We previously discovered that Pseudomonas chlororaphis NRRL B-30761 produces monorhamnolipids (R1Ls) with predominantly 3-hydroxydodecenoyl-3-hydroxydecanoate (C12:1-C10) or 3-hydroxydodecanoyl-3-hydroxydecanoate (C12-C10) as the lipid moiety under static growth conditions only. We have now cloned, sequenced, and analyzed in silico the gene locus of NRRL B-30761 containing the putative coding sequences of rhamnosyltransferase chain A (rhlA Pch , 894 bps), rhamnosyltransferase chain B (rhlB Pch , 1272 bps), and N-acyl-homoserine lactone-dependent transcriptional regulatory protein (rhlR Pch , 726 bps). The putative gene products RhlAPch (297 amino acid residues or a.a.), RhlBPch (423 a.a.), and RhlRPch (241 a.a.) only have between 60 and 65% a.a. identities to their respective closest matched homologs in P. aeruginosa. Polymerase chain reaction (PCR)-based assay did not detect the presence of rhamnosyltransferase C gene (rhlC) in P. chlororaphis, suggesting a genetic basis for the lack of dirhamnose-lipid (R2L) synthesis in this organism. We thus genetically constructed an R2L-synthesizing P. chlororaphis by expressing a rhamnosyltransferase C (rhlC) gene of P. aeruginosa using an expression vector (pBS29-P2-gfp) containing a Pseudomonas syringae promoter. The R2L/R1L ratio is 2.4 in the rhamnolipid (RL) sample isolated from the genetically engineered (GE) P. chlororaphis [pBS29-P2-rhlC], in contrast to undetectable R2L in the GE P. chlororaphis [pBS29-P2-gfp] control cells based on LC-MS analysis. The critical micelle concentrations of the R2L and R1L samples from GE P. chlororaphis [pBS29-P2-rhlC] and the control [pBS29-P2-gfp] cells were ca. 0.1 mM, and their minimum surface tensions were ca. 26 mN/m with no significant difference.

Sophorolipid (SL) purified from fermentation broth of Candida bombicola grown on oleic acid and glucose substrates was embedded at 0%, 9%, 17%, and 29% (%-total weight of final product) in solvent-cast films of poly(L-lactic acid) (PLLA), poly(ε-caprolactone), and poly(hydroxybutyrate) (PHB). Growth-inhibition activity of the SL-biopolymers against Propionibacterium acnes, a causative agent of acne vulgaris skin condition, is dependent on the SL contents of the films; the degree of inhibition as determined from the width of the zone of inhibition in agar-plate assays follows the order of SL-PCL>SL-PLLA>SL-PHB. The release of SL from the films into aqueous medium after a 4-d shaking at 25°C showed that SL-PLLA (30.1±1.7wt% SL released) most readily released the embedded SL, followed by SL-PHB (11.4±4.3wt%) and SL-PCL (4.3±1.4wt%). Thermal properties as determined by differential scanning calorimetry showed that SL decreases the heat of fusion (ΔH) and the melting temperature (Tm) of the biopolymers, indicating for the first time its usefulness as a plasticizer to prevent crystallization. In summary, the study shows the feasibility of controlling the release of antimicrobial SL by varying the type of biopolymer used, with the added advantage of SL functioning as a plasticizer to improve the physical properties of the film in term of lower crystallinity. Future research could benefit the agricultural sector via new developments as varied as antimicrobial food packaging and algal bloom mitigation.

This paper reports the incorporation of an alpha-methylene unit into fatty acid skeletons. Since the new olefin is conjugated with the carboxylate, it is susceptible to 1,4-(Michael) additions. We have used multifunctional thiols and amines for additions at the methylene. The resulting products possess clusters of functionality grouped at one end of a hydrophobic tail. These structural patterns will be of use in the design of new types of bio-based surfactants and polymers. One particularly promising pattern of functionality that can be obtained through oxidation and reduction chemistry is a 2-fatty 1,2,3-propanetriol, or a lipophilized glycerol moiety.

Candida bombicola is well-studied for the production of a biosurfactant, the sophorolipids. In this paper, the cloning of a glucosyltransferase gene using polymerase-chain-reaction (PCR) technique is described. Degenerative primer-pairs were first designed based on the highly conserved amino-acid sequences of several selected yeast glucosyltransferases. Using these primers, an amplified sequence (amplicon) of 700 base-pair from C. bombicola was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional target-specific PCR primers were designed for use in subsequent rounds of 3′- and 5′-extension using DNA walking technique to eventually obtain a C. bombicola genomic sequence containing an open-reading-frame putatively identified as a glucosyltransferase (gtf-1). The gene was subcloned in Saccharomyces cerevisiae for expression and functional characterization. Quantitative RT-PCR confirmed the expression of gtf-1 in the recombinant S. cerevisiae. In vitro assay with the sonicated cells of the recombinant yeast confirms the presence of glucosylation activity on sterol and hydroxy fatty acid substrates. This study reports for the first time the cloning and characterization of a broad-specificity lipid glucosylation gene from C. bombicola, and the functional activity of its gene product.

Spurred by improvements in catalysts as well as new experimental designs, lipid researchers have attained some impressive new outcomes by applying olefin metathesis to fatty acid substrates. Monomers for polyamides, environmentally-responsive coatings, and blends of biofuels are among the bio-derived materials obtained. This reaction is a powerful way to reconfigure the C 18 skeleton into its component subunits such as alkenes or carboxylates, or to add functionality via a cross-metathesis partner, thus continuing progress toward realizing the potential of plant oils as feedstocks for the biorefinery.

In this study, the stability of oil-in-water (O/W) emulsions prepared with structured lipid (SL) were evaluated in which the SL was produced through lipase-catalyzed interesterification between soybean oil and rice bran oil. After interesterification, the major TAG species in the SL were PLP (22.5 %), PLL/OOLn (21.8 %), LPL (16.1 %), and LLS/PLO (16.1 %), and the total amount of tocopherol and tocotrienol was 20.9 mg/100 g of SL. Sophorolipid was used as an emulsifier for preparing SL-based O/W emulsions, and the effect of pH (pH 5.8, 7 and 7.2) on stability was studied by analyzing the fat globule size. From the results, SL-based O/W emulsions showed similar stabilities to those prepared with Tween 20 at the neutral environment. In the oxidation study, any antioxidant addition of propyl gallate (PG), ascorbic acid 6-palmitate (AP) or quercetin hydrate (Que) distinctively prevented peroxide formation on the SL-based O/W emulsion throughout the 23 days of storage while AP was less effective to lower TBARS values than PG and Que.

Rhamnolipid (RL) and poly(hydroxyalkanoate) (PHA) are high-value bioproducts of Pseudomonas chlororaphis NRRL B-30761. 3-hydroxyacyl-ACP:CoA transacylase (PhaG) is an enzyme influencing the precursor flow to RL and PHA syntheses. We have cloned, analyzed and knocked-out the phaG gene (phaGPc30761) of P. chlororaphis to study its effect on RL and PHA syntheses. The phaGPc30761 had 99% identities to that of P. fluorescens O6. We used oligo-mediated recombineering to obtain two phaG-knockouts (phaG::Tn5(#1) and (#5)). The parental and the two knockouts produced mono-RLs (Rh1-C10-C12:1 and Rh1-C10-C12:0 congeners) at similar crude yields of 0.68–0.87g/L. Under these conditions, all three strains produced medium-chain-length PHA (PHAMCL) detectable only in lyophilized whole cells by GC/MS; the repeat-units (RUs) of the PHAMCL were predominantly β-hydroxydecanoate (C10:0; 37mol%), β-hydroxydodecanoate (C12:0; 28mol%), and β-hydroxydodecenoate (C12:1; 31mol%). We also studied PHA production under shaking conditions. All three strains produced only small amounts of PHAMCL when grown on gluconate or glucose. On gluconate, the PHAMCL contained predominantly the C10:0 (39–40mol%) and C12:0 (52–53mol%) RUs. On glucose, the predominant RUs were β-hydroxyoctanoate (C8:0; 18–25mol%), C10:0 (41–46mol%), and C12:0 (29–32mol%). On oleic acid, however, all three strains produced large amount (0.23–0.31g/L) of PHAMCL; C8:0 (35mol%), C10:0 (26mol%), C12:0 (13mol%), and β-hydroxytetradecenoate (C14:1; 20mol%) were the predominant RUs. On caprylic acid, the three strains produced PHAMCL having C8:0 (77–82mol%) as the predominant RU. PHA's with different compositions can thus be selectively produced by varying the carbon source.

Lipid TechnologyVolume 25, Issue 5 p. 97-98 ContentsFree Access Contents: Lipid Technology 5/2013 First published: 14 May 2013 https://doi.org/10.1002/lite.201390004AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume25, Issue5May 2013Pages 97-98 RelatedInformation

Methyl 17-hydroxy stearate was converted to methyl octadec-16-enoate using copper sulfate adsorbed on silica gel. This compound served as a useful substrate for the olefin metathesis reaction. As a result, several fatty acids with novel functional groups at the ω-end were prepared: a glyceryl ether attached at the 18-carbon, an aromatic fatty acid from eugenol, and a ferrocenyl fatty acid. By employing the unsaturated fatty alcohol, other groups were introduced, namely the terminal fluoride, bromide, and iodide were prepared, as was a thiol derivative. The penultimate and omega olefins reported here should serve as building blocks that allow fatty acids to make a greater contribution to a range of emerging technological areas.

Cyanophycin (CGP) is a bacterial bioproduct having a straight-chain poly(aspartic acid) as a backbone with arginine pendant groups attached to it. It has many potential industrial applications in the areas of water softening, hydrogel, metal–ion chelation, and nutriceuticals. Biotechnological production of CGP employs as producing strains the recombinant organisms that express heterologous cyanophycin synthase (cph) gene. A systematic study of fermentation parameters influencing CGP synthesis by a recombinant Escherichia coli expressing a cphA of Synechocystis sp. showed that high aeration conditions as provided by 400 rpm stirrer speed and 1.0 L/min air flow in a Sixfors vessel (450 mL culture working-volume) resulted in high yields of cell biomass and crude CGP product. Glycerol substrate was found to yield 1.8-times higher crude CGP than glucose did under similar conditions. With glycerol as substrate, we found that a simplified fermentation scheme consisting of a straight 48-h fermentation at 37 °C (without a 30–37 °C temperature-shifting induction step) yielded compatible or higher amounts of crude CGP as those obtained under various temperature-shifting conditions. By studying the effects of glycerol concentration on CGP yields and analyzing glycerol consumption patterns, we demonstrated that substrate-to-product conversion could be increased by at least 15% and that costly leftover of unused substrate could be alleviated. The results yielded valuable information for optimization of fermentative production of CGP using glycerol that could be obtained as surplus coproduct from biodiesel production.

This paper reports the synthesis of a series of novel compounds where carboxylic acids have been linked to a phenol through amidomethyl units. For instance, one, two, or three fatty acids have been selectively appended to the phenol in yields above 75%. The fatty acid used was oleic acid, which was subsequently epoxidized. Other functional groups, such as amino acids, can be incorporated in these compounds. Examples of monomers that are suitable for polymerization were also prepared: one acrylamide, one styrene derivative, and two types of AB2 diamino acids, all of which contain oleic units that are sites for epoxidation and crosslinking. Fatty acid aryl ethers were prepared using hydroxy fatty acids. These molecules are intended to serve as augmented analogues of epoxidized vegetable oil. Their amide groups should lead to intermolecular aggregation through hydrogen bonding, and the option to covalently include other functional groups may permit tuning of the macroscopic materials properties of films, coatings, or solids constructed from them.

Dendrimers consisting of glycerol and dicarboxylic acid units were synthesized using a convergent or “outside-in” strategy. The multimeric arms, or dendrons, are joined to a central core in the final step using the azide/alkyne click reaction. The combination of these approaches allows the preparation of dendrimers with variable and controlled degrees of substitution at their periphery. For example, a single protected amino acid has been situated at the periphery, with all other substituents being hydrophobic. In another example, all the peripheral substituents are protected amino acids. The identity of the dendrimers is unambiguous due to the purification and characterization of all the synthetic intermediates. Functional groups have also been selectively incorporated along the arms (dicarboxylic acids) and at the cores.

Sophorolipids (SLs) are known to possess antimicrobial properties towards many species (particularly Gram-positive, or Gram(+)) of bacteria. However, these properties can only be exerted if the SLs can be introduced to the bacterial cells in an acceptable manner. Propionibacterium acnes is the common bacterial cause of acne. It is a Gram(+) facultative anaerobe that is susceptible to the antimicrobial effects of SLs. In this study we demonstrated that different biopolymer matrices could be used to produce SL composite films that exert various antimicrobial efficiencies against P. acnes. Increasing SL concentrations in poly-3-hydroxybutyrate (PHB) and PHB-co-10%-3-hydroxyhexanoate (PHB/HHx) resulted in noticeably improved (PHB/HHx was best) antimicrobial activity based on the size of the zones of inhibition using an overlay plating technique on synthetic growth medium. However, increasing concentrations of SLs in PHB and PHB/HHx films also increased film opacity, which diminishes the appeal for use especially in visible (facial) areas. Pectin and alginate improved the transparent character of SL composite films while also acting as successful carriers of SLs to P. acnes. The lactone form of the SLs proved to exhibit the best antimicrobial action and in concert with either pectin or alginate biopolymers provided a comparatively transparent, successful means of utilizing SLs as a renewable, environmentally benign anti-acne solution.

Hydroxy fatty acids (FAs), which were isolated from glycolipids that can be prepared fermentatively from fats and oils, have been synthetically modified to contain azide and alkyne functional groups. These particular functional groups were chosen because they can participate in a copper-catalyzed reaction that combines them to form a 1,4-triazole, known as a "click" reaction, which has been widely used in a variety of fields but remains underutilized in FA chemistry. Depending on the starting hydroxy FA, these groups can be close to the carboxy unit (using 3-hydroxydecanoate) and hence the polar glycerol group, or distant from it (using 17-hydroxyoctadecanoate). These structural alternatives will impart different properties to the triacylglycerols that are subsequently prepared from the modified FA. Finally, the click reaction was used to conjugate triacylglycerols to each other and to other molecules such as a glycolipid or a protected amine.

Oligomeric fatty acid esters, known as estolides, have been studied for close to a century. It has only been in the last few years, however, that efficient methods for preparing estolides have been developed that also allow some control over their molecular structures. By varying the bio-based feedstocks and reaction conditions, the functional properties, particularly those geared toward lubrication, can be modified. More recently, synthetic advances have permitted stepwise construction of estolides with functional groups (hydrophilic or reactive) positioned at specific sites within the chain.

Using ester-forming reactions such as carbodiimide coupling and a modified Yamaguchi symmetrical anhydride method, a variety of estolides based on 17-hydroxy oleic and 17-hydroxy stearic acid have been prepared. These hydroxy fatty acids are produced in good yields from hydrolysis of sophorolipids, which are in turn derived from fermentation of fats and oils. Since the estolides are formed one unit, or ester bond, at a time, their length and sequence can be precisely controlled. The key to this control is the use of protecting groups at either the carboxylic or hydroxy end of the starting hydroxy fatty acids. Two mono-protected dimers, for example, when combined in a fragment-condensation approach, give a tetramer with no "contamination" from estolides of other lengths. This methodology opens the way to functionalized estolides, and several variants were prepared: hybrid estolides, containing non-fatty acid moieties such as amino acids; polymerizable estolides, containing a norbornene unit; and non-linear estolides that extend from a branched core such as glycerol or pentaerythritol. With the benzoyl chloride-mediated symmetrical anhydride method, yields for individual coupling steps ranged from 75 to 93%.