Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of  Plasmodium stages depends on generation of hydroxylated-PQ metabolites (OH-PQm), which, undergoing further reactions, results in production of H2O2.Primaquine (PQ) is a widely used anti-malaria drug, but its mechanism of action is unclear. Here, Camarda et al. show that PQ’s activity against liver and sexual  Research into the elusive PQ mode of action has mainly focused on the identification of the biochemical basis for its side effect of hemolytic toxicity in patients with G6PD deficiency1. Earlier studies led to the identification of OH-PQm, generated from hepatic phase I metabolism2, as potential culprits for toxicity4. The most relevant species in this context include the 5-hydroxy (5-HPQ) and 5,6-dihydroxy  primaquine metabolites  from spinach , PQ quinoneimines were shown to be enzymatically reduced5—but despite intensive research efforts the exact enzyme(s) and metabolic pathway(s) involved in the process remain to be fully elucidated.Since its development in the 1950s by the US Army, an estimated 200 million people have been administered PQtes Fig. . These s7. A link between drug efficacy and metabolism through CYP2D6 is supported by recent animal and clinical studies, including the association of CYP2D6 poor metaboliser phenotype status with primaquine failure in controlled human malaria infections with P. vivax8. The need for CYP2D6 metabolism to generate key OH-PQm10 supports the assumption that these metabolites contribute to drug efficacy; however, hitherto there has been no direct demonstration of the link between these metabolites and anti-parasitic activity. Any explanation of PQ action needs to address the exquisite selectivity against dormant and active liver stage parasites and the ability of very low drug doses and subsequent low systemic exposures to kill gametocyte stages of P. falciparum clinically11. This study attempts to reconcile these questions.Similarly, the mechanism of action of primaquine against the malaria parasite is also largely unknownP. falciparum gametocytes and liver stages. By assessing parasite viability in the presence or absence of metabolic conversion we show that OH-PQm full cidal potential requires further enzymatic catalysis. We identify the CYP2D6 redox partner, cytochrome P450 NADPH:oxidoreductase (CPR), as required for OH-PQm to exert full gametocytocidal activity, independently of CYP2D6 status. We also show that OH-PQm activity against P. falciparum liver stage development is comparable or better than PQ and confirm a dispensable role for CYP2D6 activity. Finally, we substantiate the critical role of H2O2 production in anti-parasitic activity and propose a CPR-mediated biochemical model to account for PQ and OH-PQm mechanism of action. These data answer long-held questions of why PQ displays parasite stage-specific activity and why PQ displays transmission blocking activity at such low doses.Here OH-PQm in their hydroxyquinoline or quinoneimine form have been synthesised and assessed for their activity against P. falciparum liver stage development, we used an advanced in vitro model that consistently mimics liver physiology and supports complete maturation of P. falciparum extra-erythrocyte forms (EEFs) in the liver12. In this in vitro system, human primary hepatocytes are organised into colonies surrounded by supportive stromal cells  and infected with P. falciparum (NF54 strain) sporozoites12. To evaluate the role of CYP2D6 in PQ and OH-PQm activity, we selected two hepatocyte lots, NON and YEM. YEM is a low metaboliser based on genotype analysis, it has a variation in the CYP2D6 allele (*4/*4); this was confirmed by incubation with the CYP2D6 probe debrisoquine13 and measuring the metabolite 4-hydroxidebrisoquine by mass spectrometry (MS)  as compared to NON hepatocytes (extensive metaboliser PQ IC50 0.40 μM). Conversely, all OH-PQm showed comparable IC50 values in the two hepatocyte lots 14. As shown in Fig. p value < 0.001 for all pairs by Mann Whitney test, Supplementary Table We next explored the anti-gametocyte activity of OH-PQm and parental PQ through coupled in vitro metabolism-gametocytocidal assays. In these assays, we first incubated PQ and OH-PQm with human liver microsomes (HLM) to mimic bioactivation and then tested for gametocytocidal activity against a p < 0.05 for 5,6-DPQ and 6OHPQQI, and p < 0.001 for all other compounds  as expected, reverting towards the levels observed in control samples without CYP2D6  component of HLM and CYP2D6 baculosome preparations. We therefore measured initial reaction rates of human recombinant CPR (huCPR)17 with PQ and OH-PQm  , confirming that huCPR catalysis mediates OH-PQm gametocytocidal activity. Cognisant of the putative role of H2O2-producing redox-active PQ metabolites in PQ haemolytic toxicity in G6PDH-deficient individuals18, we indirectly assessed the contribution of reactive oxygen species to the gametocytocidal activity of huCPR-treated PQ metabolites by using sodium pyruvate as a H2O2 scavenger19. As shown in Fig. p < 0.01; Mann Whitney, Supplementary Table These results prompted us to hypothesise that OH-PQm might be direct substrates of CPR, the CYPs redox partner required for electron transfer from NAPDH2O2 generation during drug incubation with human CPR (huCPR). To this end, O2 consumption during incubations was recorded using a closed O2 electrode and H2O2 was quantified via oxygen release after catalase addition. As shown in Fig. 2O2 production, whereas negligible amounts of H2O2 were detected when PQ was used as a substrate. When sodium pyruvate was added to CPR reactions, H2O2 production was completely abolished   could account for PQ stage-specificity. For EEFs this association is obvious with parasite residency within the hepatocyte; for gametocytes this is less obvious as circulating gametocytes are rarely in juxtaposition to a potential source of metabolism. However, nt) Fig. . These r50) of OH-PQm. Dose-response huCPR reactions were performed prior to GC-LUC assays. As shown in Fig. 50 of 15 ± 0.4 nM. Conversely, parental PQ shows an IC50 of 2.59 ± 0.77 μM, in strong agreement with the measured enzyme kinetics. These results support a catalytic mechanism of action for PQ metabolites via CPR-mediated redox cycling that is in line with expected drug metabolite exposure after clinically relevant PQ doses29.Next, we determined gametocyte inhibition sensitivity profiles  involving two host enzymes and has implications not only for bioactivation but also for pharmacodynamics. The elucidation of a catalytic (e.g. 1 nM) MoA for OH-PQm  nuclear magnetic resonance spectrometer. Solvents are indicated in the text. The data for 1H NMR spectra are reported as follows: chemical shifts were described in parts per million  downwards from an internal reference of trimethylsilane. 13C NMR spectra were measured on a Brucker AMX400 (100 MHz) and are reported in terms of chemical shift  relative to residual solvent peak. MS and High resolution mass spectrometry (HRMS) were recorded on a VG analytical 7070E machine, Frisons TRIO spectrometers or Agilent QTOF 7200 using chemical ionisation (CI) or electron ionisation (EI). Micromass LCT mass spectrometer used electron spray ionisation (ESI).32 as described below.The synthesis of metabolites was based on modifications of literature procedures  was dissolved in Tetrahydrofuran . A solution of sodium hypophosphite  in 10 mL water was added to the reaction. The flask was purged with nitrogen. 10% palladium on carbon (0.7 g) was added to the reaction mixture and purged with nitrogen. The reaction was allowed to stir vigorously for 10 min. A celite pad was prepared and the reaction mixture was filtered through the pad of celite and washed through with 20 mL chloroform. Sodium hydroxide  was added to the filtrate. The filtrate was extracted with chloroform (3 × 100 mL) and the combined organic extracts were washed with water (2 × 50 mL), dried over magnesium sulphate and filtered. The chloroform was removed under reduced pressure to obtain the pure product  as a red-orange solid; Rf = 0.15, 30 % ethyl acetate in hexane; 1H NMR  δ 8.62 , 8.33 , 7.35 , 6.73 , 4.87 , 3.96  and 3.89 ; 13C NMR  δ 149.6, 145.6, 141.5, 133.8, 133.2, 129.9, 124.5, 121.6, 99.1, 61.6 and 56.8; IR νmax (neat)/cm−1 3443, 3337 (NH), 3192 (CH) and 1623 (C=C); HRMS calculated for C11H12N2O2 [M + H]+ 205.0972 found 205.0977.For the synthesis of 5,6-dimethoxyquinolin-8-amineRf = 0.43, 20% ethyl acetate in hexane; 1H NMR  δ 7.88–7.81 , 7.76–7.70 , 4.22–4.10 , 3.72 , 2.02–1.74  and 1.70 ; 13C NMR  δ 168.4, 134.1, 132.1, 123.3, 50.6, 38.1, 37.2, 27.1 and 26.5; HRMS Calculated for C13H14BrNO2 [M + H]+ 296.0281 found 296.0280. 5,6-Dimethoxy-8-aminoquinoline  was added to a dry 50 mL two neck flask along with a dry magnetic bar. A dry condenser and equilibrating dropping funnel were attached to the flask and the second neck was sealed with a rubber septum. The system was purged with dry argon and sealed with a balloon attached to the dropping funnel. 1-Phthalimido-4-bromopentane  was added directly to the reaction via syringe and the reaction was then heated to 150 °C to produce a dark paste. Triethylamine  was added to the reaction via the dropping funnel over a 1.5 h period. The reaction was left to stir for a further 1.5 h. Additional 1-phthalimido-4-bromopentane  was added directly to the reaction via syringe. Triethylamine  was added to the reaction via the dropping funnel over 30 min. The reaction was left to stir for a further 2 h. 1-Phthalimido-4-bromopentane  was added to the reaction directly via syringe. Triethylamine  was added to the reaction via the dropping funnel over 30 min. The reaction was left to stir for a further 2 h and monitored by thin layer chromatography (TLC). The reaction was cooled, diluted with acetone and filtered. The acetone was removed, resulting in a dark oil which was dissolved in chloroform (100 mL). The organic layer was washed with water (3 × 50 mL), dried over magnesium sulphate and filtered. The crude product was pre-absorbed onto silica and purified via flash chromatography to obtain the product  as a yellow oil; Rf = 0.3, 30% Ethyl Acetate in Hexane; 1H NMR  δ 8.53 , 8.27 , 7.84–7.78 , 7.72–7.66 , 7.33 , 6.41 , 5.89 , 3.99 , 3.86 , 3.74 , 3.71–3.63 , 1.99–1.62  and 1.30 ; 13C NMR  δ 168.6, 150.0, 144.8, 141.9, 134.0, 133.7, 132.2, 131.3, 129.8, 124.6, 123.3, 121.7, 94.3, 61.6, 57.0, 48.1, 38.1, 34.1, 25.5 and 20.8; IR νmax (neat)/cm−1 3390 (NH), 2964, 2936 (CH) and 1707 (C=O); HRMS Calculated for C24H25N3O4 [M + H]+ 420.1923 found 420.1922.For the synthesis of 4-bromo-1-phthalimidopentane, potassium phthalimide  was dissolved in acetone. 1,4-Dibromopentane  was added to the mixture which was heated to reflux for 24 h. The reaction was cooled and filtered. The acetone was removed under reduced pressure and the crude product was purified via flash chromatography resulting in (14.62 g 91%) a clear light yellow oil. 31, 5,6 dimethoxy-phthaloyl primaquine  was dissolved in 25 ml of ethanol in a 50 mL round bottom, a condenser was attached and the system was purged with nitrogen. Sixty-five percent hydrazine monohydrate  was added to the reaction and refluxed (at 100 °C) for 6 h. A solid precipitate was observed. The reaction was cooled and filtered. The ethanol was removed and 30% potassium hydroxide (100 mL) was added to the residue. The mixture was extracted with diethyl ether (3 × 50 mL). The combined organic layers were washed with water (50 mL), dried over magnesium sulphate and filtered. A solution of 89% phosphoric acid  in ethanol (5 mL) was added drop wise to the ether solution. A red/orange precipitate was observed. The solvent was removed under vacuo and the precipitate was recrystallised with ethanol to obtain an orange/red solid ; 1H NMR  δ 8.57 , 8.23 , 7.47 , 6.55 , 6.02 , 3.94 , 3.75 , 3.80–3.71 , 2.80 , 1.79 –1.55  and 1.23 ; 13C NMR  δ 149.8, 144.6, 141.5, 132.6, 130.0, 129.2, 123.8, 122.0, 94.0, 60.9, 56.5, 46.9, 38.9, 33.1, 24.1 and 20.4; HRMS Calculated for C24H25N3O4 [M + H]+ 420.1923 found 420.1922.For the synthesis of 5,6 dimethoxy primaquine32. Ten milligram of 5,6 dimethoxy primaquine was dissolved in 1 mL of 48% hydrogen bromide solution (ca 6 M) and sealed under argon in a sealed tube. The reaction was heated to 120 °C on a pre-heated mantle for 20 mins. Under a flow of nitrogen, 5 mL of water was added and the product(s) were purified via HPLC. For the general procedure 2, 10 mg of 5,6 dimethoxy primaquine was dissolved in 1 mL of 48% hydrogen bromide solution and the solution was allowed to stir for 6 h. Excess reagent was removed under vacuum to give a brown solid that was stored under nitrogen.Two general procedures were used for demethylation, as indicated. The general procedure 1 was based on a modified procedure of Allahyari et al.−1.HPLC conditions were as follows: a Phenomenex Jupiter Proteo 90 A column, 250 × 10 mm, 10 Micron was used for purification using a gradient system based on the following conditions; Initial (time 0) solvent mix 5% acetonitrile, 95% 0.05% trifluoracetic acid (TFA) in water; 20 mins, 25% acetonitrile, 75% 0.05% TFA in water; 20.10 min 5% acetonitrile, 95% 0.05% TFA in water; flow rate was 5 ml min14H19N3O2 [M + H] + found 262.33). The metabolite was stored under nitrogen in a sealed tube.All samples were dried by blowing off the solvent under a flow of nitrogen 5,6-dihydroxyprimaquine  was prepared according to general procedure 2; this compound was very unstable rapidly oxidising to 5-hydroxy quinoneimine (5-HPQ) in solutions exposed to air. (MS for CFor the synthesis of 5-hydroxy primaquine (5-HPQ) general procedure 1 along with HPLC purification and drying was used (see HPLC conditions above). 5-Hydroxy 6-Methoxy Primaquine = 10.06 min. The 5-hydroxy 6-methoxy primaquine was very unstable as it readily oxidises to the quinoneimine form. LCMS has shown that this is present within the reaction mixture, however, upon isolation and re-evaluation using the HPLC conditions, the retention time at 10.06 min corresponding to the product is no longer observed. Once the quinoneimine has formed from 5-OH primaquine, it is readily converted to the 6-hydroxy form  by a demethylation reaction (through reaction with water).1H NMR  δ 9.03 , 8.59 , 7.96 , 6.99 , 4.22 , 3.07 , 2.11–1.95 , 1.88–1.72  and 1.58 ; MS for C15H19N3O2 [M + H]+ found 274.38.For the synthesis of Primaquine quinone-imine (PQQI), general procedure 1 provides the -hydroxy primaquine which can be allowed to oxidise to the quinoneimine in aqueous solution. HPLC purification and drying (see above) were used for isolation. The quinoneimine was purified with two HPLC purification runs. Retention time: = 7.76 min 1H NMR  δ 8.86 , 8.40 , 7.75 , 6.12 , 4.11 , 3.05 , 1.92–1.74  and 1.42 ; MS for C14H17N3O2 [M + H]+ found 260.35.For the synthesis of 6-hydroxy primaquine quinone imine (6OHPQQI), general procedure 2 along with HPLC purification and drying (see section 1.1.3) was used. This compound could also be produced by reaction of the 6-methoxy quinoneimine in aqueous solution followed by HPLC purification.  Retention time: = 12.55 min. + . Compounds  were prepared in 40 μl reaction buffer containing NADP+ as per kit protocol. The total volume was brought to 100 μl with reaction buffer and reactions incubated at 37 °C for 2 h. For CYP2D6 inhibition, we first determined paroxetine inhibitory profile using Vivid CYP2D6 Blue kit (Life Technologies) according to manufacturer’s instructions. For gametocytocidal assays, master mixes without compounds were pre-incubated with paroxetine (10 μM) for 15 min at 37 °C before addition of compounds and further incubation 37 °C for 2 h. Recombinant huCPR17 was used for in vitro redox cycling of compounds. Reactions were performed as described above with minor modifications. A master mix without baculosomes was prepared as above; huCPR were added in 10 μl of reaction buffer  and reactions started by adding 40 μl of compound/NADP+ mix. For H2O2 scavenging, sodium pyruvate (10 mM) was used, without pre-incubation. All experiments contained control reactions with solvent (MeOH:water 50:50) only. After incubations, reaction mixes were spun down, supernatants collected, diluted 2:3 and seeded into 96-well plates for GC-LUC assay.Human liver microsomes  and CYP2D6-expressing baculosomes from the Vivid CYP450 kit (Life Technologies) were used for compound metabolic conversions as per manufacturer’s instructions with minor modifications. For HLM, compounds  were incubated in the presence of NAPDH Regeneration System Solution A and B (BD Biosciences) at 37 °C for 2 h in a total volume of 100 μl of phosphate reaction buffer. For CYP2D6 baculosomes reaction, a 2× master mix (50 μl) was prepared containing reaction buffer, baculosomes, regeneration system and NADPP. falciparum 3D7A33 transgenic derivative 3D7elo1-pfs16-CBG99 was used34, specifically expressing the CBG99 luciferase in gametocytes. Parasites were cultured35 in human 0 + erythrocytes at 5% haematocrit under 5% CO2, 2% O2, 93% N2. Cultures were grown in complete medium (CM) containing RPMI 1640 medium (Gibco) supplemented with 25 mM Hepes (VWR), 50 μg mL−1 hypoxanthine, 0.25 mM NaHCO3, 50 μg mL−1 gentamicin sulfate, and 10% pooled heat inactivated AB + human serum. Gametocyte viability was evaluated by the GC-LUC assay34. Parasites were quickly harvested in 2× CM before addition to 96-well plates containing control, CYP2D6- or huCPR-treated compounds  in aqueous solution and incubatied at 37 °C for 72 h. Drug-treated gametocytes were then transferred to 96-well white plate; D-Luciferin, 1 mM in 0.1 M citrate buffer pH 5.5 (Promega), was added in a 1:1 ratio and luminescence measurements were recorded after 10 min on a FLUOstar Omega plate reader (BMG Labtech). Viability was expressed as % viability as compared to solvent treated controls.A 36. Briefly, soft lithography techniques were used to pattern rat tail type I collagen (Corning) into 500 µm diameter islands on the surface of glass bottom 96-well plates. Cryopreserved primary human hepatocytes (Bioreclamation IVT) were thawed and pelleted through centrifugation at 100 g for 6 min, assessed for viability using trypan blue exclusion (70–90% viability) and 10,000 hepatocytes were seeded onto the collagen islands in serum-free DMEM (Dulbecco’s Modified Eagle’s medium) with 1% Penstrep. Two to three hours later, cells were washed with DMEM containing 1% Penstrep, and media was replaced with hepatocyte culture media. The following day, each well was infected with 70,000 fresh P. falciparum sporozoites. Three hours later, cultures were washed with DMEM containing 3% Penstrep and 0.1% Fungizone, and 7000 3T3 J2 mouse fibroblasts were added to establish the co-culture. Drug was administered during daily media changes for 3 days. The impact on hepatocyte infection was measured by enumeration of exoerythrocytic forms on day 3.5 post infection, through staining for PfHSP70 and visualisation with a Nikon Eclipse Ti fluorescent microscope.Micropatterned hepatocyte-fibroblast co-cultures were established as previously described2O2 production measurements were performed using the Oxytherm system and O2 View software package v.2.06 (Hansatech Instruments Ltd). Compounds ability to generate hydrogen peroxide after in vitro metabolism or huCPR reaction, in the presence of regeneration system or 100 μM NADPH as indicated, was assessed indirectly by measuring catalase-mediated oxygen release. Once the kinetic trace for oxygen concentration within the mixture had reached a plateau for at least 3 min, catalase  was added and data recorded for a further 6 min. Oxygen concentration was recorded as nmol mL−1. To allow for easier comparison of individual traces, the x axis was adjusted by defining the addition of catalase as t = 0, and the corresponding y axis value defined as 0 nmol mL−1.Oxygen consumption and H2, 5 mM KCl, 0.8 mM Na2HPO4) using 2 ml syringe connect with a 25GA needle. The bone marrow cells were washed twice and resuspended with cold Ringer’s solution. Cells were lysed by sonication and the protein concentration of crude extracts measured. Oxygen measurements were performed at 37 ˚C in 0.4 ml samples containing Ringer’s solution pH 7.4, 8.5 mg ml−1 bone marrow extracts, 1X regeneration system (Thermo Scientific), 30 µM PQ metabolites, 30 µM NADP+. The assay mixture without NADP+ was pre-incubated in the Oxytherm’s chamber at 37˚C while recording oxygen content. Then, the reaction was initiated by addition of NADP+. After 30, 10 µl of 5 mg ml−1 catalase (prepared in 50 mM potassium phosphate buffer pH 7.0) was added to release O2 from H2O2. Experiments with red blood cells were performed as above with same amount of protein extracts.For bone marrow experiments, femurs were dissected from mice following schedule 1 procedure. The schedule 1 procedure was undertaken with local  and national (Home Office licence) authorisation. Bone marrow cells were flushed out with cold Ringer’s solution pH 7.4  at 25 °C. The activity assay of 0.3 ml contained 0.3 M potassium phosphate (pH 7.7), 25 nM huCPR, 100 µM NADPH, and various concentrations of primaquine derivatives . Reactions were initiated by adding NADPH to a final concentration of 100 µM after equilibrating the assay mixture with all other components at 25 °C for 2 min. Data were recorded every 5 s over 4 min. The Michaelis–Menten equation was used to determine Km and kcat values.Kinetic measurements were determined following Tsukamoto et al.www.graphpad.com). Significance was calculated by two-tailed Mann Whitney test. One-way or two-way ANOVA was used as appropriate. Extended statistics tables for each analysis are provided as Additional Information.Statistical analyses were done using GraphPad Prism version 5.04 and version 7 software (GraphPad Software, San Diego, California, USA, Further information on research design is available in the Supplementary InformationReporting SummarySource Data


Green organic solvents such as lactate esters have broad industrial applications and favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. However, to date, the direct microbial biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated.ldhA) to convert pyruvate to lactate, a propionate CoA-transferase (pct) to convert lactate to lactyl-CoA, and an alcohol acyltransferase (AAT) to condense lactyl-CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a pyruvate-to-lactate ester module and an alcohol  module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as a proof-of-study, we re-modularized the pathway into (1) the upstream module to generate the ethanol and lactate precursors and (2) the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found that AAT is the most rate-limiting step in biosynthesis of lactate esters likely due to its low activity and specificity toward the non-natural substrate lactyl-CoA and alcohols.In this study, we present a microbial conversion platform for direct biosynthesis of lactate esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting of a lactate dehydrogenase (E. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.We have successfully established the biosynthesis pathway of lactate esters from fermentable sugars and demonstrated for the first time the direct fermentative production of lactate esters from glucose using an  Solvents are widely used as primary components of cleaning agents, adhesives, and coatings and in assisting mass and heat transfer, separation and purification of chemical processes . HoweverLactate esters are platform chemicals that have a broad range of industrial applications in flavor, fragrance, and pharmaceutical industries . These eG = + 5 kcal/mol) in aqueous solutions and often encounter significant challenge due to self-polymerization of lactate [G = − 7.5 kcal/mol) in an aqueous phase environment at room temperature and atmospheric pressure [In industrial chemical processes, lactate esters are currently produced by esterification of lactic acid with alcohols using homogenous catalysts  under high temperature reaction conditions . However lactate . Alternapressure –16. Thispressure , 11, 17,pressure , and  into an engineered The substrate specificity of AATs is critical to produce target esters . For exaThe results show that most of the strains could produce different types of lactate esters with external supply of alcohols Fig. b, c. EcJDue to the presence of endogenous acetyl-CoA, we also produced acetate esters in addition to lactate esters Fig. . Among tVAAT could produce 6 out of 6 target lactate esters including ethyl, propyl, butyl, isobutyl, isoamyl, and benzyl lactate. Since EcJW104 achieved the highest titer of lactate esters in all cases, it was selected for establishing the biosynthesis pathway of lactate esters from glucose.Taken altogether, VAAT and ATF1 are the most suitable AATs for biosynthesis of lactate esters and acetate esters, respectively. Among the library of 12 esters  with the ethanol (pCT24) and isobutanol (pCT13) modules, respectively. By co-transforming pJW005/pCT24 and pJW005/pCT13 into the modular cell EcDL002, we generated the production strains, EcJW201 and EcJW202, for evaluating direct conversion of glucose to ethyl and isobutyl lactate esters, respectively.We characterized EcJW201 and EcJW202 together with the parent strain, EcDL002, as a negative control in high cell density cultures. The results show EcJW201 and EcJW202 produced ethyl  decreased by 6% and 10%, respectively, upon cell exposure to 5 g/L ethyl lactate. On the other hand, isoamyl lactate was the most toxic among the lactate esters, where cell exposure to only 0.5 g/L ester resulted in 18% and 15% reduction in the growth rate (0.41 ± 0.02 L/h) and OD600 (0.40 ± 0.03), respectively  and cell titer  the downstream module carrying pct and VAAT for converting lactate into lactyl-CoA and condensing lactyl-CoA and ethanol  the upstream module carrying nol Fig. a. We connol Fig. b. To evaThe results show that EcJW204, carrying the upstream module with a low copy number plasmid P15A origin) and the downstream module with a high copy number plasmid RSF1030 origin) induced by 0.01 mM of IPTG, achieved the highest titer of ethyl lactate. As compared to EcJW201, EcJW204 achieved 4.96-fold (an increase from 2.24 to 11.10 ± 0.58 mg/L), 5.50-fold (from 0.04 ± 0.00 to 0.22 ± 0.02 mg/gDCW/h), and 54.0-fold (from 0.01 ± 0.00 to 0.54 ± 0.04 mg/g glucose) improvement in titer, specific productivity, and yield of ethyl lactate, respectively  and 1.67- to 2.59-fold increase in lactate production (from 1.06 ± 0.09 to 1.77 ± 0.37 g/L ~ 2.75 ± 0.09 g/L)   and 1.27- to 2.07-fold (from 36.46 ± 3.86 to 46.22 ± 1.33 mg/L ~ from 21.96 ± 0.84 to 45.40 ± 1.20 mg/L), respectively  and three synthetic RBSs for VAAT expression (synRBSVAAT#1-3)  derived from pJW012 of the best performer EcJW204 using a combination of three synthetic RBSs for Pct expression  was overexpressed in the ldhA-deficient modular cell of our study, it is anticipated that d-(−)-lactate and the associated d-(−)-lactate esters were mainly produced. To date, production of optically pure d-(−)- [l-(+)-form [E. coli [pct from C. propionicum [Megasphaera elsdenii [d-(−)-lactate into d-(−)-lactyl-CoA in polylactic acid (PLA) production in E. coli and their catalytic activity toward l-(+)-lactate has also been demonstrated [In principle, the lactate ester platform can be controlled to produce enantiomers with broad industrial applications. Since the endogenous e d-(−)-  and l-(+(+)-form  of lacta[E. coli  has beenpionicum  and Megaelsdenii , 30 has nstrated , 46. ThuE. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.In this study, we have successfully developed a microbial lactate ester production platform and demonstrated for the first time the microbial biosynthesis of lactate esters directly from fermentable sugars in an E. coli TOP10 strain was used. To generate the lactate ester production strains, the modules, including (i) the pyruvate-to-lactate ester modules (pJW002-006), (ii) the upstream and/or downstream modules (pJW007-pJW028), and (iii) the alcohol modules (pCT24 or pCT13), were transformed into the engineered modular E. coli chassis cell, EcDL002 [The list of strains used in this study is presented in Table  EcDL002 .Table 1AThe list of plasmids and primers used in this study are presented in Tables ldhA gene, encoding d-lactate dehydrogenase, amplified from E. coli MG1655 genomic DNA using the primer pair DL_0032/DL_0033, (ii) the pct gene, encoding propionate CoA-transferase, amplified from Clostridium propionicum genomic DNA using the primer pair DL_0034/DL_0035, and (iii) the backbone amplified from pETite* using the primer pair DL_0001/DL_0002 [ATF1 gene amplified from pDL004 for pJW002, the ATF2 gene amplified from pDL005 for pJW003, the SAAT gene amplified from pDL001 for pJW004, the VAAT gene amplified from pDL006 for pJW005, or the atfA gene amplified from pCT16 for pJW006, using the primer pair DL_0015/DL_0016, and (iii) the backbone amplified from pETite* using the primer pair DL_0013/DL_0002. The genes ATF1 and ATF2 are originated from Saccharomyces cerevisiae [SAAT, VAAT, and atfA are derived from Fragaria ananassa [F. vesca [Acinetobacter sp. ADP1 [A library of pyruvate-to-lactate ester modules with five divergent AATs was constructed to screen for an efficient AAT for production of lactate esters via two rounds of cloning. First, the pyruvate-to-lactyl-CoA module (pJW001) was constructed by assembling three DNA fragments: (i) the /DL_0002 . Then, trevisiae , whereasananassa , F. vescF. vesca , and Acisp. ADP1 , respectA library of upstream and downstream modules was constructed to improve ethyl lactate biosynthesis through a combinatorial pathway optimization strategy using three different plasmids: (i) pACYCDuet-1 (P15A origin of replication), (ii) pETDuet-1 (ColE1 origin), and (iii) pRSFDuet-1 (RSF1030 origin), having the plasmid copy numbers of 10, 40, and 100, respectively .ldhA gene amplified from pJW001 using the primer pair JW_0001/JW_0002, (ii) the ethanol module containing pdc and adhB genes amplified from pCT24 using the primer pair JW_0003/JW_0004, and (iii) the backbone amplified from pACYCDuet-1 for pJW007, from pETDuet-1 for pJW008, or from pRSFDuet-1 for pJW009 using the primer pair JW_0005/JW_0006.The upstream modules (pJW007-009) were constructed by assembling three DNA fragments: (i) the pct gene amplified from pJW001 using the primer pair JW_0007/JW_0008, (ii) the VAAT gene amplified from pJW005 using the primer pair JW_0009/JW_0010, and (iii) the backbone amplified from pACYCDuet-1 for pJW010, pETDuet-1 for pJW011, or pRSFDuet-1 for pJW012 using the primer pair JW_0011/JW_0012.The downstream modules (pJW010-012) were constructed by assembling three DNA fragments: (i) the The combined upstream and downstream modules (pJW013-015) were constructed by assembling two DNA fragments: (i) the upstream module amplified from pJW007 using the primer pair JW_0001/JW_0004 and (ii) the backbone containing the downstream module amplified from pJW010 for pJW013, pJW011 for pJW014, or pJW012 for pJW015 using the primer pair JW_0005/JW_0006.For tighter regulation of biosynthetic pathway of ethyl lactate, we constructed the upstream and downstream modules with tunable promoters and RBSs.T7) was added between the multiple cloning site 1 (MCS1) and MCS2 of the pACYCDuet-1 backbone to create the first intermediate plasmid, pJW016, by assembling three DNA fragments: (i) the ldhA gene amplified from pJW001 using the primer pair JW_0013/JW_0014, (ii) the linker containing TT7 terminator amplified from pETite* using the primer pair JW_0015/JW_0016, and (iii) the backbone amplified from pACYCDuet-1 using the primer pair JW_0017/JW_0018. Next, the original T7 promoter (PT7) in MCS2 of pJW016 was replaced with the PAY1 (BBa_J23100) promoter and PAY3 (BBaJ23108) promoter to generate two second-intermediate plasmids, pJW017 and pJW018, respectively, by assembling two DNA fragments: (i) the ethanol module amplified from pCT24 under the PAY1 promoter for pJW017 or PAY3 promoter for pJW018 using the primer pair JW_0019/JW_0020 or JW_0021/JW_0020, respectively, and (ii) the backbone amplified from pJW016 using the primer pair JW_0022/JW_0012 or JW_0023/JW_0012, respectively. Last, the final four synthetic operons (pJW019-022) were constructed by assembling two DNA fragments: (i) the ethanol module amplified from pCT24 with the synthetic RBS sequences with predicted translation initiation rates of 0.33 a.u. for pJW019 and pJW021 and 0.03 a.u. for pJW020 and pJW022 using the primer pairs JW_0024/JW_0020, JW_0025/JW_0020, JW_0026/JW_0020, and JW_0027/JW_0020, respectively, and (ii) the backbone amplified from pJW017 for pJW019, pJW017 for pJW020, pJW018 for pJW021, and pJW018 for pJW022 using the primer pairs JW_0028/JW_0012, JW_0029/JW_0012, JW_0030/JW_0012, and JW_0031/JW_0012, respectively. The PAY1 and PAY3 promoter sequences were obtained from the iGEM Anderson promoter library  and the strength of promoters were assigned as PAY3 = 0.5 × PAY1. The RBS Calculator v2.0 [AY1 (or PAY3) promoter and pdc start codon  were constructed via three rounds of cloning. First, the T7 terminator  the pct gene amplified from pJW001 using the primer pair JW_0013/JW_0032, (ii) the linker containing TT7 terminator from pETite* using the primer pair JW_0033/JW_0034, and (iii) the backbone from pRSFDuet-1 using the primer pair JW_0017/JW_0018. Then, the original RBS in MCS1 of pJW023 was replaced with synthetic RBSs of various strengths to generate the second-intermediate plasmids, pJW024-026, by assembling two DNA fragments: (i) the pct gene amplified from pJW001 with the synthetic RBS sequences with predicted translation initiation rates at 90, 9000, or 90000 a.u. for pJW024, pJW025, or pJW026 using the primer pair JW_0035/JW_0036, JW_0037/JW_0036, or JW_0038/JW_0036, respectively, and (ii) the backbone amplified from pJW023 using the primer pair JW_0039/JW_0040 for pJW024, JW_0041/JW_0040 for pJW025, or JW_0042/JW_0040 for pJW026, respectively. Last, the final nine downstream modules (pJW027-035) were constructed by assembling a combination of two DNA fragments: (i) the VAAT gene amplified from pDL006 with the synthetic RBS sequences predicted with translation initiation rates of 90, 9000, or 90000 a.u. for pJW027/pJW030/pJW033, pJW028/pJW031/pJW034, or pJW029/pJW032/pJW035 using the primer pair JW_0043/JW_0010, JW_0044/JW_0010, or JW_0045/JW_0010, respectively, and (ii) the backbone amplified from pJW024, pJW025, or pJW026 for pJW027-029, pJW030-032, or pJW033-035 using the primer pair JW_0046/JW_0012, JW_0047/JW_0012, or JW_0048/JW_0012, respectively. The RBS Calculator v2.0 [T7 promoter and pct (or VAAT) start codon  were constructed via three rounds of cloning. First, the Ttor v2.0 , 55 was 7-035 werFor molecular cloning, seed cultures, and protein expression analysis, the Luria–Bertani (LB) medium, comprising 10 g/L peptone, 5 g/L yeast extract, and 5 g/L NaCl, was used. For high cell density cultures, pre-cultures of bioreactor batch fermentations, and growth inhibition analysis of lactate esters, the M9 hybrid medium [600nm) of 3 in M9 hybrid medium containing appropriate concentration of isopropyl-beta-d-thiogalatopyranoside (IPTG) and antibiotics. The resuspended cultures were distributed into 15-mL polypropylene centrifuge tubes  with a working volume of 5 mL and grown for 24 h (h) on a 75° angled platform in a New Brunswick Excella E25 at 37 °C, 200 rpm. The tubes were capped to generate anaerobic condition. All high cell density culture studies were performed in biological triplicates.For seed cultures, 2% (v/v) of stock cells were grown overnight in 5 mL of LB with appropriate antibiotics. For pre-cultures, 1% (v/v) of seed cultures was transferred into 100 mL of LB medium in 500-mL baffled flasks. For main cultures, pre-cultures were aerobically grown overnight , centrifuged , and resuspended to yield an optical density measured at 600 nm  dual 1.5-L fermentation system at a working volume of 1 L M9 hybrid medium. The seed and pre-cultures were prepared as described in high cell density cultures in LB and M9 hybrid media, respectively. For main cultures, 10% (v/v) of pre-cultures were inoculated into fermentation cultures. During the fermentation, to achieve high cell density, dual-phase fermentation approach , 56, aer®  to prevent evaporation of lactate esters and incubated at 37 °C with continuous shaking using a BioTek Synergy HT microplate reader . OD600nm was measured at 20-min intervals. Growth inhibition studies of lactate esters were performed twice in biological triplicates (n = 6).Seed cultures of EcDL002 were prepared as described in high cell density cultures. 4% (v/v) of seed cultures was inoculated into 100 µL of the M9 hybrid media, containing various concentrations (0.5–40 g/L) of lactate esters including ethyl-, propyl-, butyl-, isobutyl-, isoamyl-, or benzyl lactate, in a 96-well microplate. Then, the microplate was sealed with a plastic adhesive sealing film, SealPlate600nm of 0.6–0.8 with 0.5 mM of IPTG. After 4 h of induction, cells were collected by centrifugation and resuspended in 100 mM of sodium phosphate buffer (pH7.0) at the final OD600nm of 10. Cell pellets were disrupted using a probe-type sonicator  on ice–water mixture. The resulting crude extracts were mixed with 6× sodium dodecyl sulfate (SDS) sample buffer, heated at 100 °C for 5 min, and then analyzed by SDS–polyacrylamide gel electrophoresis . Protein bands were visualized with Coomassie Brilliant Blue staining.Seed cultures were prepared as described in high cell density cultures. 1% (v/v) of seed cultures subsequently inoculated in 500-mL baffled flasks containing 100 mL of LB medium. Cells were aerobically grown at 37 °C and 200 rpm and induced at an ODThe optical density was measured at 600 nm using a spectrophotometer . The dry cell mass was obtained by multiplication of the optical density of culture broth with a pre-determined conversion factor, 0.48 g/L/OD.2SO4 was used at a flow rate of 0.6 mL/min. Detection was made with the reflective index detector (RID) and UV detector (UVD) at 220 nm.Glucose, lactate, acetate, ethanol, isobutanol, isoamyl alcohol, and benzyl alcohol were quantified using the Shimadzu HPLC system  equipped with the Aminex HPX-87H cation exchange column  heated at 50 °C. A mobile phase of 10 mN HAll esters were quantified by GC/MS. For GC/MS analysis, analytes in the supernatants were extracted with dichloromethane (DCM), containing pentanol as an internal standard, in a 1:1 (v/v) ratio for 1 h at 37 °C, 200 rpm in 15-mL polypropylene centrifuge tubes. After extraction, supernatant–DCM mixtures were centrifuged and 5 μL of DCM extracts were injected into a gas chromatograph (GC) HP 6890 equipped with the mass selective detector (MS) HP 5973. For the GC system, helium was used as the carrier gas at a flow rate of 0.5 mL/min and the analytes were separated on a Phenomenex ZB-5 capillary column (30 m × 0.25 mm × 0.25 μm). The oven temperature was programmed with an initial temperature of 50 °C with a 1 °C/min ramp up to 58 °C. Next, a 25 °C/min ramp was deployed to 235 °C and then finally held a temperature of 300 °C for 2 min to elute any residual non-desired analytes. The injection was performed using a splitless mode with an initial injector temperature of 280 °C. For the MS system, a selected ion monitoring (SIM) mode was deployed to detect analytes.The SIM parameters for detecting lactate esters were as follows: (i) for pentanol, ions 53.00, 60.00, and 69.00 detected from 5.00 to 7.70 min, (ii) for ethyl lactate, ions 46.00, 47.00, and 75.00 detected from 7.70 to 10.10 min, (iii) for propyl lactate, ions 59.00, 88.00, and 89.00 detected from 10.10 to 11.00 min, (iv) for isobutyl lactate, ions 56.00, 57.00, and 59.00 detected from 11.00 to 11.60 min, (v) for butyl lactate, ions 75.00, 91.00, and 101.00 detected from 11.60 to 12.30 min, (vi) for isoamyl lactate, ions 46.00, 73.00, 75.00 from 12.30 to 14.50 min, and (vii) for benzyl lactate, ions 45.00, 91.00, and 180.00 from 14.50 to 15.08 min. The SIM parameters for detecting acetate esters were as follows: (i) for ethyl acetate, ions 45.00, 61.00, and 70.00 detected from 4.22 to 5.35 min, (ii) for propyl acetate, ions 57.00, 59.00, and 73.00 detected from 5.35 to 6.40 min, (iii) for pentanol, ions 53.00, 60.00, and 69.00 detected from 6.40 to 6.60 min, (iv) for isobutyl acetate, ions 56.00, 61.00, and 73.00 detected from 6.60 to 7.70 min, (v) for butyl acetate, ions 57.00, 71.00, and 87.00 detected from 7.70 to 9.45 min, (vi) for isoamyl acetate, ions 58.00, 70.00, and 88.00 detected from 9.45 to 13.10 min, and (vii) for benzyl acetate, ions 63.00, 107.00, and 150.00 from 13.10 to 15.82 min.t test.Statistical analysis was performed with SigmaPlot v.14 using the two-tailed unpaired Student’s Additional file 1: Table S1. Summary of high cell density cultures of EcJW101, EcJW102, EcJW103, EcJW104, and EcJW105 with addition of glucose and various alcohols after 24 h. The subscripts i and f are referred to the initial (0 h) and final (24 h) time of the culture, respectively. Table S2. Summary of titer, specific productivity, and yield of esters in high cell density cultures of EcJW101, EcJW102, EcJW103, EcJW104, and EcJW105 with addition of glucose and various alcohols after 24 h. The acyl acetate and acyl lactate columns correspond to the acyl alcohols added. For example, with the exogenous addition of ethanol, the acyl acetate, and acyl lactate columns represent ethyl acetate, and ethyl lactate, respectively. Table S3. (A) Summary of high cell density cultures of EcDL002, EcJW201, and EcJW202 after 24 h. (B) Summary of bioreactor batch fermentation of EcJW201 after 18 h. The subscripts i and f are referred to the initial (0 h) and final (24 h) time of the culture, respectively. Table S4. Summary of high cell density cultures of EcJW106-108 and EcJW203-208 with different concentrations of IPTG  after 24 h. The subscripts i and f are referred to the initial (0 h) and final (24 h) time of the culture, respectively. Table S5. Summary of titer, specific productivity, and yield of esters in high cell density cultures of EcJW106-108 and EcJW203-208 with different concentrations of IPTG  after 24 h. Table S6. Summary of high cell density cultures of EcJW209-212 with or without addition of ethanol (2 or 10 g/L) after 24 h. (A) OD600, pH, glucose, lactate, and ethanol. (B) Titer, specific productivity, and yield of esters. The subscripts i and f are referred to the initial (0 h) and final (24 h) time of the culture, respectively. Table S7. Summary of high cell density cultures of EcJW213-221 after 24 h. (A) OD600, pH, glucose, lactate, and ethanol. (B) Titer, specific productivity, and yield of esters. The subscripts i and f are referred to the initial (0 h) and final (24 h) time of the culture, respectively. Table S8. Summary of high cell density cultures of EcJW109-117 after 24 h. (A) OD600, pH, glucose, lactate, and ethanol. (B) Titer, specific productivity, and yield of esters. The subscripts i and f are referred to the initial (0 h) and final (24 h) time of the culture, respectively.Additional file 2: Figure S1. Expression of the recombinant enzymes in engineered E. coli strains. The positions corresponding to the overexpressed proteins are indicated by arrowheads. Lane M represents protein ladder while lanes T, S, and I are referred to total, soluble, and insoluble proteins, respectively. ①~③, Pyruvate-to-lactate ester module; ④~⑤, Ethanol module; ⑥~⑩, Isobutanol module. Protein sizes were predicted with their amino acids sequences. Figure S2. Effect of lactate esters on cell growth. (A) Specific growth rates of EcDL002 with or without addition of lactate esters. (B) logP values of characterized lactate esters. The values were obtained from http://www.thegoodscentscompany.com. (C–H) Growth curves of EcDL002 with or without addition of (C) n-ethyl lactate (NEL), (D) n-propyl lactate (NPL), (E) n-butyl lactate (NBL), (F) i-butyl lactate (IBL), (G) i-amyl lactate (IAL), and (H) benzyl lactate (BZL). Figure S3. Design of (A) upstream module and (B) downstream module of the ethyl lactate pathway. The RBS Calculator v2.0 software was used to generate synthetic RBS sequences. For the upstream, four synthetic RBS sequences were generated with predicted translation initiation rates at 0.33 and 0.03 between the PAY1 or PAY3 promoter and pdc start codon. For the downstream, six synthetic RBS sequences were generated with predicted translation initiation rates at 90, 9000, and 90000 a.u. between the PT7 promoter and pct or VAAT start codon. Figure S4. (A) Correlation between ester production and the amount of added ethanol in high cell density cultures of EcJW209-212. (B) Correlation between ester production and the RBS strength for VAAT expression in high cell density culture of EcJW213-221.


Ofloxacin and ketoconazole (10 μg/mL) were used as reference standards for antibacterial and antifungal activity, respectively. Compounds 11 and 12 showed notable antibacterial and antifungal activities at higher concentrations (125–200 μg/mL), whereas benzo[d]thiazole derivatives 13 and 14 were found to display significant antibacterial or antifungal activity (50–75 μg/mL) against the Gram+ve, Gram–ve bacteria, or fungal cells used in the present study. In addition, a correlation between calculated and determined partition coefficient (log P) was established which allows future development of compounds within this series to be carried out based on calculated log P values. Moreover, compounds 13 and 14 show that the optimum logarithm of partition coefficient (log P) should be around 4.A new series of 1,3-thiazole and benzo[ Compounds 13 and 14 sharing as a common feature the presence of a 4-hydroxyphenyl substituent at the 2-position of the benzo[d]thiazole nucleus displayed promising antibacterial activities (MIC 50–75 μg/mL) against all tested culture strains used in the present study, indicating that a structural modification of 1,3-thiazole heterocyle to benzannelated 1,3-thiazole derivatives positively influenced the antimicrobial activity. In contrast, the antimicrobial results observed for compound 15 (MIC 125 μg/mL) was clearly lower, pointing out that a monohydroxysubstitution is most favorable among the newly developed and tested series , as shown in 13–14 .The results show that correlations between lipophilic properties and MICs against different microbial species are not statistically significant a,b. The 1H-NMR spectra were recorded on a Bruker WC 300 spectrometer with tetramethylsilane (TMS) as internal standard. Chemical shifts are reported in ppm downfield from internal tetramethylsilane used as reference. 1H-NMR signals are reported in order: multiplicity , number of protons, and approximate coupling constants in Hertz. 13C-NMR spectra were recorded on a Bruker DPX 400 Avance (100 MHz) instrument. Chemical shifts are reported in ppm downfield from internal tetramethylsilane used as reference. Elemental analyses were performed on Perkin-Elmer 240B and 240C instruments. Analyses  indicated by the symbols of elements were within ±0.4% of the theoretical values. Chromatographic separations were done using a Chromatotron Model 7924 (Harrison Research) with 4-mm layers of silica gel 60 PF containing gypsum (Merck). EI-mass spectra were recorded using Finnigan MAT CH7A (70 eV), Finnigan MAT 711 (80 eV), or Kratos MS 25 RF (70 eV). +FAB-MS spectra were recorded on Finnigan MAT CH5DF instrument / glycerol). The following abbreviations are used: N,N-Dimethylformamide, DMF; EtOH, ethanol; Et2O, diethyl ether; MeOH, methanol; Me2SO, dimethyl sulfoxide; Ph, phenyl; Th, thiazole; Benz, benzene; BTH, benzothiazole.Melting points are uncorrected and were determined in open capillaries in a Buechi 512 Dr. Tottoli apparatus. 1, 1.49 g, 6.5 mmol), propionthioamide , and K2CO3  were dissolved in dry DFM (30 mL). The mixture was stirred for 4 h at 100–120 °C, cooled, and the solvent was removed under reduced pressure. The solid residue was recrystallized from EtOH. Yield 75%; mp 66–67 °C; 1H-NMR δ ppm = 1.34 , 2.99 , 3.79 , 6.99 , 7.74 , 7.87 ; EI-MS m/z (%) 220 .4-Methoxyphenacyl bromide , benzothioamide , and K2CO3  were dissolved in dry DMF (30 mL). The mixture was stirred for 2 h at 100–120 °C, cooled, and the solvent was removed under reduced pressure. The solid residue was recrystallized from EtOH. Yield 73%; mp 96.5–98 °C; 1H-NMR δ ppm = 3.82 , 6.68–6.90 , 7.35–7.38 , 7.44–7.48 , 7.84 , 7.86–7.90 ; EI-MS m/z (%) 268 .4-Methoxyphenacyl bromide , 4-methoxybenzothioamide , and K2CO3  were dissolved in dry DMF (30 mL). The mixture was stirred for 2 h at 100–120 °C, cooled, and the solvent was removed under reduced pressure. The solid residue was recrystallized from EtOH. Yield 73%; mp 93.5–95 °C; 1H-NMR δ ppm = 3.85 , 6.93–6.97 , 7.36–7.39 , 7.44–7.74 , 7.85 , 7.88–7.92 ; EI-MS m/z (%) 268 .Phenacyl bromide (o-aminothiophenol were added to polyphosphoric acid (15 g) and the mixture was refluxed for 4 h at 220 °C. The reaction mixture was cooled and poured in ice cold 10% sodium carbonate solution. The precipitate was filtered and recrystallised from methanol thiazole derivatives 14 and 15 with significantly improved in vitro antibacterial activity. A relationship could not be established between the compounds partition coefficient and the antibacterial activity, although an optimum log P of approximately 4 was observed which could be used to generate further structural optimization among this series. These results, combined with the potential benefits or at least differences in pharmacokinetics make the title 1,3-thiazole and benzo[d]thiazole classes interesting leads for future study of their mechanism of action and detailed structure-activity relationship studies. One can conclude that a comprehensive study by using new potent compounds of this series should be conducted in order to obtain more significant correlations between the chemical structures of the compounds and their antimicrobial activity. The new compounds


Background and Objectives: Radiotherapy uses ionizing radiation for cancer treatment. One of the side effects of radiotherapy is peripheral neuropathy. After irradiation, the first stage of neuropathy involves electrophysiological, biochemical and histopathological variations, while the fibrosis of soft tissues surrounding the exposed nerve occurs in the second stage. The present study aimed to examine the radioprotective effects of melatonin against ionizing radiation-induced sciatic nerve damage. Materials and Methods: Sixty male Wistar rats were assigned to four groups: C (Control + Vehicle), M (Melatonin), R (Radiation + Vehicle), MR (Radiation + Melatonin). Their right legs were irradiated with a 30 Gy single dose of gamma rays. Then, 100 mg/kg melatonin was administered to the animals 30 min before irradiation once daily (5 mg/kg) until the day of rats’ sacrifice. Their exposed nerve tissues were assessed using the sciatic functional index (SFI) and histological evaluation. Results: Four, 12 and 20 weeks post irradiation, the SFI results showed that irradiation led to partial loss of motor nerve function after 12 and 20 weeks. Histological evaluation showed the various stages of axonal degeneration and demyelination compared to the C and M groups. Scar-like tissues were detected around the irradiated nerves in the R group at 20 weeks, but were absent in the MR group. The SFI and histological results of the R group showed partial nerve lesion. However, in all cases, treatment with melatonin prevented these effects. Conclusions: Results showed that melatonin has the potential to improve functional and morphological features of exposed sciatic nerves. This could possibly improve the therapeutic window of radiotherapy. Radiotherapy is a treatment modality for cancer using ionizing radiation. It is administered to about 60% of cancer patients during the course of their treatment . Despite2− to hydrogen peroxide (H2O2), thereby preventing further production of free radicals /NPL. Toe spread factor (TSF): [experimental toe spread (ETS) − normal toe spread (NTS)]/NTS. Intermediary toe spread factor (ITF): [experimental intermediary toe spread (EIT) − normal intermediary toe spread (NIT)]/NIT.These parameters are presented in Applying the Bain et al. equation, we obtain: SFI = −38.3 (PLF) + 109.5 (TSF) + 13.3 (ITF) − 8.8 . Here, SFive rats in each group were sacrificed 4, 12 and 20 weeks after irradiation. Their sciatic nerves were isolated from surrounding tissues by separating the femoral biceps and gluteal muscles using blunt dissection, as shown in p value < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS software version 22 . Data were presented as the mean ± standard deviation (SD). Differences between groups were analyzed using two-way ANOVA, in addition to Tukey’s multiple comparison tests. Mann-Whitney test was used for nonparametric histopathological comparisons at each time point. p < 0.001). The average SFI values after 12 and 20 weeks were −20.51 ± 1.10 and −35.06 ± 2.00 in the R group, −6.98 ± 1.00 and −7.37 ± 0.35 in the C group as well as −7.06 ± 1.08 and −6.86 ± 0.58 in the M group, respectively. However, no significant effect was observed after 4 weeks (p > 0.05). After 12 and 20 weeks, the average SFI values were −8.13 ± 0.80 and −7.40 ± 1.01 in the MR group, respectively (p < 0.001). There was no significant difference between the SFI values of the C group compared with those of the MR group (p > 0.05). Only the R group was time-dependent (p < 0.001), as shown in Evaluation of the SFI showed that the R group had higher effects compared with the C and M groups after 12 and 20 weeks (p < 0.005) in terms of vasculature changes, degenerative changes, necrotic changes and swelling in myelin sheath. Scar-like tissues were detected significantly around the nerves in the R group compared with the C and M groups at 20 weeks post irradiation (p < 0.005), while treatment with melatonin prevented these effects in the MR group (p < 0.01). No significant finding was observed between the C and M groups and the MR group (p > 0.05), except for swelling at 4 weeks and degenerative changes, necrotic changes and swelling in myelin sheath at 12 weeks. There was time dependence in the R group (p < 0.05), hence suggesting melatonin as an efficient protective agent in all pathological conditions as shown in Four, 12 and 20 weeks post irradiation, the histological evaluation showed statistically significant differences between the R group and the C and M groups (p < 0.001). The average values of the C group were 54.61 ± 1.61 µm, 53.02 ± 0.83 µm and 55.95 ± 1.79 µm, respectively. In the M group, they were 53.64 ± 0.98 µm, 55.35 ± 0.94 µm and 57.09 ± 1.03 µm, respectively. In the R group, they were 62.09 ± 5.13 µm, 42.86 ± 3.59 µm and 38.18 ± 2.77 µm. Diameter measurements were significantly increased in the R group compared to the C and M groups at 4 weeks (p < 0.001), while they were significantly reduced in the R group compared to the C and M groups at 12 and 20 weeks (p < 0.001). Treatment with melatonin reversed these effects (p < 0.001). The mean values of the MR group were 56.83 ± 1.08 µm, 51.35 ± 4.13 µm and 55.30 ± 1.53 µm, respectively. There was no significant difference between the C and M groups (p > 0.05). Melatonin as well as radiation effects were both time-dependent (p < 0.01), except for in the MR group between 4 and 20 weeks (p > 0.05), as shown in Four, 12 and 20 weeks post irradiation, the axon as well as myelin sheath diameters in the R group were significantly different compared with those in the C and M groups (Radiotherapy is aimed at delivering the maximum radiation dose to kill tumor cells with minimal complications to normal tissues. However, normal tissues surrounding cancer cells are inevitably exposed to radiation, leading to early and late effects ,33. PeriRadioprotectors can be used to reduce the risk of side effects after radiotherapy . An apprOur results showed that irradiation led to partial loss of motor nerve function in terms of the SFI after 12 and 20 weeks. This finding agrees with previous studies which showed that the irradiation of peripheral nerves led to sensory and motor destruction ,8,34,35.Histopathological examination is traditionally considered a descriptive approach. However, in recent years, several studies have shown that it is possible to conduct morphometric or quantitative analysis in histological sections . HistoloRadiation-induced neuropathy may be mostly unavoidable in radiotherapy for cancer patients. However, melatonin has the ability to prevent radiotherapy-induced complications via its anti-apoptotic and anti-inflammatory effects as well as its ability to regulate physiological and pathological processes ,66. It iResults from this study have shown that melatonin has the potential to improve functional and morphological features of irradiated sciatic nerves. Hence, melatonin could be used to protect against radiotherapy-induced complications. Optimal and safe doses of melatonin should be administered over a long period of time for effective protection of the peripheral nerve tissues. This could go a long way in improving the therapeutic window of radiotherapy.


Herein, the Ti-doped LiNi0.8Co0.1Mn0.1O2 compounds are successfully synthesized. The Li(Ni0.8Co0.1Mn0.1)0.99Ti0.01O2 sample exhibits the best electrochemical performance. Under the voltage range of 2.7–4.3 V, it maintains a reversible capacity of 151.01 mAh·g−1 with the capacity retention of 83.98% after 200 cycles at 1 C. Electrochemical impedance spectroscopy (EIS) and differential capacity profiles during prolonged cycling demonstrate that the Ti doping could enhance both the abilities of electronic transition and Li ion diffusion. More importantly, Ti doping can also improve the reversibility of the H2-H3 phase transitions during charge-discharge cycles, thus improving the electrochemical performance of Ni-rich cathodes.Although LiNi The Ti doping can keep the layered structure materials with one phase. Among them, the Li(Ni0.8Co0.1Mn0.1)0.99Ti0.01O2 sample exhibits the best electrochemical performance. Within a voltage window of 2.7–4.3 V, the Li(Ni0.8Co0.1Mn0.1)0.99Ti0.01O2 sample maintains a reversible capacity of 151.01 mAh·g−1 with 83.98% capacity retention after 200 cycles, corresponding 0.08% decay per cycle. The improved electrochemical performance can be ascribed to the enhanced abilities of electronic transition and Li ion diffusion. More importantly, the reversibility of H2-H3 phase transitions and the lossless H3 phase during prolonged cycling can also improve the electrochemical performance of Ni-rich cathodes.In this work, the structural and electrochemical performances of Ti-doped LiNi0.8Co0.1Mn0.1O2 (denoted as NCM) spherical material was synthesized by solid-state method. Stoichiometric ratio of LiOH·H2O and Ni0.8Co0.1Mn0.1(OH)2 precursor were mixed and ground in an agate mortar. Then, under oxygen atmosphere, the mixtures were precalcined at 480°C for 5 h and calcined at 830°C for 12 h with a heating rate of 5°C·min−1. Next, the sample was cooled to room temperature naturally in a tube furnace.The pristine LiNi16H36O4Ti was dissolved into 60 mL absolute ethanol, then stirring continuously in water bath at the temperature of 60°C. Secondly, the LiOH·H2O were added to the solution and kept stirring for about 30 min, subsequently the Ni0.8Co0.1Mn0.1(OH)2 precursor was added. Afterward, the mixed solution was dried overnight at 120°C. Finally, the Ti-doped samples were obtained by calcining under the same condition as NCM.The 0.5, 1, 2 mol% Ti-doped NCM  samples were prepared by the following steps. Firstly, the stoichiometric amount of C−1 and operated at 40 kV and 40 mA. Scanning electron microscopy  was performed to observe the particle morphologies of samples. The microstructures of the NCM and Ti-1 samples were examined by high-resolution transmission electron microscopy .The crystalline structures of all the samples were determined by X-ray diffraction  using Cu Kα radiation in the 2θ range of 10–90° with a scan rate of 5°·min−2 with an electrode diameter of 12 mm. 1 M LiPF6 dissolved in a solution of dimethyl carbonate (DMC), ethylene carbonate (EC), and ethyl methyl carbonate (EMC) (volume ratio = 1:1:1) was used as the electrolyte. All the cells were assembled in an Ar-filled glovebox where the moisture and oxygen content is under 0.1 ppm by using a lithium metal anode and separator (Celgard 2500 polyethylene). Then, the cells were silenced before electrochemically tests. Electrochemical tests were performed on NEWARE BTS7.6 battery test system between 2.7 and 4.3 V vs. Li/Li+ at room temperature (25°C). The cycling performance was initially charged and discharged at 0.1 C (18 mA·g−1) with 2 cycles, then charged and discharged at 1 C in the subsequent cycles. The cyclic voltammetry (CV) was measured at a scan rate of 0.1 mV·s−1 within 2.7–4.5 V through an electrochemical workstation (Solartron 1470E). Electrochemical impedance spectroscopy (EIS) tests were conducted in the frequency range of 10−3-105 Hz.The electrochemical properties were measured using a CR2025 coin-type half cell. The working electrodes were prepared by mixing the active materials, acetylene black, and polyvinylidenedifluoride (PVDF) with a weight ratio of 8:1:1 in N-methyl-2-pyrrolidone (NMP). The resulting slurry was cast uniformly onto aluminum foil, followed by drying at 120°C in a vacuum oven for 4 h and pouched. The typical mass loading of positive electrode was about 2.3 mg·cm2 layered structure (PDF#70-4314) with R-3m space group, and no secondary phase is observed, indicating that Ti4+ is successfully incorporated into the bulk structure. Furthermore, the clear splitting of (006)/(102) and (108)/(110) couples for all samples manifest a well-developed layered structure. The partial magnified patterns in 4+. The result can also be proved by the increase of lattice parameter c and the cell volume as shown in The structure characteristics of the NCM and Ti-doped NCM samples are analyzed by XRD measurements as shown in The morphologies of the pristine NCM and Ti-1 samples are shown in −1 with the increasing of Ti amounts. However, the Ti-1 electrode shows the higher Coulombic efficiency of 83.20% than NCM electrode showing Coulombic efficiency of 80.82%, most probably owing to that suitable Ti doping can reduce the irreversible capacity.Electrochemical properties of the pristine and Ti-doped NCM samples are tested in lithium-ion half-cells at room temperature. −1 at high current rates of 10 C, while the NCM cathodes only deliver a specific capacity of 105.52 mAh·g−1. The remarkable rate property is mainly related to the slight enlargement along c axis by Ti doping, which benefits the fast Li ion diffusion. In addition, the cycling performances of all electrodes are compared at 1 C rate in the range of 2.7–4.3 V. As can be seen in −1 after 200 cycles, corresponding to 76.45% capacity retention with 0.118% decay per cycle. In comparison, the Ti-doped electrodes possess the higher capacity retention than pristine electrode, specifically, the Ti-1 electrode maintains 151.01 mAh·g−1 with 83.98% capacity retention after 200 cycles, corresponding 0.08% decay per cycle.The rate performances of the pristine and Ti-doped NCM electrodes at the current density ranging from 0.1 to 10 C are presented in To further understand the different charge-discharge behavior of the NCM and Ti-doped electrodes, the differential capacity (dQ/dV) curves are shown in ct) and the followed sloped line relates to the Warburg impedance (Zw). Moreover, the intercept with real axis (Z′) is assigned to the solution resistance (Rs). Differently, f) due to the solid electrolyte interface (SEI) film formed on the electrode during high-voltage cycling. The fitting results of the electrodes based on the equivalent circuit are summarized in ct of the NCM electrode in the fresh cell is 96.5 Ω, and the resistance increases dramatically after 200 cycles (1372 Ω). In comparison, the Ti-doped electrodes exhibit more stable values of Rct during cycling, implying better structural stability. According to the relationships between −1/2 in the low-frequency region (Li+) is calculated and the results are listed in Li+ values than that of the NCM electrode after 200 cycles, contributing to the best rate property among all electrodes. The EIS results demonstrate that the Ti-doped samples exhibit better kinetic behavior than those of the NCM, which can be related to the outstanding structural stability by the modification of Ti doping.To understand the relationship between electrochemical degradation and resistance parameters, EIS measurements are conducted for all samples in a charged state of 4.3 V after different cycle numbers. y region , the lit−1 between 2.7 and 4.5 V. To study the redox behavior associated with phase transition during battery operation, Cyclic voltammetry (CV) is employed at a scan rate of 0.1 mV·s0.8Co0.1Mn0.1O2. The Ti-doped LiNi0.8Co0.1Mn0.1O2  composites are successfully synthesized via one-step calcination method. Among them, the Li(Ni0.8Co0.1Mn0.1)0.99Ti0.01O2 sample exhibits the best electrochemical performance, it maintains a reversible capacity of 151.01 mAh·g−1 after 200 cycles at 1 C with 83.98% capacity retention. The superior electrochemical performance can be ascribed to two aspects: (1) the enhanced reversibility of H2-H3 phase transitions and the lossless H3 phase during prolonged cycling; (2) the lower electrochemical impedance and the improved Li-ion diffusion ability.To address the problem related to the structural degradations of NCM, Ti is intentionally introduced to enhance the structural stability and electrochemical performance of LiNiAll datasets generated for this study are included in the manuscript/supplementary files.LL, JieC, and HY: conceived the idea. JieC, HY, and LL: prepared all materials. JieC, HY, and TL: conducted SEM experiments. CL, JiaC, ZL, and LX: conducted XRD experiments. LL, JieC, and HY: analyzed the data. JieC and HY: wrote the manuscript. HT, ZC, and JD: commented on the manuscript. LL: supervised the implementation of the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health. These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. In contrast to O-glycosyltransferases, few C-glycosyltransferases (CGTs) have so far been characterized. Two different biosynthetic routes for C-glycosylated flavones have been identified in plants. Depending on the type of C-glycosyltransferase, flavones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyflavanone intermediate formed by a flavanone 2-hydroxylase (F2H).Saccharomyces cerevisiae, to produce some relevant CGT substrates, either the flavanones naringenin and eriodictyol or the flavones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyflavanone intermediates into the 6C-glucoside flavones isovitexin and isoorientin, and the 8C-glucoside flavones vitexin and orientin. Furthermore, we established direct glycosylation of flavones using the recently identified GtUF6CGT1 from Gentiana triflora. The ratio between 6C and 8C glycosylation depended on the CGT used. The indirect route resulted in mixtures, similar to what has been reported for in vitro experiments. In this case, hydroxylation at the flavonoid 3′-position shifted the ratio towards the 8C-glucosylated orientin. The direct flavone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides.In this study, we reconstructed the pathways in the yeast C-glycosides, and it provides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation.The current study features yeast as a promising host for production of flavone The online version of this article (10.1186/s12934-018-0952-5) contains supplementary material, which is available to authorized users. The NAR1 strain was based on the EVST28856 strain, which already had the naringenin pathway, comprising the genes AtPAL2, AmC4H, ScCPR1, At4C2L, MsCHI, and MdCHS, integrated , an ARS4/CEN6 cassette (from pEVE1968), two non-coding “stuffer sequence” cassettes (from pEVE2176 to pEVE27453), the AtCPR1 gene (from pEVE4012) cassette, and finally the EZ closing linker (from pEVE1916), which is used to close the plasmid as it fuses to pEVE4012 and pEVE4730  [PhF3′H (pEVE3999) [AtCPR1 and AmFNSII) pEVE2176 was replaced by pEVE23312. For the ERI1 strain (containing AtCPR1 and PhF3′H) pEVE27453 was replaced by pEVE3999. For the LUT1 strain , pEVE2176 and pEVE27453 were replaced by pEVE23312 and pEVE3999, respectively. The four strains were cultured for 72 h and analysed for production of the expected flavonoids.The NAR1 strain was created by in vivo assembly of a low-copy HRT vector comprising the VE23312)  and PhF3EVE3999)  substitup-coumaric acid, between 18 and 19 mg/L in the NAR1 and ERI1 strains, and 12–13 mg/L in API1 and LUT1 strains, but no other direct intermediates were detected.The NAR1 strain produced 124.91 ± 6.94 mg/L naringenin, the ERI1 strain produced 133.43 ± 1.63 mg/L eriodictyol, and the API1 strain produced 80.74 ± 11.38 mg/L apigenin  OsCYP93G2 [Sorghum bicolor) SbCYP93G3 [Zea mays) ZmCYP93G5 [C-glycosylation.In some monocots such as the cereals rice, sorghum, and maize, avonoids , and recsCYP93G2 , the sorbCYP93G3 , and themCYP93G5  were selO-glycosylating activity on 2-hydroxylated flavanones [C-glycosides, both of the 6C and 8C types, and it seems that plants acquired the ability to C-glycosylate 2-hydroxyflavanones before the monocot–dicot species split [Fagopyrum esculentum) and the UGT708D1 [Glycine max), with no reported O-glycosylation activity, were chosen for producing the four corresponding C-glycosides from either 2-hydroxynaringenin or 2-hydroxyeriodictyol.Although CGTs have been identified from the monocots rice and maize, the latter of these has been reported to also show avanones . Howeveres split . Hence, es split  from bucUGT708D1  from soyC-glucosides  and vitexin (apigenin-8C-glucoside)  or SbCYP93G3 (strain NCG3).NCG strains were analysed for naringenin and the corresponding C-glucosides isoorientin (luteolin-6C-glucoside) and orientin (luteolin-8C-glucoside), as well as other potential intermediates of this pathway. As seen in Fig. C-glycosylated orientin versus the 6C-glycosylated isoorientin. In strain ECG2, for example, a level of 67.03 ± 2.92 mg/L orientin was reached, compared to 29.19 ± 0.24 mg/L of isoorientin.ECG strains were similarly analysed for eriodictyol and the corresponding The ECG strains also accumulated products derived from naringenin via 2-hydroxynaringenin, i.e. more than approx. 50 mg/L isovitexin and 48 mg/L vitexin  and isoorientin (luteolin-6C-glucoside) of more than 60 mg/L (64.24 ± 0.51 mg/L) and 30 mg/L (31.47 ± 0.38 mg/L), respectively. Less than 4 mg/L of orientin was detected and essentially no vitexin. More than 63 mg/L of luteolin, and close to 6 mg/l apigenin accumulated in the culture broth  in S. cerevisiae. Figure S4. Quantification of vitexin and isovitexin. Table S1. Synthetic and codon optimized genes, used in this study. Table S2. Main plasmids used in this study. Table S3. Indirect biosynthetic pathway. Table S4. Additional intermediates of the indirect biosynthetic pathway. Table S5. Direct biosynthetic pathway.


Penicillium oxalicum HP7-1 isolated by our laboratory secretes RSDG with suitable properties but at low production levels. Here, we screened and identified novel regulators of RSDG gene expression in P. oxalicum through transcriptional profiling and genetic analyses.Raw-starch-digesting glucoamylases (RSDGs) from filamentous fungi have great commercial values in starch processing; however, the regulatory mechanisms associated with their production in filamentous fungi remain unknown. Penicillium oxalicum HP7-1 transcriptomes in the presence of glucose and starch, respectively, used as the sole carbon source were comparatively analyzed, resulting in screening of 23 candidate genes regulating the expression of RSDG genes. Following deletion of 15 of the candidate genes in the parental P. oxalicum strain ∆PoxKu70, enzymatic assays revealed five mutants exhibiting significant reduction in the production of raw-starch-digesting enzymes (RSDEs). The deleted genes , were the first report to regulate RSDE production of P. oxalicum. Further analysis revealed that ∆POX01907 lost the most RSDE production (83.4%), and that POX01907 regulated the expression of major amylase genes, including the RSDG gene POX01356/PoxGA15A, a glucoamylase gene POX02412, and the α-amylase gene POX09352/Amy13A, during the late-stage growth of P. oxalicum.POX01907 encoding a transcription factor in controlling the production of RSDE, regulating the expression of an important RSDG gene POX01356/PoxGA15A, in P. oxalicum. These results provide insight into the regulatory mechanism of fungal amylolytic enzyme production.Our results revealed a novel essential regulatory gene The online version of this article (10.1186/s13068-018-1345-z) contains supplementary material, which is available to authorized users. Starch is an important renewable carbohydrate biosynthesized in large quantities through plant photosynthesis. Starch biorefinery can provide a variety of useful chemicals, including biofuels. In traditional starch biorefinery, amylases, including α-amylase EC 3.2.1.1) and glucoamylase (EC 3.2.1.3), are used to hydrolyze starch to glucose  database) were generated from each sample, with > 90% mapped into the genome of P. oxalicum wild-type strain HP7-1  involved in the citric acid cycle (TCA cycle). The transcript levels of these DEGs, except for POX02392 and POX03958, were upregulated in ∆POX01907 by 105.6%–255.3%  and 61 were upregulated (1.0 < log2 fold change < 4.5)   reported to regulate cellulase gene expression in P. oxalicum /Myb domains [SANT/Myb833–881  and SANT/Myb1086–1134 ] , respectively. The functions of PDE_09981 in P. oxalicum 114-2 and AN8076.2 in A. nidulans FGSC A4 (XP_681345.1) are unknown.The POX01907 protein contains 1794 amino acids and two SANT  was added and mixed. Phenol–chloroform was used to remove proteins. Total DNA was separated and collected by centrifugation at 11,300g for 10 min a Trizol RNA kit  was used to extract total RNA according to manufacturer instructions.Extraction of total DNA and RNA from eviously . BrieflyP. oxalicum was performed according to methods reported by Zhao et al. [PoxKu70 protoplasts, and selected transformants were further confirmed by PCR and/or Southern blot using specific primer pairs and/or probes  used as the integrative locus in the genome, 1.2 kb of a DNA fragment encoding the bleomycin-resistance gene, and 7.8 kb of the complementary gene containing the promoter, coding region, and terminator. These four DNA fragments were amplified by PCR using specific primer pairs .Equal amounts of fresh spores from 8 spores from P. oxalicum strains, including the parental strain ∆PoxKu70 and the deletion mutant ∆POX01907, were inoculated into 100 mL of glucose or starch liquid medium, respectively, and cultured at 28 °C for 12 h–72 h. The hypha was harvested using a vacuum filter every 12 h and dried to a constant weight at 50 °C.Fresh 1.0 × 10P. oxalicum strains was used to construct cDNA libraries, with each cDNA having an average length of 100 bp. The constructed cDNA libraries were subjected to evaluation using an Agilent 2100 Bioanalyzer  and an ABI StepOnePlus real-time PCR system , and subsequently sequenced using an IIIumina HiSeq 4000 system . After quality control, the generated clean reads were mapped onto the P. oxalicum HP7-1 genome and functionally annotated using BWA v0.7.10-r789 (http://sourceforge.net/projects/bio-bwa/files/) and Bowtie2 v2.1.0 [RNA-seq analysis was performed according to methods described by Zhao et al. . Total R2 v2.1.0 . Gene-ex2 v2.1.0 , and DEG2 v2.1.0 . Pearson∆POX01907 and the parental strain ∆PoxKu70 was performed as previously described [PstI . After separation on an 0.8% agarose gel, the generated DNA fragments were transferred to a Hybond-N+ nylon membrane . The probe used for Southern hybridization was amplified with the primers POX01907-probe-F and POX01907-probe-R . Each qPCR comprised a 20-μL volume, including 2.0 μL of the template for first-stand cDNA, 10 μL of SYBR Premix ExTaq II, 0.8 μL of 10 μM primer (either forward or reverse), and 6.4 μL of sterile water, subjected to initial denaturation for 3 min at 98 °C, followed by 40 cycles of 10 s at 98 °C and 30 s at 58 °C. Fluorescent signals were investigated at the end of each extension step at 80 °C according to the method described by Zhang et al. [RT-qPCR was used to analyze differences in the expression levels of amylase genes between the deletion mutant ∆d method . Total RP. oxalicum strains, including the deletion mutant ∆POX01907, the complementary strain CPOX01907, and the parental strain ∆PoxKu70, was added to 0.1 M citric acid/disodium hydrogen phosphate buffer (pH 4.5) containing 1.0% (w/v) raw cassava flour as the substrate, and the mixture was incubated at 65 °C for 30 min. Inactivated crude enzyme extract was used as the blank control. The generated reducing sugars were measured using the 3,5-dinitrosalicylic acid method [RSDE activity and concentration were measured as described previously . Brieflyd method  at 540 nP. oxalicum strains was measured using a Bradford assay kit  according to manufacturer instructions.Intracellular enzyme concentration in https://www.ncbi.nlm.nih.gov/) website following BlastP analyses (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). The phylogenetic tree was constructed using MEGA 7.0 software [POXO1907 homologs were downloaded from the NCBI (software  with a nP < 0.05 or P < 0.01) among samples was calculated using Student’s t test.Microsoft Excel  was used for the statistical analysis of all experimental data associated with enzyme production and gene transcription. Significance .All transcriptomic data are available from the SRA database (Accession Number SRP116594). DNA sequences of Additional file 1: Table S1. Summary of RNA-seq reads obtained for Penicillium oxalicum HP7-1.Additional file 2: Figure S1. Pearson’s correlation coefficient of the transcriptomes of Penicillium oxalicum HP7-1 among three biological replicates. P. oxalicum HP7-1 was cultivated in media containing glucose or soluble corn starch for 4 h after a shift from glucose.Additional file 3: Table S2. List of 916 differentially expressed genes in Penicillium oxalicum HP7-1 grown in the presence of starch as compared with that in the presence of glucose.Additional file 4: Table S3. List of 23 candidate regulatory genes determined in this study that regulate raw starch-degrading enzymes production in P. oxalicum HP7-1.Additional file 5: Figure S2. Confirmation of deletion of 11 candidate genes derived from ∆PoxKu70 and the complementary strain. a PCR analysis. M, 1-kb DNA marker; lanes 1–3, three transformants for each candidate gene; lane 4, ∆PoxKu70; and lane 5, ddH2O. The top panel shows amplification of the region to the left of the target gene, the middle panel shows amplification of the region to the right of the target gene, and the bottom panel shows amplification of the region of the target gene. b Southern hybridization analysis of the deletion mutant ∆POX01907. M, 1-kb DNA marker; lane 1, ∆PoxKu70; lane 2, ∆POX01907-7; lane 3, ∆POX01907-9; and lane 4, ∆POX01907-15. c PCR confirmation of the complementary strain CPOX01907. M, 1-kb DNA marker; lane 1, CPOX01907; lane 2, ∆PoxKu70; and lane 3, ddH2O. The top panel shows amplification of the bleomycin-resistance gene, and the bottom panel shows amplification of complementary cassette.Additional file 6: Table S4. Primers used in this study.Additional file 7: Figure S3. Pearson’s correlation analysis of the transcriptomes of Penicillium oxalicum strains ∆POX01907 and ∆PoxKu70 grown in medium containing soluble corn starch as the carbon source.Additional file 8: Table S5. List of 1003 genes differentially expressed in ∆POX01907 as compared with the parental strain ∆PoxKu70 grown on soluble corn starch as the sole carbon source.


Antrodia cinnamomea (AC), an edible Taiwanese mushroom, has been recognized as a valuable natural resource with vast biological and medicinal benefits. Recently, the hypoglycemic and anti-diabetic effects of AC were mentioned in several studies. However, no studies have investigated α-glucosidase inhibitors from AC fruiting bodies (ACFB) as they relate to type 2 diabetes (T2D) treatment. The purpose of this study was to gain evidence of potent α-glucosidase inhibitory effects, as well as isolate, identify and characterize the active compounds of ACFB. The MeOH extract of ACFB demonstrated potent α-glucosidase inhibitory activity, and possessed high pH stability (pH 2–11) and thermostable properties at 40–50 °C. Further purification led to the isolation of eight constituents from ACFB, identified as: 25S-antcin K (1), 25R-antcin K (2), dehydrosulphurenic acid (3), 25S-antcin I (4), 25S-antcin B (5), 25R-antcin B (6), dehydroeburicoic acid (7) and eburicoic acid (8). Notably, the ACFB extract and its identified compounds, except 1, 4, and 6 demonstrated a greater effect (EC50 = 0.025–0.21 mg/mL) than acarbose (EC50 = 0.278 mg/mL). As such, these active compounds were determined to be new potent mushroom α-glucosidase inhibitors. These active compounds were also identified on the HPLC fingerprints of ACFB. The incidence of diabetes mellitus (DM), a chronic metabolic disorder, has been dramatically increasing and reducing people’s quality of life worldwide . People aGIs may be obtained from several common natural sources, including medicinal herbs, microbial conversion, edible and medicinal mushrooms ,18,19,20Antrodia cinnamomea (AC), an edible Taiwanese mushroom, was recognized early on for its valuable use in Chinese folk medicine to treat diarrhea, hypertension, allergies, abdominal pain, food and drug intoxication, skin itching and tumorigenic diseases ,,1, 2 [7  2 [7 [8  also shotivities . However18-AR-II column under the same conditions. The eight isolated compounds were successfully recognized in the HPLC fingerprints of ACFB extract at the retention times (RT) of 7.282 min (1), 7.543 min (2), 20.896 min (3), 21.122 min (4) 22.069 min (5), 22.084 min (6) 31.425 min (7), and 32.467 min (8)  .Almost all of the active inhibitors could be clearly observed in the HPLC fingerprints of the crude extract. These results indicate that they should be major constituents of ACFB extract. As such, HPLC analysis coupled with inhibitory assay may be a convenient, rapid and precise method to standardize ACFB extracts from different sources in order to select good material for T2D management.50 (mg/mL) and inhibition (%), as presented in 3), dehydroeburicoic acid (7) and eburicoic acid (8), demonstrated the greatest effect against α-glucosidase due to their small EC50 values , and greatest max inhibition at 0.25 mg/mL . The compounds 25R-antcin K (2) and 25S-antcin B (5) were also potent inhibitors since their activity was stronger than that of the control, while compound 1 showed the weakest activity.To evaluate the most potent aGI compounds, all identified compounds and acarbose were tested for activity over a large concentration range of 0.02–0.25 μg/mL. Activity was expressed as EC8) ≥ dehydroeburicoic acid (7) ≥ dehydrosulphurenic acid (3) ≥ 25R-antcin K (2) ≥ 25S-antcin B (5) ≥ acarbose ≥ 25S-antcin K (1), where “≥” indicates that there is no significantly higher inhibitory activity between the two inhibitors, based on Duncan’s multiple range test. The results indicate that almost all inhibitors isolated from ACFB extract showed higher activity than acarbose. As such, this edible medicinal mushroom may have potential as a safe and natural source of aGIs for effective management of T2D.The potent inhibitory activities of the tested inhibitors, in order of decreasing activity, are as follows: Eburicoic acid  possessed high pH stability with great relative activity of 80–117%. Compound 1 demonstrated high stability in acidic pH treatment (pH 2–4) but very weak activity in the pH range treatment from 5–11, while compound 2 showed good activity in the alkaline pH but weak activity in acidic condition treatment. It was suggested that pH stability is an important characteristic that should be considered when evaluating aGIs. A potential aGI should have acceptable or high pH stability, especially at acidic pH, since the pH in the gastrointestinal tract (stomach) is normally acidic [Euonymus laxiflorus Champ which showed low relative activity of 48% at pH 4 and aGIs from Dalbergia tonkinensis which demonstrated an acceptable relative activity of 80–83% at pH 2–4, while fermented nutrient broth and fermented squid pens by Paenibacillus sp. had potent relative activities of 86–97% (pH 1–4) and 89–95% (pH 2–4), respectively. In this study, ACFB extract and its purified compounds, except compound 2, demonstrated comparable or higher stability than those of previous reports with a relative activity of 85–110% at an acidic pH of 2–4.To determine pH stability of y acidic . Recentl2 and 7 showed good thermal stability at all the treated temperatures with high relative activity of 96–108 °C. However, ACFB extract and other identified compounds were only stable at 40–50 °C with relative activity of 70.2–93.3%. The results suggest that ACFB should be not treated higher than 50 °C to obtain aGIs for use in folk medicine or to prepare the extract for purification, since it may reduce significant inhibitory activity. In comparison, aGIs from ACFB except 2 and 7 demonstrated lower thermal stability (22.7–93.3%) than those of aGIs from E. laxiflorus Champ. (75–100%) [Paenibacillus sp. (91–99%) [The thermal stability of aGIs from ACFB was evaluated by pre-treating samples to a range of temperatures 40–100 °C) for 30 min, before inhibition was tested at 37 °C by bioassay. The results are presented in 75–100%)  or squid00 °C forA. cinnamomea were collected from the mountains of Kavulungan, Taitung, Taiwan in March 2017. Saccharomyces cerevisiae α-glucosidase and acarbose were purchased from Sigma Chemical Co., St. Louis City, MO, USA. The substrate pNPG was purchased from Sigma Aldrich, St. Louis, MO, USA. Solvents, reagents and other chemicals were obtained at the highest grade availableThe fruiting bodies of p-NPG (10 mmol/L) was added to each mixture to start a reaction. The mixture was kept at 37 °C for 30 min before 100 μL Na2CO3 (1 mol/L) was added to stop the reaction. The final mixture was then measured at 410 nm (A). The control group was also prepared as described above but with the use of 50 μL, 0.1 mol/L potassium phosphate buffer (pH 7) instead of the sample solution; its absorbance was also recorded at 410 nm (B). The α-glucosidase inhibition (%) was estimated using the following equation:The α-glucosidase inhibition was performed following the assay methods described by Nguyen et al. (2018) , with sl50 value, which is defined as the concentration of inhibitor that inhibits 50% of enzyme activity [Inhibition activity was also expressed as an ECactivity . The cru5D5N (pyridine-d5) as solvent. Silica gel 60  were used for column chromatography, and pre-coated silica gel (Merck 60 F-254) plates were used for TLC. The spots on TLC were detected by spraying with an anisaldehyde-sulfuric acid solution and then heating at 100 °C. HPLC separations were performed on a Shimadzu LC-2040C series apparatus  with a photodiode array detector, equipped with a 250 × 4.6 mm i.d. preparative Cosmosil 5C18 AR-II column .General experimental procedures: High resolution electronic ionization mass spectrometry (HREIMS) data were measured using a Shimadzu IT-TOF HR mass spectrometer . Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AC-400 FT-NMR  using CA. cinnamomea fruiting bodies (200 g) were extracted five times with MeOH (10 L) at 50 °C for 12 h, then concentrated under reduced pressure. The MeOH extract (45.2 g) was separated by silica gel flash column  with a gradient solvent system of CH2Cl2 100% to MeOH 100%, to provide 12 fractions (ACFB.1–ACFB.12). ACFB.3 (423.2 mg) was purified by preparative HPLC  to produce 25S-antcin K  and 25R-antcin K . ACFB.5 (865.2 mg) was purified by preparative HPLC  to produce four subfractions (ACFB.5.1–ACFB.5.4). ACFB.5.2 (232.1 mg) was further purified by recycled preparative HPLC  to produce dehydrosulphurenic acid  and 25S-antcin I . ACFB.6 (765.2 mg) was purified by preparative HPLC  to produce two subfractions: ACFB.6.1 and ACFB.6.2. ACFB.6.1 was further purified by recycled preparative HPLC  to produce 25S-antcin B  and 25R-antcin B . ACFB.9 (450.6 mg) was purified by preparative HPLC  to produce dehydroeburicoic acid  and eburicoic acid .Extraction and isolation: Dried 18-AR-II, 5 μm, 250 × 4.6 mm i.d.) was used, while eluting at a flow rate of 1.0 mL/min at 35 °C. The mobile phase consisted of water containing 0.1% phosphoric acid and acetonitrile (ACN), using a gradient program of 40−50% ACN from 0−12 min, 50−60% ACN from 12−17 min, 60−95% ACN from 17−26 min and 95−100% ACN from 26−50 min. The real-time UV absorption was detected at 210 nm. Each isolated compound was accurately weighed and dissolved in MeOH; the terminate concentration was ca. 1.0 mg/mL. ACFB extract was dried under vacuum, accurately weighed to about 10 mg, then dissolved in MeOH, in a 1.0 mL volumetric flask. The sample solutions were all filtered with 0.45 μm PVDF membrane filter  before use. The injection volumes of the compound and ACFB were 1 μL and 10 μL, respectively.Separation column   with sli50 values was analyzed with the use of SAS  version 9.4, provided by SAS Institute Taiwan Ltd. , using Duncan’s multiple range test (α = 0.01). All tests were performed in triplicate (n = 3).The differences between the means of inhibition (%) and ECR-antcin K (2), dehydrosulphurenic acid (3), 25S-antcin B (5), dehydroeburicoic acid (7) and eburicoic acid (8), were successfully isolated and identified from ACFB, showing stronger α-glucosidase inhibitory effect and higher activity (EC50 = 0.025–0.21 mg/mL) than acarbose (EC50 = 0.278 mg/mL). Notably, these novel compounds were investigated for the first time for their α-glucosidase inhibitory effect in this study. The results of this study contributed to the catalogue of novel biological activities of AC, as well as its constituents. The results also suggest that ACFB is a highly rich, safe and natural source of bioactive constituents that may be developed as drugs or health foods with potential antidiabetic effects.The MeOH extract of ACFB was investigated for the first time for its potent in-vitro antidiabetic effect, characterized using an α-glucosidase inhibitory activity assay. ACFB demonstrated high pH stability (pH 2–11) and thermostable properties at 40–50 °C. Five active compounds, including 25


Several subunits are upregulated  and downregulated (e.g. Kvβ) in cancer, while other subunits have been functionally implicated as oncogenes  and tumour suppressor genes  based on in vivo studies. The strengthening link between ion channel auxiliary subunits and cancer has exposed these subunits as potential biomarkers and therapeutic targets. However further mechanistic understanding is required into how these subunits contribute to tumour progression before their therapeutic potential can be fully realised.Several superfamilies of plasma membrane channels which regulate transmembrane ion flux have also been shown to regulate a multitude of cellular processes, including proliferation and migration. Ion channels are typically multimeric complexes consisting of conducting subunits and auxiliary, non-conducting subunits. Auxiliary subunits modulate the function of conducting subunits and have putative non-conducting roles, further expanding the repertoire of cellular processes governed by ion channel complexes to processes such as transcellular adhesion and gene transcription. Given this expansive influence of ion channels on cellular behaviour it is perhaps no surprise that aberrant ion channel expression is a common occurrence in cancer. This review will focus on the conducting and non-conducting roles of the auxiliary subunits of various Ca A . A 2+ chith Cavα . Cavα1 sn cancer . Researc2.11 DI-DII intracellular linker region ), which localise to the brain i) [2+]i alone, however the required membrane potential (V1/2 = 168 mV at [Ca2+]i = 0) or [Ca2+]i  are out of physiological range i but enhancing activation at high [Ca2+]i [1 hyperpolarises channel activation to such an extent (−140 mV in LNCaP prostate cancer cells) that BK channels open without the need for increased [Ca2+]i at resting membrane potentials [Large conductance Ca[Ca2+]i) . BK chanal range . BK chanal range , pain moal range  and blooal range . BK chanal range ,186. BK sitivity , BKβ2 hytivation , BKβ3 detivation  and BKβ4 [Ca2+]i . BKγ sub [Ca2+]i . BKγ1 hytentials .1 implicated. There are conflicting reports on the involvement of BKγ1  in cancer. BKγ1 is upregulated in the MDA-MB-456 breast cancer cell line and in metastatic secondary breast cancer tumours compared to the primary tumour of a single patient 1-4) [2+]i [2+]i also increases ICl and accelerates current onset 1-4) ,291. IntLCA]1-4) . CaCCs d4) [2+]i . Increasnt onset . CaCCs ant onset , smooth nt onset  and fluint onset . Expresset [CaCC ,300. MorCC [CaCC . CLCA1 cCC [CaCC . Despiteor Best1 . Insteadepletion .in vivo [in vivo [Cl independent, as inhibiting ICl has a further anti-migratory effect in cells expressing CLCA2 as well as having an anti-migratory effect in cells not expressing CLCA2 [in vivo via adhesive interactions between endothelial CLCA and β4 integrin expressed on circulating cancer cells [CLCAs have a well-documented tumour-suppressive role . CLCA1 iin vivo . CLCA2 ein vivo . Express[in vivo ,312,315.[in vivo . At a ce[in vivo , decreas[in vivo , inhibit[in vivo , induces[in vivo ,318, inh[in vivo  and indu[in vivo . The abing CLCA2 . Sequestng CLCA2 ,321,322.ng CLCA2 ,322. Deser cells ,324. Simer cells ,326, sug6vβs, or downregulated, e.g. Kvβs, in tumours and thus may represent novel cancer biomarkers. in vitro and in vivo experimentation has further implicated various auxiliary subunits in tumour formation and progression, such as Navβ1 and α2δ1 (Vβ3/4, may function as tumour suppressors. Clearly, it is important from a treatment perspective to understand the mechanistic function of ion channel auxiliary subunits, including the extent that they contribute to cancer progression through potentiating ion conductance or via non-conducting signalling. For example, α2δ1- and α2δ2-induced Ca2+ influx may promote hepatocellular carcinoma cell sphere formation and pancreatic adenoma proliferation respectively [Vα-dependent, NaVβ1-mediated process outgrowth and the extent of glibenaclamide-induced inhibition of SUR2-mediated cancer cell proliferation correlating with the mRNA expression of Kir6.2 [vβ1 and KChIP3 [vβs [Many ion channel auxiliary subunits are upregulated, e.g. Caand α2δ1 . Howeverectively ,83. Othef Kir6.2 ,249. Valf Kir6.2 . On the d KChIP3 ,172. VarIP3 [vβs ,278,316.The authors declare that they have no conflicts of interest.