Reductase Reaction Research Articles

Metabolic and enzymatic engineering approach for the production of 2-phenylethanol in engineered Escherichia coli

2-Phenylethanol, known for its rose-like odor and antibacterial activity, is synthesized via exogenous phenylpyruvate by the sequential reaction of phenylpyruvate decarboxylase (PDC) and aldehyde reductase. We first targeted ARO10, a phenylpyruvate decarboxylase gene from Saccharomyces cerevisiae, and identified a suitable aldehyde reductase gene. Co-expression of ARO10 and yahK in E. coli transformants yielded 1.1 g/L of 2-phenylethanol in batch culture. We hypothesized that there might be a bottleneck in PDC activity. The computer-based enzyme evolution was utilized to enhance production. The introduction of an amino acid substitution in ARO10 (ARO10 I544W) stabilized the aromatic ring of the phenylpyruvate substrate, increasing 2-phenylethanol yield 4.1-fold compared to wild-type ARO10. Cultivation of ARO10 I544W-expressing E. coli produced 2.5 g/L of 2-phenylethanol with a yield from glucose of 0.16 g/g after 72 h. This approach represents a significant advancement, achieving the highest yield of 2-phenylethanol from glucose using microbes to date.

NADPH-dependent peroxidase activity of antibodies in patients with schizophrenia

IntroductionThe development of oxidative stress in patients with schizophrenia is associated with changes in the level of activity of antioxidant enzymes. It is likely that catalytically active antibodies (abzymes) can take on these functions. Abzymes are antibodies with enzymatic activity. Catalase and SOD activity of abzymes was previously detected in patients with schizophrenia. But NADPH-dependent peroxidase activity has not been studied. The present work discusses the protective role of abzymes against reactive oxygen species within the pathogenesis of schizophrenia.ObjectivesThe aim of the study was to investigate the NADPH-dependent peroxidase activity of IgG in patients with paranoid schizophrenia in the exacerbation phase and in the remission phase.MethodsA total of 124 patients were examined during the work. Of them, 82 patients with paranoid schizophrenia (F20.0) had a mean age of 33.6±5.12 years (52 males, 30 females), disease duration averaged 8.9± 4.62 years. Patients with schizophrenia included 42 patients with acute schizophrenia and 40 patients with schizophrenia in therapeutic remission. The control group included 42 sex- and age-matched patients. IgG was purified by affinity chromatography on columns with proteinsepharose on an AKTA purifier chromatograph (GE). The homogeneity of isolated IgG preparations was checked by Lemilly electrophoresis in a gradient of 4-18% PAAG. Gel filtration under pH-shock conditions was performed on a Superdex-200 HR 10/30 column. NADPH-dependent peroxidase activity of IgG was determined on a SPECORD M-40 spectrophotometer (Carl Zeiss) at 340 nm by NADPH oxidation in the conjugated glutathione reductase reaction of tertiary butyl hydroperoxide reduction. Statistical processing of data was performed in Statistica 12.0 program.ResultsIt was proved that IgG from patients with schizophrenia had NADPH-dependent peroxidase activity, and this activity is an intrinsic property of the investigated antibodies. The NADPH-dependent peroxidase activity in IgG patients in the exacerbation stage was increased 3-fold (p=0.0001) compared to the studied activity in the group of healthy individuals, and it was increased 2-fold (p=0.017) in the group of patients in therapeutic remission compared to the activity in healthy individuals. Also NADPH-dependent IgG peroxidase activity in patients in remission was 1.7 times lower than in patients during the exacerbation period (p=0.012).ConclusionsIt was established for the first time that abzymes from patients with schizophrenia and healthy individuals have NADPH-dependent peroxidase activity and can decompose lipo and hydroperoxides. We hypothesize that these abzymes help cope with generalized oxidative stress. Under the influence of neuroleptic therapy in patients in remission, the level of oxidative stress and NADPH-dependent peroxidase activity of abzymes decrease.Disclosure of InterestNone Declared

Cloning and Functional Characterization of NADPH-Cytochrome P450 Reductases in Aconitum vilmorinianum

Diterpenoid alkaloids (DAs) are major pharmacologically active ingredients of Aconitum vilmorinianum, an important medicinal plant. Cytochrome P450 monooxygenases (P450s) are involved in the DA biosynthetic pathway, and the electron transfer reaction of NADPH-cytochrome P450 reductase (CPR) with P450 is the rate-limiting step of the P450 redox reaction. Here, we identified and characterized two homologs of CPR from Aconitum vilmorinianum. The open reading frames of AvCPR1 and AvCPR2 were found to be 2103 and 2100 bp, encoding 700 and 699 amino acid residues, respectively. Phylogenetic analysis characterized both AvCPR1 and AvCPR2 as class II CPRs. Cytochrome c and ferricyanide could be reduced with the recombinant proteins of AvCPR1 and AvCPR2. Both AvCPR1 and AvCPR2 were expressed in the roots, stems, leaves, and flowers of A. vilmorinianum. The expression levels of AvCPR1 and AvCPR2 were significantly increased in response to methyl jasmonate (MeJA) treatment. The yeasts co-expressing AvCPR1/AvCPR2/SmCPR1 and CYP76AH1 all produced ferruginol, indicating that AvCPR1 and AvCPR2 can transfer electrons to CYP76AH1 in the same manner as SmCPR1. Docking analysis confirmed the experimentally deduced functional activities of AvCPR1 and AvCPR2 for FMN, FAD, and NADPH. The functional characterization of AvCPRs will be helpful in disclosing molecular mechanisms relating to the biosynthesis of diterpene alkaloids in A. vilmorinianum.

First report of bacterial leaf streak in sweet corn caused by Pantoea ananatis in southeast China.

Sweet corn (Zea mays convar. saccharata var. rugosa) is a popular vegetable crop in southeast China. During the spring seasons of 2018-2021, a serious outbreak of bacterial leaf streak was observed in sweet corn variety Yuetian28 in the field in Guangzhou, Guangdong Province. The disease incidence was 50%-70%. Infected leaves initially displayed long, chlorotic streaks parallel to veins at the V5-V6 stage, and then turned white or brown and dried out over the course of disease development. In severe infections, leaf lesion coalesced to form large irregular blight areas (Fig. S1A). To investigate this disease, we collected 0.5 cm2 samples of infected leaves from four plants after surface sterilization and rinsed them three times with sterile distilled water. We placed all leaf samples on nutrient agar (NA) medium and incubated them at 28℃ for 48 hours. Bright-yellowish colonies were observed near the edges of the samples. We picked the colonies and re-streaked them onto NA medium three times to obtain pure cultures. Four isolates, GZ2201, GZ2202, GZ2203, and GZ2204, were selected for further study. All isolates were gram-negative rods and were negative for oxidase, urease, nitrate reductase reactions, and gelatin liquefaction. They were positive for catalase, citrate utilization, indole production, and the Voges-Proskauer test. We sequenced the 16S rDNA, rpoB, leuS, and gyrB sequences using previously reported primers (Brady et al. 2008) and deposited the sequences in GenBank (accession nos. ON740665 to ON740668 for 16S rDNA; ON755167 to ON755170 for rpoB; ON755171 to ON755174 for leuS; and OP227136 to OP227139 for gyrB). The sequences share >98% identity with sequences from Pantoea ananatis type strain LMG2665 (GenBank JFZU01) indicating that the causal pathogen of bacteria leaf streak of sweet corn is P. ananatis (Fig. S1B). Phylogenetic analysis of gyrB, leuS, and rpoB concatenated sequence showed that the four isolates clustered with P. ananatis (Fig S2). To test the pathogenicity of the isolates of P. ananatis on the sweet corn variety Yuetian28, we inoculated plants at the V3 stage by syringe infiltration of bacterial suspension (108 CFU/ml) (Kini et al. 2020) or sterile distilled water as a negative control. Inoculated plants were placed in a growth chamber at 28 ℃, 60% relative humidity, 16-h/8-h light-dark cycle. After 7 days of incubation, chlorotic streaks resembling the original symptoms developed on inoculated plants (Fig. S1D), while control plants remained symptomless (Fig. S1C). We successfully re-isolated bacteria from the inoculated plants and confirmed their identity by sequencing of 16S rDNA, rpoB, leuS, and gyrB. P. ananatis was previously reported to cause leaf spot disease in maize grown in Argentina, Ecuador, and China (Alippi et al. 2010; Toaza et al. 2021; Cui et al. 2022). To our knowledge, this is the first report of P. ananatis causing leaf streak in sweet corn in southeast China. Further research on P. ananatis management is needed to help control disease spread.

Dynamic Assembly of Cascade Enzymes by the Shape Transformation of a DNA Scaffold

AbstractWithin cells, the close spatial arrangement of cascade enzymes facilitates the channeling of intermediates and enhances cascade reaction efficiency. Reconfigurable DNA nanostructures, owing to their structural controllability and precise spatial addressability, are promising tools for mimicking such processes. In this study, a 3D DNA origami scaffold, with a dynamic shape transformation from its open boat form to a closed hexagonal prism induced by toehold‐mediated strand displacement, is designed to investigate the enzyme cascade reaction of xylose reductase and xylitol dehydrogenase from D‐xylose metabolic pathway. Enzymes are assembled on the DNA scaffold in its open state, which is subsequently closed by the assistance of DNA sequence‐specific closing keys. The enzyme cascade efficiency is much higher in the static encapsulated closed state than in the open state due not only to the enzyme proximity but also the environmental factors of 3D DNA structure. These results provide novel insights into controlling enzyme cascade reactions by inducing the shape transformation of DNA nanostructures and how environmental factors affect the action of multi‐enzyme complexes in the cell.

First Report of New Bacterial Leaf Blight of Rice Caused by Pantoea ananatis in Southeast China.

In autumn 2020, leaf blight was observed on rice (Oryza sativa L., variety Zhongzao39, Yongyou9, Yongyou12, Yongyou15, Yongyou18, Yongyou1540, Zhongzheyou8, Jiafengyou2, Xiangliangyou900 and Jiyou351) in the fields of 17 towns in Zhejiang and Jiangxi Provinces, China. The disease incidence was 45%-60%. Initially, water-soaked, linear, light brown lesions emerged in the upper blades of the leaves, and then spread down to leaf margins, which ultimately caused leaf curling and blight during the booting-harvest stage (Fig. S1). The disease symptoms were assumed to be caused by Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial blight. 63 isolates were obtained from the collected diseased leaves as previously described (Hou et al. 2020). All isolates showed circular, smooth-margined, yellow colonies when cultured on peptone sugar agar (PSA) medium for 24h at 28℃. The cells were all gram-negative and rod-shaped with three to six peritrichous flagella; positive for catalase, indole, glucose fermentation and citrate utilization, while negative for oxidase, alkaline, phenylalanine deaminase, urease, and nitrate reductase reactions. 16S rRNA gene sequence analysis from the 6 isolates (FY43, JH31, JH99, TZ20, TZ39 and TZ68) revealed that the amplified fragments shared 98% similarity with Pantoea ananatis type strain LMG 2665T (GenBank JFZU01) (Table S3). To further verify P. ananatis identity of these isolates, fragments of three housekeeping genes including gyrB, leuS and rpoB from the 6 isolates were amplified and sequenced, which showed highest homology to LMG 2665T with a sequence similarity of 95%-100% (Table S3). Primers (Brady et al. 2008) and GenBank accession numbers of gene sequences from the 6 isolates are listed in Table S1 and Table S2. Phylogenetic analysis of gyrB, leuS and rpoB concatenated sequences indicated that the 6 isolates were clustered in a stable branch with P. ananatis (Fig. S2). Based on the above morphological, physiological, biochemical and molecular data, the isolates are identified as P. ananatis. For pathogenicity tests, bacterial suspension at 108 CFU/mL was inoculated into flag leaves of rice (cv. Zhongzao39) at the late booting stage using clipping method. Water was used as a negative control. The clipped leaves displayed water-soaked lesions at 3 to 5 days after inoculation (DAI); then the lesion spread downward and turned light brown. At about 14 DAI, blight was shown with similar symptoms to those samples collected from the rice field of Zhejiang and Jiangxi provinces (Fig. S1). In contrast, the control plants remained healthy and symptomless. The same P. ananatis was re-isolated in the inoculated rice plants, fulfilling Koch's postulates. In the past decade, P. ananatis has been reported to cause grain discoloration in Hangzhou, China (Yan et al. 2010) and induce leaf blight as a companion of Enterobacter asburiae in Sichuan province, China (Xue et al. 2020). Nevertheless, to the best of our knowledge, this is the first report of P. ananatis as the causative agent of rice leaf blight in southeast China. This study raises the alarm that the emerging rice bacterial leaf blight in southeast China might be caused by a new pathogen P. ananatis, instead of Xoo as traditionally assumed. Further, the differences of occurrence, spread and control between two rice bacterial leaf blight diseases caused by P. ananatis and Xoo, respectively need to be determined in the future.

INVESTIGATION OF THE BIOLOGICAL ACTIVITY OF SEA BUCKTHORN MEAL FLAVONOIDS USING SPECIFIC BIOTEST SYSTEMS

The need to expand the raw material base for obtaining flavonoids is due to the wide spectrum of their biological activity. The purpose of this work is to study the biological activity of a complex of bioflavonoids, quercetin and rutin on specific enzyme biotest systems in vitro. The objects of the study were: a complex of bioflavonoids from fat-free sea buckthorn meal Hippophae rhamnoides L. and individual flavonoids rutin and quercetin isolated from it. The study was carried out by methods of detecting the biological activity of substances using specific enzyme biotest systems in vitro. It was revealed that rutin and a complex of bioflavonoids have antioxidant properties – the rate of glutathione reductase reaction increased by 64 and 51% of control, respectively, and catalase – by 15%. Quercetin exhibits antimicrobial activity and also reduces the rate of the enzymatic iNOS reaction by 24% of the control, which indicates the anti-inflammatory properties of this sample. Rutin and a complex of bioflavonoids from sea buckthorn meal increased the iNOS reaction rate by 14 and 28%, respectively, which indicates the immunostimulating properties of these samples. In the course of a microbiological study, it was found that all samples have weak bacteriostatic activity against gram-positive and gram-negative bacteria Staphylococcus aureus ATCC 6538 (209-P) and Pseudomonas aeruginosa ATCC 9027. Fungistatic activity was confirmed against the yeast-like fungi Candida albicans ATCC 10231 of quercetin and the complex. The results obtained make it possible to consider a complex of bioflavonoids, quercetin and rutin as promising active substances in antioxidant, antimicrobial, fungistatic and anti-inflammatory drugs.

The 5,6,7,8-Tetrahydro-2-Naphthoyl-Coenzyme A Reductase Reaction in the Anaerobic Degradation of Naphthalene and Identification of Downstream Metabolites.

Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-coenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD+-dependent β-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent β-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a β'-hydroxy-β-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate cis-2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene.IMPORTANCE Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via in vitro enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of β-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi.org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.

Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping-Electron Paramagnetic Resonance Approach.

Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read‐out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter‐copper electron transfer catalyzed by CuNiRs. Cryoreduction‐EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox‐center containing proteins (homo and hetero metal ions).

Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping‐Electron Paramagnetic Resonance Approach

AbstractRedox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read‐out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter‐copper electron transfer catalyzed by CuNiRs. Cryoreduction‐EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox‐center containing proteins (homo and hetero metal ions).

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Timing of NO Binding and Protonation in the Catalytic Reaction of Bacterial Nitric Oxide Reductase as Established by Time-Resolved Spectroscopy

Abstract Membrane-integrated nitric oxide reductases (NOR) catalyze the formation of nitrous oxide (N2O) from two NO molecules using two protons and two electrons at a heme/non-heme iron binuclear center. Despite extensive efforts, the mechanism underlying the NOR-catalyzed reaction has been poorly understood due to the rapidity of the reaction. Here, we utilized a photosensitive caged NO compound as a trigger for the NOR reaction to characterize the NO reduction mechanism by time-resolved visible absorption spectroscopy. We showed that the NOR reaction consists of three steps. One NO molecule binds to the reduced binuclear center to form a non-heme Fe(II)-NO species in the 1st phase (microsecond timescale), followed by a migration of NO to form the other chemical species, possibly 5-coordinate heme b3-NO, in the 2nd phase (timescale of tens of microseconds). Then, the NO bound to heme reacts with a second NO molecule in the 3rd phase (millisecond timescale), in which protonation and electron transfer promote N-N bond formation and N-O bond cleavage to yield N2O. These findings led us to propose a revised trans mechanism for NO reduction by NOR.

Single Crystal X-ray Structural Analysis and Electronic Theory of Sugar Molecules

Rare sugar is a monosaccharide that there are trace amounts in nature, natural sugars present in large amounts in nature world is referred to as a naturally monosaccharide. Recently, the isomerase called D-tagatose-3-epimerase (DTE) was discovered, and it has a possibility to synthesize quantity of the rare sugar from a natural sugar through the enzymatic reaction. And all of hexoses can be synthesized by four kinds of enzymatic reactions (oxidoreductase, aldose isomerase, aldose reductase and DTE reactions). In this study, single-crystal X-ray structure analysis of a sugar alcohol and its derivative have been performed in order to determine the absolute coordinates of these sugar molecules. We have also investigated the electronic state calculation by means of DV-Xα method using the obtained data of the absolute coordinates. We also consider that mechanism of the enzymatic reaction and intermolecular energy properly such as a hydrogen bond in order to synthesize the supramolecular rare sugars (SRSs) which can be controlled structures and the hydrogen bonds.

Effects of MCHM on yeast metabolism.

On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether (PPH) were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHM's toxicological data were limited, an issue that has been addressed by different studies focused on understanding the immediate and long-term effects of MCHM on human health and the environment. Using S. cerevisiae as a model organism we study the effect of acute exposure to crude MCHM on metabolism. Yeasts were treated with MCHM 550 ppm in YPD for 30 minutes. Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture. The extracts were then analyzed by direct injection ESI-MS and by GC-MS. The metabolomics analysis was complemented with flux balance analysis simulations done with genome-scale metabolic network models (GSMNM) of MCHM treated vs non-treated control. We integrated the effect of MCHM on yeast gene expression from RNA-Seq data within these GSMNM. A total of 215 and 73 metabolites were identified by the ESI-MS and GC-MS procedures, respectively. From these 26 and 23 relevant metabolites were selected from ESI-MS and GC-MS respectively, for 49 unique compounds. MCHM induced amino acid accumulation, via its effects on amino acid metabolism, as well as a potential impairment of ribosome biogenesis. MCHM affects phospholipid biosynthesis, with a potential impact on the biophysical properties of yeast cellular membranes. The FBA simulations were able to reproduce the deleterious effect of MCHM on cellular growth and suggest that the effect of MCHM on ubiquinol:ferricytochrome c reductase reaction, caused by the under-expression of CYT1 gene, could be the driven force behind the observed effect on yeast metabolism and growth.

Effect of fcl gene for butenyl-spinosyn biosynthesis and growth of Saccharopolyspora pogona

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.

Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

Metabolic flux analysis for metabolome data validation of naturally xylose-fermenting yeasts

BackgroundEfficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts.ResultsA metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts.ConclusionsThis study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.

Exogenously applied salicylic acid maintains redox homeostasis in salt-stressed Arabidopsis gr1 mutants expressing cytosolic roGFP1

Exogenous salicylic acid (SA) can be used for chemical hardening to alleviate oxidative stress in plants exposed to salinity. The treatment of 5-week-old Arabidopsis thaliana plants with increasing doses of SA alters the ascorbate (ASC) and glutathione (GSH) pools, and modulates their redox status and the activity of several antioxidant enzymes, such as ascorbate peroxidase (APX) and glutathione reductase (GR). To investigate the role of GR in the maintenance of cytoplasmic redox homeostasis after hardening by SA, wild type (WT) and gr1 mutant plants, expressing the cytoplasmic redox-sensitive green fluorescent protein (c-roGFP1), were pre-treated with 10−7 and 10−5 M SA for 2 weeks and subsequently exposed to 100 mM NaCl. The redox status of the salt-stressed WT plants became more oxidized, which was prevented by pretreatment with 10−5 M SA. The gr1 mutants showed more positive redox potential than WT plants, which could be reversed by treatment with 10−5 M SA. In mutants, the increased GSH levels may have compensated for the deleterious effect of GR deficiency and stabilized the redox potential in plants exposed to salinity. The ASC regeneration in WT plants shifted from the GSH-dependent dehydroascorbate reductase (DHAR) reaction to the NAD(P)H-dependent monodehydroascorbate reductase (MDHAR) activity during chemical hardening, which contributed to the preservation of the GSH pool in plants under salt stress. Our results suggest that the maintenance of GSH levels and redox homeostasis by SA-mediated hardening play a major role in priming and defending against salt stress.

H2 drives metabolic rearrangements in gas-fermenting Clostridium autoethanogenum

BackgroundThe global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions.ResultsHere, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model. CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a fourfold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by fivefold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood–Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity.ConclusionsOur quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

Ferredoxin: the central hub connecting photosystem I to cellular metabolism

Ferredoxin (Fd) is a small soluble iron-sulfur protein essential in almost all oxygenic photosynthetic organisms. It contains a single [2Fe-2S] cluster coordinated by four cysteine ligands. It accepts electrons from the stromal surface of PSI and facilitates transfer to a myriad of acceptors involved in diverse metabolic processes, including generation of NADPH via Fd-NADP-reductase, cyclic electron transport for ATP synthesis, nitrate reduction, nitrite reductase, sulfite reduction, hydrogenase and other reductive reactions. Fd serves as the central hub for these diverse cellular reactions and is integral to complex cellular metabolic networks. We describe advances on the central role of Fd and its evolutionary role from cyanobacteria to algae/plants. We compare structural diversity of Fd partners to understand this orchestrating role and shed light on how Fd dynamically partitions between competing partner proteins to enable the optimum transfer of PSI-derived electrons to support cell growth and metabolism.