Optimization of the Isopentenol Utilization Pathway for Isoprenoid Synthesis in Escherichia coli.

Engineering microbes to produce isoprenoids can be limited by the competition between product formation and cell growth because biomass and isoprenoids are naturally derived from central metabolism. Recently, a two-step synthetic pathway was developed to partially decouple isoprenoid formation from central carbon metabolism. The pathway used exogenously added isopentenols as substrates. In the present study, we systematically optimized this isopentenol utilization pathway in Escherichia coli by comparing enzyme variants from different species, tuning enzyme expression levels, and using a two-stage process. Under the optimal conditions found in this study, ∼300 mg/L lycopene was synthesized from 2 g/L isopentenol in 24 h. The strain could be easily modified to synthesize two other isoprenoid molecules efficiently (248 mg/L ß-carotene or 364 mg/L R-(-)-linalool produced from 2 g/L isopentenol). This study lays a solid foundation for producing agri-food isoprenoids at high titer/productivity from cost-effective feedstocks.

Fungal growth, patulin accumulation and volatile profile in 'Fuji Mishima' apples under controlled atmosphere and dynamic controlled atmosphere.

The objective of this study was to evaluate fungal and patulin contamination, together with its correlation with the volatile compounds (VCs), in 'Fuji Mishima' apples (up to 25% decayed) under controlled atmosphere (CA) and dynamic controlled atmosphere with respiratory quotient (DCA-RQ) of 1.3 combined with different partial pressures of carbon dioxide (0.8, 1.2, 1.6 and 2.0 pCO2). Fruits were stored under the above conditions for 8 months at 0.5 °C plus 7 days shelf life at 20 °C. Toxigenic fungi and patulin accumulation were found in apples from all treatments. Penicillium expansum was the most prevalent species. For all storage conditions, patulin concentrations were above the maximum level allowed in Brazil (50  µg  kg-1) with an exception of DCA-RQ1.3 + 0.8 kPa CO2. This condition, with lower pCO2, showed the lowest patulin accumulation, below the legal limit. The CA provided the highest patulin concentration (166 µg  kg-1). It was observed that fungal growth could also contribute to changes in the volatile composition. Styrene and 3-methyl-1-butanol are considered P. expansum markers in some apple cultivars and were detected in the samples. However, it was not possible to identify volatile organic compounds (VOCs) that are biomarkers from P. expansum, because there were other fungi species present in all samples. In this study, styrene, n-decanoic acid, toluene, phenol and alpha-farnesene were the compounds that showed the most positive correlation with patulin accumulation. On the other hand, a negative correlation of patulin with acids has been shown, indicating that in treatments with a higher patulin concentration there were less acidic compounds.

Metabolomics reveals the role of isopentenyl group in coumarins metabolism.

Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7-Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7-ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC-ESI-QTOF-MS (ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-MS)-based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N-acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase.

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.

Electrophysiological and Behavioral Responses of an Ambrosia Beetle to Volatiles of its Nutritional Fungal Symbiont.

Ambrosia beetles (Coleoptera: Scolytinae) cultivate their fungal symbiont within host substrates as the sole source of nutrition on which the larvae and adults must feed. To investigate a possible role for semiochemicals in this interaction, we characterized electrophysiological and behavioral responses of Xylosandrus germanus to volatiles associated with its fungal symbiont Ambrosiella grosmanniae. During still-air walking bioassays, X. germanus exhibited an arrestment response to volatiles of A. grosmanniae, but not antagonistic fungi Beauveria bassiana, Metarhizium brunneum, Trichoderma harzianum, the plant pathogen Fusarium proliferatum, or malt extract agar. Solid phase microextraction-gas chromatography-mass spectrometry identified 2-ethyl-1-hexanol, 2-phenylethanol, methyl benzoate and 3-methyl-1-butanol in emissions from A. grosmanniae; the latter two compounds were also detected in emissions from B. bassiana. Concentration-responses using electroantennography documented weak depolarizations to A. grosmanniae fungal volatiles, unlike the comparatively strong response to ethanol. When tested singly in walking bioassays, volatiles identified from A. grosmanniae elicited relatively weak arrestment responses, unlike the responses to ethanol. Xylosandrus germanus also exhibited weak or no long-range attraction to the fungal volatiles when tested singly during field trials in 2016-2018. None of the fungal volatiles enhanced attraction of X. germanus to ethanol when tested singly; in contrast, 2-phenylethanol and 3-methyl-1-butanol consistently reduced attraction to ethanol. Volatiles emitted by A. grosmanniae may represent short-range olfactory cues that could aid in distinguishing their nutritional fungal symbiont from other fungi, but these compounds are not likely to be useful as long-range attractants for improving detection or mass trapping tactics.

Effects of Solvents on the Glycerolysis Performance of the SBA-15 Supported Lipases.

In this study, Candida antarctica lipase B (CALB), Rhizomucor miehei lipase (RML) and Lecitase® Ultra (LU) were immobilized onto the mesoporous silica SBA-15. The glycerolysis performance of the obtained supported lipases (lipase@SBA-15) in solvent systems was carefully investigated. LU@SBA-15 exhibited good glycerolysis performance in solvent-free system, with diacylglycerols (DAG) content and triacylglycerols (TAG) conversion at 52.4 and 98.6% respectively obtained after 12 h reaction at 60°C. CALB@SBA-15 showed good glycerolysis activity in tert-pentanol and tert-butanol systems, with TAG conversion over 90% obtained. In addition, the present CALB@SBA-15 exhibited selectivity for monoacylglycerols (MAG) production, with glycerol to TAG molar ratio increased to 3:1, MAG content over 80% and TAG conversion over 99% could be obtained from both tert-pentanol and tert-butanol systems. However, RML@SBA-15 showed low glycerolysis activity neither in solvent nor in solvent-free systems. The present results favor the practical enzymatic design for MAG and DAG production.

Immobilization of Eversa® Transform via CLEA Technology Converts It in a Suitable Biocatalyst for Biolubricant Production Using Waste Cooking Oil.

The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants.

Esterification of Fructose-oleic Acid by tert-Butanol/Dimethyl Sulfoxide and by 2-Methyl-2-butanol/Dimethyl Sulfoxide.

In this study two different strategy were followed to obtain a D-fructose-oleic acid ester. One of the strategies has been well established enzymatic synthesis of an ester bond. The other strategy excluded the biocatalyst and only used a mixture of two organic solvents as the reaction media, 2-methyl-2-butanol / dimethyl sulfoxide or tert-butanol / dimethyl sulfoxide for the production of D-fructose-oleic acid ester. Ester products obtained were characterised by using FT-IR, NMR, by MS. Product yield was also assessed by HPLC. Results of structural analyses and yield measurement indicated that two approaches produced almost identical ester products.

Sucrose-mediated heat-stiffening microemulsion-based gel for enzyme entrapment and catalysis.

Formation of a thermally stiffening microemulsion-based gel showing a nanoconfinement effect of carbohydrates in terms of microviscosity and hydrodynamic diameter of the reverse micelle (specifically with sucrose) is reported. The advantage of this gel as an efficient batch bioreactor for entrapped enzymes (horseradish peroxidase and thermophilic α-glucosidase) was shown, and illustrated its potential biocatalytic application at high temperatures.

Synthesis of Ribose - Oleic Acid Esters in the Presence- and Absence of Candida antarctica Lipase B.

D-ribose-oleic acid esters were produced with or without a biocatalyst, using in the same organic media, dimethyl sulfoxide (DMSO): tert-butanol (TBU) or 2-methyl-2-butanol (2M2B). The yield of the ester product was above 90% in both of the reactions. The biocatalyst used was lipase B of Candida antarctica. Molecular characterization was performed by using all the analytical methods available: IR, 1H-NMR and 13C-NMR, HSQC, and ESI-MS.

Transfer kinetics of labeled aroma compounds from liquid media into coffee beans during simulated wet processing conditions.

The transfer kinetics of three labelled compounds (butanal, 2-phenyethanol, isoamyl acetate) was studied from a liquid medium into the coffee beans during simulated wet processing using four media (M) (M1: contained dehulled beans, M2: contained demucilaginated beans, M3: contained depulped beans, M4: contained depulped beans with yeast). Trials were carried out at 25 °C, under agitation and for five time periods (0, 6, 12, 24 and 48 h), and then the labelled volatiles were analyzed by SPME-GC-MS. The three labelled molecules were transferred into the coffee beans with different mass transfer rates; reaching at 12hrs in the M4, 0.2 ± 0.03, 11.2 ± 0.66 and 1.3 ± 0.04 µg/g of coffee respectively for butanal, 2-phenyethanol and isoamyl acetate. The parchment resistance significantly affected the mass transfer of the 2-phenylethanol. Butanal and isoamyl acetate underwent metabolic reactions, which decreased their amount in the coffee beans. Furthermore, an interaction between molecules and the yeast was observed and decreased significantly the butanal's transfer.

Anti-echinococcal activity of menthol and a novel prodrug, menthol-pentanol, against Echinococcus multilocularis.

Alveolar echinococcosis is one of the most dangerous parasitic zoonoses. This disease, widely distributed in the northern hemisphere, is caused by the metacestode stage of the tapeworm Echinococcus multilocularis. All surgical and non-surgical patients should perform chemotherapy with benzimidazoles, mainly with albendazole. However, the efficacy of albendazole is variable due to its deficient pharmacokinetic properties. Therefore, the need to find new therapeutic alternatives for the treatment of alveolar echinococcosis is evident. Menthol is a natural compound of low toxicity, used in industries such as cosmetics and gastronomy and generally recognized as safe by the Food and Drug Administration. In addition, menthol has important pharmacological effects and is effective against a wide variety of organisms. The development of prodrugs allows improving the pharmacokinetic properties of the parental drug. To improve lipophilicity and therefore the bioavailability of menthol, a novel prodrug called menthol-pentanol was developed by masking the functional polar group of menthol by linking n-pentanol by a carbonate bond. The aim of the current work was to evaluate the in vitro and in vivo efficacy of menthol and menthol-pentanol against E. multilocularis. Menthol-pentanol had a greater protoscolicidal effect than menthol. In addition, the prodrug demonstrated a similar clinical efficacy to albendazole. The increase in lipophilicity of the prodrug with respect to menthol was reflected in an increase in its antiparasitic activity against E. multilocularis. Thus, menthol-pentanol appears as a promising candidate for further evaluation as a potential alternative for the treatment of alveolar echinococcosis.

Effects of Carriers on Spray-dried Flavors and Their Functional Characteristics.

BACKGROUND AND OBJECTIVE: Encapsulation is an effective method to keep the quality of and avoid changes in flavors or essential oils due to oxidation, heating, volatilization, or chemical interactions. This study aims to microencapsulate key flavorings by traditional wall materials, namely, gum arabic (GA), maltodextrin (MD) and sodium caseinate (SC) and evaluate the effects of different wall materials on the properties of the flavor microcapsules. MATERIALS AND METHODS: The emulsions of flavor compounds (linalool, citral, orange oil, allyl caproate and isoamyl acetate) were prepared using GA, SC and MD as carriers with different concentrations and then encapsulated in powder form by a spray dryer. Physical properties, including encapsulation efficiency, viscosity, emulsion stability and moisture, were studied before and after the spray drying process. Moreover, the morphology and extent of the nonenzymatic browning (NEB) of powder particles were performed using a scanning electron microscope (SEM) and a chroma meter. The effect of encapsulation on flavor retention and chemical composition was evaluated using gas chromatography-mass spectrometry (GC-MS). RESULTS: Orange oil shows the maximum retention efficiency (84.5-97.9%), whereas isoamyl acetate is less retained during drying (44.4-72.5%) compared with other volatile compounds. Increasing the concentration of GA provides the highest retention for all aroma compounds and improves the viscosity and the emulsion stability of spray-dried powder. The presence of MD as the main encapsulating agent enables the formation of several homogeneous capsules with a good spherical shape and a smooth surface, according to SEM. The NEB is observed intensively in encapsulated samples containing citral and orange oils, whereas the least browning occurs in isoamyl acetate microcapsules. CONCLUSION: Wall material formulation affects the retention, morphology and physical properties of the encapsulated flavors, which can be used in food or nutraceutical powder premixes.

Systematic Investigation of Lipophilicity Modulation by Aliphatic Fluorination Motifs.

Optimization of compound lipophilicity is a key aspect of drug discovery. The aim of this work was to compare the lipophilicity modulations induced by 16 distinct known and novel fluoroalkyl motifs on three parent models. Fifty fluorinated compounds, with 28 novel experimental aliphatic log P values, are involved in discussing various lipophilicity trends. As well as confirming known trends, a number of novel lipophilicity-reducing motifs are introduced. Tactics to reduce lipophilicity are discussed, such as "motif extensions" and "motif rearrangements", including with concomitant extension of the carbon chain, as well as one- and two-fluorine 'deletions' within perfluoroalkyl groups. Quantum chemical log P calculations (SMD-MN15) based on solvent-dependent three-dimensional (3D) conformational analysis gave excellent correlations with experimental values, superior to Clog P predictions based on 2D structural motifs. The availability of a systematic collection of data based on a small number of parent molecules illustrates the relative lipophilicity modulations of aliphatic fluorination motifs.

Kraft lignin grafted with isopentenol polyoxyethylene ether and the dispersion performance.

In order to develop a biomass-based superplasticizer, kraft lignin (KL) was grafted with isopentenol polyoxyethylene ether (TPEG) to prepare a novel macromonomer (KL-TPEG). It was shown that the retention ratio of the aliphatic CC bond increased from 81.07% to 90.20% with the increase of m(TPEG)/m(KL). When the grafting ratio was increased, the average number of TPEG grafted on one KL molecule was approximately 1.4, 3.1, 4.6, 6.2 and 7.6. The star-like structure was also confirmed by FT-IR, 1H NMR and GPC. KL-TPEG had favorable surface activity and dispersion stability on the cement particles. It was illustrated that the shear stress and shear viscosity of the cement slurries with KL-TPEG were significantly less than that of blank slurry. The dispersion-retention ability of KL-TPEG on the slurry was also gradually enhanced with the increase of the grafting ratio of TPEG. It was seldom reported on the biomass-based TPEG. Through modified with KL, the rheology behavior and the dispersion-retention ability of TPEG was greatly improved, and the cost of TPEG was also reduced, thus this study not only promoted the development of biomass-based macromonomer, but also helped for the high value utilization of lignin.

Immobilization of lipase onto novel constructed polydopamine grafted multiwalled carbon nanotube impregnated with magnetic cobalt and its application in synthesis of fruit flavours.

Surface modification of multiwalled carbon nanotubes (MWCNTs) could enhance the features of the nanomaterial as carrier for enzyme immobilization. In this strategy, magnetic MWCNTs were fabricated by incorporating them with cobalt and functionalization was carried out by aminated polydopamine. The surface modified MWCNTs were then used as a carrier for the immobilization of Candida rugosa lipase (CRL) via covalent binding using glutaraldehyde. The immobilized CRL maintained high activity, which was 3-folds of free CRL. The immobilized CRL exhibited excellent thermal resistance as validated by TGA and DTA technique and was found to be active in a broad range of pH and temperatures in comparison to free CRL. Systematic characterization via FT-IR spectroscopy, CD spectroscopy, SEM, TEM and confocal laser scanning microscopy confirmed the presence of CRL on the modified MWCNTs. Immobilized CRL presented an exquisite recycling performance as after ten consecutive reuses it retained around 84% of its initial hydrolytic activity and further showed high yield enzymatic synthesis of ethyl butyrate and isoamyl acetate having characteristic pineapple and banana flavour demonstrating 78% and 75% ester yield, respectively. The present work provides a novel perspective for lipase catalyzed biotechnological applications by adding a magnetic gain to intrinsic features of MWCNTs.

Stereocontrolled Synthesis of Tetrafluoropentanols: Multivicinal Fluorinated Alkane Units for Drug Discovery.

A stereodivergent synthesis of four diastereomeric 2,3,4,5-tetrafluoropentanols is disclosed. X-ray crystallographic analysis reveals conformations that manifest sequential stereoelectronic gauche effects (σC-H/C → σC-F*), thereby generating topological diversity via subtle C(sp3)-H to C(sp3)-F exchange. Two representative tetrafluoro arrays have been incorporated into truncated analogues of Gilenya for the management of relapsing remitting multiple sclerosis. These closely similar multivicinal fluoroalkanes have notably different physicochemical profiles and were found to be stable in the presence of human microsomes.

Cell free biosynthesis of isoprenoids from isopentenol.

Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-d-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far.