Medium Chain Length Research Articles

Structure and chain conformation characterization of arabinoglucan from by-product of peanut oil processing

A neutral polysaccharide (NPP) from peanut sediment of aqueous extraction process was purified via anion-exchange and gel-filtration chromatography. The weight-average molecular weight and polydispersity index were 3.36 × 104 Da and 1.06. Composition of glucose (82.66 %, molar percentage) and arabinose (17.34 %) suggested an arabinoglucan structure. Multiple medium-length chains consisting of many 1,4-linked α-Glcp and a few 1,5-linked α-Araf maintained the main chain structure. The backbone was substituted at O-6 and O-3 positions, attached by side chains consisting of two to six α-Glcp and terminated with Araf and Glcp. Degree of branching was 42.50 %. Aggregates formed in NPP aqueous solution. They were eliminated by DMSO combining with sonication. Consequently, the average radius of gyration (Rg), hydrodynamic radius (Rh), and Rg/Rh ratio were 17.0 nm, 5.8 nm and 2.93, respectively, indicating extended rigid chain conformation. The backbone substituted at O-3 and short branching chains probably together induced this conformation.

Alterations in Mediterranean mussel (Mytilus galloprovincialis) composition exposed to cyanotoxins as revealed by analytical pyrolysis

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are biotoxins produced by cyanobacteria species that, due to climate change and water eutrophication, proliferate together with an increase of the associated cyanotoxins. Both toxins are usually found in the aquatic environment and filter feeding organisms such as mussels are particularly exposed which may be the cause of metabolic alterations. In this work, pyrolysis gas chromatography mass spectrometry (Py-GC/MS) is used to evaluate compositional changes in mussel (M. galloprovincialis) after exposure for 14 days to cyanotoxins of Chrysosporum ovalisporum (CYN), Microcystis aeruginosa (MC-LR) and to a combination of both toxins. The pyrolysis of mussel flesh produced complex chromatograms, with up to 100 different compounds, but very similar between treatments. Major groups found were N compounds (pyridine, N-alkyl molecules) (18.2 % ± 1.5) and peptide/protein derived compounds that include alkyl indols and diketopiperazines (DKPs) (17.7 % ± 2.4), series of medium chain length (C14-C22) saturated, mono and polyunsaturated fatty acids (45.1 % ± 5.0), steroids (7.5 % ± 0.6) and aromatics with undetermined origin (8.9 % ± 1.1). A chemometric treatment of the chromatographic data allowed the discrimination between the different mussel populations exposed to cyanotoxins. The results are discussed in terms of the probable effects exerted by the biotoxins in mussel composition and the possible metabolic pathways affected.

P.584 The actions of lumateperone and single nucleotide polymorphism (SNP) converge on the glutamate-NO-cGMP pathway

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are biotoxins produced by cyanobacteria species that, due to climate change and water eutrophication, proliferate together with an increase of the associated cyanotoxins. Both toxins are usually found in the aquatic environment and filter feeding organisms such as mussels are particularly exposed which may be the cause of metabolic alterations. In this work, pyrolysis gas chromatography mass spectrometry (Py-GC/MS) is used to evaluate compositional changes in mussel (M. galloprovincialis) after exposure for 14 days to cyanotoxins of Chrysosporum ovalisporum (CYN), Microcystis aeruginosa (MC-LR) and to a combination of both toxins. The pyrolysis of mussel flesh produced complex chromatograms, with up to 100 different compounds, but very similar between treatments. Major groups found were N compounds (pyridine, N-alkyl molecules) (18.2 % ± 1.5) and peptide/protein derived compounds that include alkyl indols and diketopiperazines (DKPs) (17.7 % ± 2.4), series of medium chain length (C14-C22) saturated, mono and polyunsaturated fatty acids (45.1 % ± 5.0), steroids (7.5 % ± 0.6) and aromatics with undetermined origin (8.9 % ± 1.1). A chemometric treatment of the chromatographic data allowed the discrimination between the different mussel populations exposed to cyanotoxins. The results are discussed in terms of the probable effects exerted by the biotoxins in mussel composition and the possible metabolic pathways affected.

Characterization and in vitro digestion properties of cassava starch and epigallocatechin-3-gallate (EGCG) blend

In this study, characteristics and in vitro digestibility of cassava starch and epigallocatechin-3-O-gallate (EGCG) blend were investigated. The structure changes of the starch during digestion were also evaluated. X-ray diffraction (XRD) and thermogravimetric analysis (TGA) revealed that starch-EGCG blend had more ordered crystal structure and stronger thermal stability as compare to the native starch. Iodine binding analysis suggested the incorporation of starch and EGCG. Starch-EGCG blend retained strong ability of DPPH radical scavenging after storage, indicating that starch could protect EGCG from oxidation. EGCG greatly inhibited the enzymatic hydrolysis of starch during digestion, and the starch could keep EGCG controlled-released. The addition of EGCG decreased the rate of granule collapse and molecular weight reduction of the starch during digestion. The hydrolysis of long and medium length chains of the starch was restrained greatly, and the short chains were protected from hydrolyzing by blending with EGCG during digestion.

Influence of inulin and oligofructose on the sensory properties and antioxidant activity of apple jelly

The objective of this study was to analyse the influence of inulin with different degrees of polymerization (DP values) and oligofructose preparation on the sensory properties and antioxidant activity of apple jelly. It has been determined that both the addition of inulin (independently of DP) as well as oligofructose significantly modifies colour and sensory properties and influence antioxidant activity of apple jelly. It has been observed that the manner in which water is bound by the applied preparations may have a significant impact on the analysed properties of jelly. In terms of taste, the highest scores were awarded to the desserts with addition of long-chain inulin and in terms of the overall sensory evaluation those with addition of preparation with medium length chain. The variability of the sensory properties depended on the type and level of the additive, and this impact varied between individual preparation types. The conducted study has enabled the conclusion that inulin may pose an attractive ingredient of desserts with health-promoting properties.

Efficient Exciton Diffusion in Micrometer-Sized Domains of Nanographene-Based Nonfullerene Acceptors with Long Exciton Lifetimes in Blend Films with Conjugated Polymer.

Phase-separated structures in photoactive layers composed of electron donors and acceptors in organic photovoltaics (OPVs) generally exert a profound impact on the device performance. In this study, nonfullerene acceptors (NFAs) where a heteronanographene central core was furnished with branched alkoxy chains of different lengths, TACIC-EH, TACIC-BO, and TACIC-HD, were prepared to adjust the aggregation tendency and systematically probe the relationships of film structures with photophysical and photovoltaic properties. The side-chain length showed negligible effects on the absorption properties and energy levels of TACICs. In addition, regardless of the chain length, all TACIC films exhibited characteristically long singlet exciton lifetimes (1330-2330 ps) compared to those in solution (≤220 ps). Using a conjugated polymer donor, PBDB-T, the best OPV performance was achieved with TACIC-BO that contained medium-length chains, exhibiting a power conversion efficiency (PCE) of 9.92%. TACIC-HD with the longest chains showed deteriorated electron mobility due to the long insulating alkoxy groups. Therefore, the PBDB-T:TACIC-HD-based device revealed a low charge collection efficiency and PCE (8.21%) relative to the PBDB-T:TACIC-BO-based device, but their film morphologies were analogous. Meanwhile, TACIC-EH with the shortest chains showed low solubility and formed micrometer-sized large aggregates in the blend film with PBDB-T. Although the charge collection efficiency of PBDB-T:TACIC-EH was lower than that of PBDB-T:TACIC-BO, the efficiencies of exciton diffusion to the donor-acceptor interface were sufficiently high (>98%) owing to the elongated singlet exciton lifetime of TACIC-EH. The PCE of the PBDB-T:TACIC-EH-based device remained moderate (7.10%). Therefore, TACICs with the long singlet exciton lifetimes in the films provide a clear guideline for NFAs with low sensitivity of OPV device performance to the blend film structures, which is advantageous for large-scale OPV production with high reproducibility.

Recovery of the PHA Copolymer P(HB-co-HHx) With Non-halogenated Solvents: Influences on Molecular Weight and HHx-Content.

Biodegradable and biocompatible polyhydroxyalkanoates (PHAs) are promising alternatives to conventional plastics. Based on the chain length of their monomers they are classified as short chain length (scl-) or medium chain length (mcl-) PHA polymers. The type of monomers, the composition and the molecular weight (MW) define the polymer properties. To accelerate the use of PHA as a bulk material, the downstream associated costs need to be minimized. This study focuses on the evaluation of non-halogenated solvents, especially acetone as a scl-PHA non-solvent, for the recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) – P(HB-co-HHx) – with an mcl-HHx content >15 mol% and a MW average (Mw) < 2 × 105 Da. Solvents and precipitants were chosen regarding zeotrope formation, boiling point differences, and toxicity. Non-halogenated solvent-precipitant pairs were evaluated regarding the MW characteristics (MWCs) of the extracted polymer. Acetone and 2-propanol as a low toxic and zeotropic solvent-precipitant pair was evaluated at different extraction temperatures and multiple extraction times. The extraction process was further evaluated by using impure acetone for the extraction and implementing a multi-stage extraction process. Additionally, P(HB-co-HHx) extracted with three different solvents was characterized by 1H and 13C-APT NMR. The screening of precipitants resulted in a negative influence on the MWCs by ethanol precipitation for extractions with acetone and ethyl acetate, respectively. It was observed, that extractions with acetone at 70°C extracted a higher fraction of PHA from the cells compared to extractions at RT, but the Mw was decreased by 9% in average. Acetone with a 2-propanol fraction of up to 30% was still able to extract the polymer 95% as efficient as pure acetone. Additionally, when acetone and ethyl acetate were used in a multi-stage extraction process, a two-stage process was sufficient to extract 98–99% of the polymer from the cells. 1H and 13C-APT NMR analysis confirmed the monomer fraction and structure of the extracted polymers and revealed a random copolymer structure. The presented strategy can be further developed to an ecological and economically feasible PHA downstream process and thus contributes to the commercialization of low-cost PHAs.

Engineering of β-mannanase from Aspergillus niger to increase product selectivity towards medium chain length mannooligosaccharides

Mannooligosaccharides (MOSs) are one of the most commonly used biomass-derived feed additives. The effectiveness of MOS varies with the length of oligosaccharides, medium length MOSs such as mannotetraose and mannopentaose being the most efficient. This study aims at improving specificity of β-mannanase from Aspergillus niger toward the desirable product size through rational-based enzyme engineering. Tyr 42 and Tyr 132 were mutated to Gly to extend the substrate binding site, allowing higher molecular weight MOS to non-catalytically bind to the enzyme. Hydrolysis product content was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. Instead of mannobiose, the enzyme variants yielded mannotriose and mannotetraose as the major products, followed by mannobiose and mannopentaose. Overall, 42% improvement in production yield of highly active mannotetraose and mannopentaose was achieved. This validates the use of engineered β-mannanase to selectively produce larger MOS, making them promising candidates for large-scale MOS enzymatic production process.

Thermo-mechanical characterization of polyurea variants

The aim of this study is to investigate the role of chain length and hard segment dispersion within the soft segment on thermo-mechanical properties of a block copolymer by formulating and comparing three different polyurea compositions. Polyurea (PU) was synthesized by reacting oligomeric poly (tetramethylene oxide) (PTMO) diamines (Versalink, Air Products) with aromatic 4,4’-methylenediphenyldiisocyanate (Isonate 143L Modified MDI, Dow Chemicals). Three Versalink prepolymers were used: VP-1000 (long chain length), VP-650 (medium chain length), and VP-250 (short chain length), where 1000, 650, and 250 refer to the molecular weight of the PTMO repeating units. PU-105 and PU-605 were synthesized by mixing VP-1000 and VP-650 with Isonate 143L. A hybrid polyurea specimen, PU-HB05, was synthesized by reacting a blend of VP-1000:VP-250 at 76:24wt% with Isonate 143L. This weight ratio was calculated in order to ensure the total proportion of hard to soft segments of PU-HB05 match that of PU-605. A comparative study on PU-605 and PU-105 was conducted to determine the effect of diamine chain length physical, morphological, and mechanical properties. Concurrently, a blend of two diamines with significantly different chain lengths (short chain length Versalink P-250 and long chain length Versalink P-1000)was synthesized with the same diisocyanate prepolymer to create a hybrid variant of polyurea, PU-HB05. The blend was created in a proportion such that the hybrid polyurea PU-HB05 has the same hard to soft domain ratio as PU-605. It was shown that inclusion of short chain length diamine can increase the soft segment Tg by almost 15 ∘C in PU-HB05 compared to PU-105. Furthermore, the modulus in the hybrid blend formulation is also increased compared to PU-105, to the levels of PU-605. However, PU-HB05 demonstrates slightly better stability against high temperatures compared to PU-605. The high strain-rate measurements (including yield-like behavior and hardening plateau) using Split Hopkinson Pressure bar (SHPB) setup, indicate that in spite of close behavior of PU-HB05 and PU-605 (due to matching hard domain proportion), PU-HB05 appears to show less hardening post-yield and generally softer response at lower temperatures due to the presence of longer diamine chains. Master curves, relaxation spectra and relaxation modulus are reported for all three variants. Finally thermal properties of the three materials are reported indicating a more amorphous structure for PU-HB05 based on its lowest thermal conductivity and low heat capacity.

Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

Novel Polyhydroxyalkanoate-Based Biocomposites Obtained by Solution Casting and Their Application for Bacteria Removal and Domestic Wastewater Purification

In this study, novel biocomposites of medium chain length polyhydroxyalkanoate (mcl-PHA) containing 75 mol% of 3-hydroxydecanoate (3-HD) incorporating torch ginger cellulose nanowhiskers (TGCN) were developed via solution casting. The result showed that the morphology and mechanical properties of mcl-PHA were not significantly affected by TGCN. However, addition of TGCN decrease the crystallization temperature and water vapour and oxygen permeability of mcl-PHA. Furthermore, this material was prepared as a filter for bacteria removal and water filtration. The efficiency of the filter was increased with an increase of film number. 90–92% of bacteria and yeast were removed when a stack of three mcl-PHA-based films was applied in the filter holder. However, the efficiency of the filter was lower than the World Health Organization standard. The filter was also used for domestic wastewater purification. The biological oxygen demand, solid content and total bacteria in wastewater were significantly decreased using mcl-PHA based filter. The parameters tested in this current study also passed water quality standards for domestic wastewater (Thailand). It can be summarized that mcl-PHA-based biocomposites are able to remove microorganisms from water. Moreover, they can be applied for water purification.

Asymmetric Open-Closed Dimer Mechanism of Polyhydroxyalkanoate Synthase PhaC.

SummaryBiodegradable polyester polyhydroxyalkanoate (PHA) is a promising bioplastic material for industrial use as a replacement for petroleum-based plastics. PHA synthase PhaC forms an active dimer to polymerize acyl moieties from the substrate acyl-coenzyme A (CoA) into PHA polymers. Here we present the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, bound to CoA. The structure reveals an asymmetric dimer, in which one protomer adopts an open conformation bound to CoA, whereas the other adopts a closed conformation in a CoA-free form. The open conformation is stabilized by the asymmetric dimerization and enables PhaC to accommodate CoA and also to create the product egress path. The bound CoA molecule has its β-mercaptoethanolamine moiety extended into the active site with the terminal SH group close to active center Cys291, enabling formation of the reaction intermediate by acylation of Cys291.

Preparation and Characterization of Films Based on a Natural P(3HB)/mcl-PHA Blend Obtained through the Co-culture of Cupriavidus Necator and Pseudomonas Citronellolis in Apple Pulp Waste.

The co-culture of Cupriavidus necator DSM 428 and Pseudomonas citronellolis NRRL B-2504 was performed using apple pulp waste from the fruit processing industry as the sole carbon source to produce poly(3-hydroxybutyrate), P(3HB) and medium-chain length PHA, mcl-PHA, respectively. The polymers accumulated by both strains were extracted from the co-culture’s biomass, resulting in a natural blend that was composed of around 48 wt% P(3HB) and 52 wt% mcl-PHA, with an average molecular weight of 4.3 × 105 Da and a polydispersity index of 2.2. Two melting temperatures (Tm) were observed for the blend, 52 and 174 °C, which correspond to the Tm of the mcl-PHA and P(3HB), respectively. P(3HB)/mcl-PHA blend films prepared by the solvent evaporation method had permeabilities to oxygen and carbon dioxide of 2.6 and 32 Barrer, respectively. The films were flexible and easily deformed, as demonstrated by their tensile strength at break of 1.47 ± 0.07 MPa, with a deformation of 338 ± 19% until breaking, associated with a Young modulus of 5.42 ± 1.02 MPa. This study demonstrates for the first time the feasibility of using the co-culture of C. necator and P. citronellolis strains to obtain a natural blend of P(3HB)/mcl-PHA that can be processed into films suitable for applications ranging from commodity packaging products to high-value biomaterials.

Insight on esterase from Pseudomonas aeruginosa strain S3 that depolymerize poly(lactic acid) (PLA) at ambient temperature

Pseudomonas aeruginosa strain S3 exhibited high esterolytic activity against poly (lactic acid) (PLA) at environmental temperature (~30 °C). A comprehensive study was conducted for optimization of physico-chemical parameters for high-throughput esterase production using Plackett–Burman Design and Central Composite Design. PLA degradation by Pseudomonas aeruginosa strain S3 was confirmed during preliminary studies using Fourier Transform Infrared Spectroscopy (FTIR) and scanning electron microscopy (SEM). The date obtained from statistical optimization demonstrated interactive action of inoculum size with peptone, yeast extract and Tween 20 was more influential towards esterase activity. The following medium constituents were optimized for maximum esterase production [(g/L) Glucose, 0.8; Peptone, 10.5; Yeast extract, 15; MgSO4.7H2O, 0.3; Sodium citrate, 4; CaCl2, 1; NaCl, 1; FeSO4.7H2O, 1; Inoculum size 75 mL and Tween 20, 3.8 mL (v/v)]. The molecular weight of purified esterase was approximately 35 kDa with apparent Km and Vmax values, i.e., Km 12.38 μM and Vmax 769.23 U/mg, respectively. The enzyme showed stability over a broad range of pH (3–10) and temperature (20–40 °C). The activity of the purified enzyme was significantly enhanced by non-polar detergents (Tween 80 and Triton-X100), glycerol as well as both mono and divalent cations (Co+2 and K+1). Hydrolysis of PLA films with purified esterase released oligomers of medium chain length (n = 6–13), reaffirming the biodegradation potential of Pseudomonas aeruginosa strain S3 against PLA. The results obtained from the study demonstrate the potential of our strain and its enzymes in development of high efficient system of PLA biodegradation and recovery processes.

Production and characterization of polyhydroxyalkanoates synthesized by E. coli Isolated from sludge soil

The petrochemical-based non-degradative polymers are causing severe environmental pollution that resulting in acute or chronic toxicity in living organisms. Hence emerging of biological polymers is significant to replace traditional polymers. Therefore, the present study aimed to isolate efficient PHAs producing bacteria from municipal sludge soil, characterize the biopolymers by FT-IR and 1H NMR and optimize growth conditions for mass production of PHAs. 10 predominant bacteria cultures were isolated from sludge soil, out of 10 single isolates has the potential to produce PHAs, and it was characterized and identified asE. coli.The PHAs produced byE. coliwas characterized by FT-IR and NMR analysis. The results show that components of PHAs such as carbonylhydroxybutyrate, hydroxyoctaoate (HO), medium chain length polyhydroxyalkanoates, and hydroxydecanoate showed their strong characteristic band at 1631 cm−1, 1548 cm−1, 1409 cm-1 respectively in the extracted sample. Further, the NMR reports show that the presence of interconnection functional groups like HC = CH bond at 3.363 ppm, CH2O–COOH bond at 2.548 ppm. The optimal growth conditions forE. coliwere optimized. Briefly, 9% of inoculum, sago molasses (as suitable carbon source), 35 °C, pH 7.5 48hrs of incubation, in which theE. coliproduce 27.1 g/L of PHAs extracted from 33.26 g/L of biomass and in percentage it was calculated as 81.47%. The entire results determine that the E. colienumerated from sludge soil has the efficiency to produce PHAs, hence it possible for commercialization by using cheap carbon sources.

Rational Engineered C-Acyltransferase Transforms Sterically Demanding Acyl Donors.

The biocatalytic Friedel–Crafts acylation has been identified recently for the acetylation of resorcinol using activated acetic acid esters for the synthesis of acetophenone derivatives catalyzed by an acyltransferase. Because the wild-type enzyme is limited to acetic and propionic derivatives as the substrate, variants were designed to extend the substrate scope of this enzyme. By rational protein engineering, the key residue in the active site was identified which can be replaced to allow binding of bulkier acyl moieties. The single-point variant F148V enabled the transformation of previously inaccessible medium chain length alkyl and alkoxyalkyl carboxylic esters as donor substrates with up to 99% conversion and up to >99% isolated yield.

Response to lethal UVA radiation in the Antarctic bacterium Pseudomonas extremaustralis: polyhydroxybutyrate and cold adaptation as protective factors.

Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly as polyhydroxybutyrate (PHB) and, to a lesser extent, medium-chain length polyhydroxyalkanoates (mclPHAs). In this work, we analyzed the role of PHAs and cold adaptation in the survival of P. extremaustralis after lethal UVA exposure. P. extremaustralis presented higher radiation resistance under polymer accumulation conditions. This result was also observed in the derivative mutant strain PHA-, deficient for mclPHAs production. On the contrary, the PHB- derivative mutant, deficient for PHB production, showed high sensitivity to UVA exposure. Complementation of the PHB- strain restored the wild-type resistance level, indicating that the UVA-sensitive phenotype is due to the lack of PHB. All strains exhibited high sensitivity to radiation when cultured under PHAs non-accumulation conditions. A slight decrease in PHB content was observed after UVA exposure in association with increased survival. The scattering of UVA radiation by intracellular PHAs granules could also result in bacterial cell protection. In addition, cold conditions improved UVA tolerance, probably depending on PHB mobilization. Results showed that PHB accumulation is crucial in the resistance to UVA in P. extremaustralis. Mechanisms involved probably entail depolymerization and light scattering acting as a screen, both conferring protection against oxidative stress.

De novo biosynthesis of 8-hydroxyoctanoic acid via a medium-chain length specific fatty acid synthase and cytochrome P450 in Saccharomyces cerevisiae

Terminally hydroxylated fatty acids or dicarboxylic acids are industrially relevant compounds with broad applications. Here, we present the proof of principle for the de novo biosynthesis of 8-hydroxyoctanoic acid from glucose and ethanol in the yeast Saccharomyces cerevisiae. Toxicity tests with medium-chain length ω-hydroxy fatty acids and dicarboxylic acids revealed little or no growth impairments on yeast cultures even at higher concentrations. The ability of various heterologous cytochrome P450 enzymes in combination with their cognate reductases for ω-hydroxylation of externally fed octanoic acid were compared. Finally, the most efficient P450 enzyme system was expressed in a yeast strain, whose fatty acid synthase was engineered for octanoic acid production, resulting in de novo biosynthesis of 8-hydroxyoctanoic acid up to 3 ​mg/l. Accumulation of octanoic acid revealed that cytochromes P450 activities were limiting 8-hydroxyoctanoic acid synthesis. The hydroxylation of both externally added and intracellularly produced octanoic acid was strongly dependent on the carbon source used, with ethanol being preferred. We further identified the availability of heme, a cofactor needed for P450 activity, as a limiting factor of 8-hydroxyoctanoic acid biosynthesis.

Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n-alkane pollution by Pseudomonas aeruginosa DN1.

The purpose of this study was to elucidate the characteristics of multiple alkane hydroxylase systems in Pseudomonas aeruginosa DN1, including two homologues of AlkB (AlkB1 and AlkB2 ), a CYP153 homologue (P450), and two homologues of Alm-like (AlmA1 and AlmA2 ). DN1 was capable of utilizing diverse n-alkanes with chain lengths from 8 to 40 C atoms as the sole carbon source, and displayed high degradation efficiency (＞85%) of crude oil and a majority of n-alkanes using gas chromatography method. RT-qPCR analysis showed that the five enzyme genes could be induced by n-alkanes ranging from medium-chain length to long-chain length which indicated the dissimilarity of expression between those genes when grown on different n-alkanes. Notably, the expression of alkB2 gene was upregulated in the presence of all of the tested n-alkanes, particularly responded to long-chain n-alkanes like C20 and C32 . Meanwhile, long-chain n-alkanes (C20 -C36 ) significantly elevated cyp153 expression level, and the expression of two almA genes was only upregulated in the presence of n-alkanes with chain lengths of 20C's and longer. Furthermore, the disruption of those genes demonstrated that AlkB2 appeared to play a key role in the biodegradation of substrates of a broad-chain length ranges, besides other alkane hydroxylase systems ensured the utilization of n-alkanes with chain lengths of from 20 to 40 C atoms. The five functional alkane hydroxylase genes make DN1 an attractive option for its versatile alkane degradation, which is primarily dependent on the expression of alkB2 . Our findings suggest that P. aeruginosa DN1 is a predominately potential long-chain n-alkane-degrading bacterium with multiple alkane hydroxylase systems in crude oil-contaminated environment.