalpy change accompanying the sphere-to-rod equilibrium. Spacers with an even number of methylenes show smaller C* values than those with an odd number of -CH(2)- units. An endothermic enthalpy change is found for even spacers whereas an exothermic enthalpy change is found for odd spacers. To the authors knowledge, this is the first time this experimental trend has been shown. Addition of MEGA10 diminishes the tendency of the aggregates to grow. An increment in the solution mole fraction of MEGA10 makes the formation of elongated micelles difficult. Microviscosity measurements provide additional information about the influence of the MEGA10 content on the sphere-to-rod transition.

Micellization and micellar growth of alkanediyl-alpha,omega-bis(dimethyldodecylammonium bromide) surfactants in the presence of medium-chain linear alcohols.

Micellization and micellar growth of cationic dimeric surfactants of the alkanedyil-alpha,omega-bis(dimethyldodecylammonium) bromide type, 12-s-12,2Br(-) (s=3, 4, 6), in the presence of various amounts of 1-butanol, 1-pentanol, and 1-hexanol have been investigated. The influence of the nature and concentration of alcohol on the cmc, on the micellar ionization degree, on the average micellar aggregation number, and on the polarity of the micellar interfacial region was investigated by using conductivity and fluorescence measurements. Subsequently, effects of alcohol addition on the surfactant concentration range where sphere to rod transitions occur were examined and information about changes in the micropolarity and in the microviscosity accompanying the morphological transition was obtained. The experimental results were explained by considering the variations in the different contributions to the Gibbs energy of micellization caused by the presence of alcohols. The study of the reaction methyl naphthalene-2-sulfonate+Br(-) in some water-alcohol 12-6-12,2Br(-) micellar solutions provided information about the characteristics of the dimeric micelles as microreactors and show the complexity of the microheterogeneous systems studied.