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1.
ACS Synth Biol ; 2022 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-34979802

RESUMO

Microbial cell factories using a single carbon source (e.g., sugars) have been used to produce a wide variety of chemicals. However, this process is often accompanied by stoichiometric constraints on carbons and redox cofactors. Here, a synthetic pathway was designed and constructed in Escherichia coli to synergistically use glucose and formate as mixed carbon sources. By optimizing this synthetic pathway via enzyme mining, protein engineering, and bioprocess approaches, the yield of pyruvate from glucose was enhanced to 94% of the theoretical glycolysis yield, reaching 1.88 mol/mol. Finally, the optimized synthetic pathway was integrated with a phosphite reductase-based NADH regeneration system in malate-producing E. coli, resulting in the conversion of glucose into l-malate with a high yield of up to 1.65 mol/mol. This synergistic carbon metabolism strategy can be used to establish carbon- and energy-efficient productive processes.

2.
Biotechnol Bioeng ; 119(1): 277-286, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34708879

RESUMO

Saccharomyces cerevisiae is an attractive chassis for the production of medium-chain fatty acids, but the toxic effect of these compounds often prevents further improvements in titer, yield, and productivity. To address this issue, Lem3 and Sfk1 were identified from adaptive laboratory evolution mutant strains as membrane asymmetry regulators. Co-overexpression of Lem3 and Sfk1 [Lem3(M)-Sfk1(H) strain] through promoter engineering remodeled the membrane phospholipid distribution, leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane. As a result, membrane potential and integrity were increased by 131.5% and 29.2%, respectively; meanwhile, the final OD600 in the presence of hexanoic acid, octanoic acid, and decanoic acid was improved by 79.6%, 73.4%, and 57.7%, respectively. In summary, this study shows that membrane asymmetry engineering offers an efficient strategy to enhance medium-chain fatty acids tolerance in S. cerevisiae, thus generating a robust industrial strain for producing high-value biofuels.

3.
Front Plant Sci ; 12: 739671, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868124

RESUMO

In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3'-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.

4.
Front Chem ; 9: 766078, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34858942

RESUMO

The excellent properties of nanomaterials have been confirmed in many fields, but their effects on plants are still unclear. In this study, different concentrations of bismuth vanadate (BV) were added to the growth medium to analyze the growth of seedlings, including taproots, lateral roots, leaf stomata, root activity, and superoxide anion O2 .- generation. Gene expression levels related to root growth were determined by quantitative PCR in Arabidopsis thaliana. The results showed that BV promoted the growth of taproots and the development of lateral roots, enhanced the length of the extension zone in roots, increased the number and size of leaf stomata and root activity, reduced the accumulation of ROS in seedlings, and changed the expression levels of genes related to polyamines or hormones. At the same time, we investigated the antibacterial activity of BV against a variety of common pathogens causing crop diseases. The results showed that BV could effectively inhibit the growth of Fusarium wilt of cotton and rice sheath blight. These results provide a new prospect for the development of nanomaterial-assisted plants, which is expected to become one of the ways to solve the problem of controlling and promoting the development of plants. At the same time, it also provides a reference for the study of the effect of BV on plants.

5.
Acta Pharmacol Sin ; 2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34931016

RESUMO

Major depressive disorder (MDD) is a common psychiatric disorder characterized by persistent mood despondency and loss of motivation. Although numerous hypotheses have been proposed, the possible pathogenesis of MDD remains unclear. Several recent studies show that a classic transporter protein, sortilin, is closely associated with depression. In the present study, we investigated the role of sortilin in MDD using a well-established rodent model of depression. Mice were subjected to chronic unpredictable mild stress (CUMS) for 6 weeks. We showed that the expression levels of sortilin were significantly increased in the prefrontal cortex and hippocampus of CUMS mice. The depressive-like behaviors induced by CUMS were alleviated by specific knockdown of sortilin in the prefrontal cortex and hippocampus. We revealed that sortilin facilitated acid sphingomyelinase (ASM)/ceramide signaling, which activated RhoA/ROCK2 signaling, ultimately causing the transformation of dendritic spine dynamics. Specific overexpression of sortilin in the prefrontal cortex and hippocampus induced depressive-like behaviors, which was mitigated by injection of ASM inhibitor SR33557 (4 µg/µL) into the prefrontal cortex and hippocampus. In conclusion, sortilin knockdown in the prefrontal cortex and hippocampus plays an important role in ameliorating depressive-like behavior induced by CUMS, which is mainly evidenced by decreasing the trafficking of ASM from the trans-Golgi network to the lysosome and reducing the ceramide levels. Our results provide a new insight into the pathology of depression, and demonstrate that sortilin may be a potential therapeutic target for MDD.

6.
Biotechnol Bioeng ; 2021 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-34936092

RESUMO

Tryptophan, an essential aromatic amino acid, is widely used in animal feed, food additives, and pharmaceuticals. Although sustainable and environmentally friendly, microbial tryptophan production from renewable feedstocks is limited by low biosynthesis and transport rates. Here, an Escherichia coli strain capable of efficient tryptophan production was generated by improving and balancing the supply of precursors and by engineering membrane transporters. Tryptophan biosynthesis was increased by eliminating negative regulatory factors, blocking competing pathways, and preventing tryptophan degradation. Promoter engineering balanced the supply of the precursors erythrose-4-phosphate and phosphoenolpyruvate, as well as the availability of serine. Finally, the engineering of tryptophan transporters prevented feedback inhibition and growth toxicity. Fed-batch fermentation of the final strain (TRP12) in a 5 L bioreactor produced 52.1 g·L-1 of tryptophan, with a yield of 0.171 g·g-1 glucose and productivity of 1.45 g·L-1 ·h-1 . The metabolic engineering strategy described here paves the way for high-performance microbial cell factories aimed at the production of tryptophan as well as other valuable chemicals.

7.
Nat Commun ; 12(1): 6886, 2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34824227

RESUMO

Microbial populations are a promising model for achieving microbial cooperation to produce valuable chemicals. However, regulating the phenotypic structure of microbial populations remains challenging. In this study, a programmed lysis system (PLS) is developed to reprogram microbial cooperation to enhance chemical production. First, a colicin M -based lysis unit is constructed to lyse Escherichia coli. Then, a programmed switch, based on proteases, is designed to regulate the effective lysis unit time. Next, a PLS is constructed for chemical production by combining the lysis unit with a programmed switch. As a result, poly (lactate-co-3-hydroxybutyrate) production is switched from PLH synthesis to PLH release, and the content of free PLH is increased by 283%. Furthermore, butyrate production with E. coli consortia is switched from E. coli BUT003 to E. coli BUT004, thereby increasing butyrate production to 41.61 g/L. These results indicate the applicability of engineered microbial populations for improving the metabolic division of labor to increase the efficiency of microbial cell factories.

8.
J Agric Food Chem ; 69(47): 14215-14221, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34786944

RESUMO

l-Ascorbic acid-2-phosphate (AsA-2P) is stable in aqueous solution and at high temperatures and is widely used in foods, pharmaceuticals, cosmetics, and fodders; however, practical application of enzymatic synthesis methods to promote industrial-scale production of AsA-2P remains a major challenge. In this study, we enhanced the phosphorylation efficiency of Pseudomonas aeruginosa acid phosphatase (APase; EC 3.1.3.2) for AsA-2P production via protein engineering. Among the mutants obtained, we selected the most efficient mutant (Var5; G125E/D135T/S136N), which exhibited an increased kcat of 18.6 s-1 and a Km for AsA of 223.9 mM. In addition, Var5 exhibited a maximum enzyme activity of 2080.4 U/L after 10 h of fermentation, which was 80% higher than the wild-type enzyme. Furthermore, under optimal conditions, Var5 showed a maximal conversion of 48.6% and achieved a final AsA-2P titer of 61.5 g/L at 8 h, which is considerably higher than that reported for other similar biocatalytic approaches. These findings demonstrate the potential of this method for the large-scale production of AsA-2P.

9.
Ann Palliat Med ; 10(10): 10779-10788, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34763439

RESUMO

BACKGROUND: Nutritional support is very important in the treatment of severe acute pancreatitis, this study aimed to investigate the effect of total parenteral nutrition (TPN) and enteral nutrition (TEN) on the prognosis of patients with acute pancreatitis. METHODS: The databases of PubMed, Embase, Cochrane Library, and Ovid were searched using the keywords acute pancreatitis, enteral nutrition, and parenteral nutrition to obtain the reports of randomized controlled trials (RCTs) published after 2000. After screening the articles according to the inclusion criteria, risk of bias of the included literatures was evaluated using the Cochrane Handbook for Systematic Reviews. The software RevMan 5.3.5 was used for analysis and the creation of a forest plot and funnel plot. RESULTS: A total of 488 literatures were preliminarily searched in this study, from which 10 articles were included into the final quantitative analysis, involving a total of 699 participants. A total of 6 literatures (n=329 participants) reported the infection rate indicators. The obtained statistic value [odds ratio (OR) =0.25, 95% confidence interval (CI): 0.10 to 0.62] showed TEN had less infection rate that TPN (P=0.003). A total of 8 studies (654 participants) reported the incidence rate indicators of multiple organ failure rate indicator, the obtained statistic value (OR =0.50, 95% CI: 0.24 to 1.08) showed no statistical difference between TEN and TPN (P>0.05). A total of 7 studies (550 participants) reported the mortality indicators. The obtained statistic value (OR =0.59, 95% CI: 0.37 to 0.94) showed TEN had less mortality than TPN (P=0.03). A total of 3 studies reported the length of hospital stay indicators. The obtained statistic value [mean difference (MD) -4.18, 95% CI: -5.07 to -3.30] showed the length of hospital stay for TEN was shorter that TPN (P<0.001). DISCUSSION: Compared with TPN, TEN can reduce the incidence of infection, reduce the development of multiple organ failure, reduce mortality, and shorten the length of hospital stay in patients with severe acute pancreatitis (SAP). However, attention should be paid to prevent the occurrence of complications during the implementation of nutritional intervention.


Assuntos
Nutrição Enteral , Pancreatite , Humanos , Pancreatite/terapia , Nutrição Parenteral , Nutrição Parenteral Total , Prognóstico
10.
Int J Mol Sci ; 22(21)2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34769318

RESUMO

Bulblet formation and development determine the quantitative and qualitative traits, respectively, of bulb yield for most flowering bulbs. For Lycoris species, however, the underlying molecular mechanism remains elusive. Here, clonal bulblets of Lycoris sprengeri (Ls) derived from the same probulb were used as explants to establish efficient and inefficient in vitro regeneration systems by adjusting the 6-benzyladenine (BA) concentrations in media. BA application did not change the biological processes among groups but led to earlier decreases in sucrose and total soluble sugar (TSS) contents. Correlation analyses showed that the BA treatments changed the interaction between carbohydrate and endogenous hormone contents during bulblet regeneration. We found that two sucrose degradation enzyme-related genes, cell wall invertase (CWIN) and sucrose synthase, exhibited exactly opposite expression patterns during the competence stage. In addition, the regeneration system that obtained more bulblets showed significantly higher expression of LsCWIN2 than those that obtained fewer bulblets. Our data demonstrate the essential role of BA in accelerating sucrose degradation and the selection of a dominant sucrose cleavage pattern at the competence stage of in vitro bulblet regeneration. We propose that a relatively active CWIN-catalyzed pathway at the competence stage might promote bulblet regeneration, thus influencing bulb yield.

11.
Sci Rep ; 11(1): 22162, 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34772992

RESUMO

Magnetoactive membrane-type acoustic metamaterials are fabricated by coating a layer of magnetic nanoparticles on the polyethylene (PE) membranes and their vibration characters are investigated experimentally. From our experiments, we discovered that, under different magnetic fields by varying the distance between a magnet and the membranes, such membranes exhibit tunable vibration eigenfrequencies (the shift towards lower frequencies), which is caused by the variation of the effective mass density and effective tension coefficient resulted from the second derivative of the magnetic field. The strong magnetic force between the layer of magnetic nanoparticles and the magnet enhances the eigenfrequency shift. A spring oscillator model is proposed and it agrees well with the experimental results. We also experimentally observed that the vibration radius, effective mass density, and effective tension coefficient of the membranes can enormously affect the eigenfrequencies of the membranes. We believe that this type of metamaterials may open up some potential applications for acoustic devices with turntable vibration properties.

12.
ACS Synth Biol ; 10(10): 2661-2671, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34609846

RESUMO

Building autonomous switches is an effective approach for rewiring metabolic flux during microbial synthesis of chemicals. However, current autonomous switches largely rely on metabolite-responsive biosensors or quorum-sensing circuits. In this study, a stationary phase promoter (SPP) and a protein degradation tag (PDT) were combined with the CRISPR interference (CRISPRi) system to construct an autonomous repression system that could shut down multiple-gene expression depending on the cellular physiological state. With this autonomous CRISPRi system to regulate one target gene, a fermenter-scale titer of shikimic acid reached 21 g/L, which was the highest titer ever reported by Escherichia coli in a minimal medium without any chemical inducers. With three target genes repressed, 26 g/L glutaric acid could be achieved with decreased byproduct accumulation. These results highlight the applicability of the autonomous CRISPRi system for microbial production of value-added chemicals.

13.
Pest Manag Sci ; 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34596946

RESUMO

BACKGROUND: The brown planthopper (BPH), Nilaparvata lugens (Stål), is the most severe pest attacking rice crops using sucking mouthparts. It causes significant damages to rice growth and food production worldwide. With the long-term and wide use of insecticides, field populations of BPH have developed resistance to many insecticides. RESULTS: Here, we showed that upregulation of an ATP-binding cassette transporter gene NlMdr49-like contributes to imidacloprid resistance in field populations of BPH. A comparative transcriptome analysis was performed to evaluate the gene expression in two field populations (JXSG18 and YNTC18). Compared with a susceptible strain (Sus), 202 upregulated genes and 170 downregulated genes were identified in both field populations. Functional enrichment analysis revealed that the differentially expressed genes (DEGs) are mainly linked to metabolic process and transmembrane transport. Among the candidate DEGs, NlMdr49-like was significantly upregulated in both field populations. Based on the genome and transcriptome of BPH, the full-length complementary DNA of NlMdr49-like was sequenced and its molecular characteristics were analyzed. Expression pattern analysis of various tissues showed that NlMdr49-like was predominantly expressed in midgut and Malpighian tubules which are important excretion organs. Knocking down NlMdr49-like reduced BPH resistance to imidacloprid, but did not affect its resistance to the other nine insecticides (chlorpyrifos, thiamethoxam, nitenpyram, dinotefuran, sulfoxaflor, triflumezopyrim, ethiprole, buprofezin and pymetrozine). Furthermore, a transgenic strain of Drosophila melanogaster overexpressing NlMdr49-like was less susceptible to imidacloprid. CONCLUSIONS: Our findings indicate that upregulation of NlMdr49-like is another mechanism contributing to imidacloprid resistance in N. lugens. This result is helpful to further understand the resistance mechanism of N. lugens to imidacloprid.

14.
Materials (Basel) ; 14(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34640253

RESUMO

In this study, we incorporated 2,3-dialdehyde nanocrystalline cellulose (DANC) into chitosan as a reinforcing agent and manufactured biodegradable films with enhanced gas barrier properties. DANC generated via periodate oxidation of cellulose nanocrystal (CNC) was blended at various concentrations with chitosan, and bionanocomposite films were prepared via casting and characterized systematically. The results showed that DANC developed Schiff based bond with chitosan that improved its properties significantly. The addition of DANC dramatically improved the gas barrier performance of the composite film, with water vapor permeability (WVP) value decreasing from 62.94 g·mm·m-2·atm-1·day-1 to 27.97 g·mm·m-2·atm-1·day-1 and oxygen permeability (OP) value decreasing from 0.14 cm3·mm·m-2·day-1·atm-1 to 0.026 cm3·mm·m-2·day-1·atm-1. Meanwhile, the maximum decomposition temperature (Tdmax) of the film increased from 286 °C to 354 °C, and the tensile strength of the film was increased from 23.60 MPa to 41.12 MPa when incorporating 25 wt.% of DANC. In addition, the chitosan/DANC (75/25, wt/wt) films exhibited superior thermal stability, gas barrier, and mechanical strength compared to the chitosan/CNC (75/25, wt/wt) film. These results confirm that the DANC and chitosan induced films with improved gas barrier, mechanical, and thermal properties for possible use in film packaging.

15.
Drug Deliv ; 28(1): 1962-1971, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34565273

RESUMO

Betulinic acid (3ß-Hydroxy-20(29)-lupaene-28-oic acid, BA) has excellent anti-cancer activity but poor solubility and low bioavailability. To improve the antitumor activity of BA, a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (PVCL-PVA-PEG) graft copolymer (Soluplus) encapsulated BA micelle (Soluplus-BA) was fabricated. The Soluplus-BA micelles presented a mean size of 54.77 ± 1.26 nm and a polydispersity index (PDI) of 0.083. The MTT assay results showed that Soluplus-BA micelles increased the inhibitory effect of BA on MDA-MB-231 cells, mainly due to the enhanced accumulation of reactive oxygen species (ROS) and the destruction of mitochondrial membrane potential (MMP). Soluplus-BA micelles induced the DNA double-strand breaks (DSBs) as the γH2AX foci increased. Moreover, Soluplus-BA also inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVECs), and inhibited the neovascularization of the chicken chorioallantoic membrane (CAM). This angiogenesis inhibitory effect may be accomplished by regulating the HIF-1/VEGF-FAK signaling pathway. The in vivo study confirmed the improved anti-tumor effect of Soluplus-BA and its inhibitory effect on angiogenesis, demonstrating the possibility of Soluplus-BA as an effective anti-breast cancer drug delivery system.

16.
Front Robot AI ; 8: 716007, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34527706

RESUMO

Image-based navigation is widely considered the next frontier of minimally invasive surgery. It is believed that image-based navigation will increase the access to reproducible, safe, and high-precision surgery as it may then be performed at acceptable costs and effort. This is because image-based techniques avoid the need of specialized equipment and seamlessly integrate with contemporary workflows. Furthermore, it is expected that image-based navigation techniques will play a major role in enabling mixed reality environments, as well as autonomous and robot-assisted workflows. A critical component of image guidance is 2D/3D registration, a technique to estimate the spatial relationships between 3D structures, e.g., preoperative volumetric imagery or models of surgical instruments, and 2D images thereof, such as intraoperative X-ray fluoroscopy or endoscopy. While image-based 2D/3D registration is a mature technique, its transition from the bench to the bedside has been restrained by well-known challenges, including brittleness with respect to optimization objective, hyperparameter selection, and initialization, difficulties in dealing with inconsistencies or multiple objects, and limited single-view performance. One reason these challenges persist today is that analytical solutions are likely inadequate considering the complexity, variability, and high-dimensionality of generic 2D/3D registration problems. The recent advent of machine learning-based approaches to imaging problems that, rather than specifying the desired functional mapping, approximate it using highly expressive parametric models holds promise for solving some of the notorious challenges in 2D/3D registration. In this manuscript, we review the impact of machine learning on 2D/3D registration to systematically summarize the recent advances made by introduction of this novel technology. Grounded in these insights, we then offer our perspective on the most pressing needs, significant open problems, and possible next steps.

17.
Adv Mater ; 33(38): e2102292, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34346108

RESUMO

Nanofluidics derived from low-dimensional nanosheets and protein nanochannels are crucial for advanced catalysis, sensing, and separation. However, polymer nanofluidics is halted by complicated preparation and miniaturized sizes. This work reports the bottom-up synthesis of modular nanofluidics by confined growth of ultrathin metal-organic frameworks (MOFs) in a polymer membrane consisting of zwitterionic dopamine nanoparticles (ZNPs). The confined growth of the MOFs on the ZNPs reduces the chain entanglement between the ZNPs, leading to stiff interfacial channels enhancing the nanofluidic transport of water molecules through the membrane. As such, the water permeability and solute selectivity of MOF@ZNPM are one magnitude improved, leading to a record-high performance among all polymer nanofiltration membranes. Both the experimental work and the molecular dynamics simulations confirm that the water transport is shifted from high-friction-resistance conventional viscous flow to ultrafast nanofluidic flow as a result of rigid and continuous nanochannels in MOF@ZNPM.

18.
ACS Synth Biol ; 10(8): 1966-1979, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34337931

RESUMO

The recycle and reutilization of food wastes is a promising alternative for supporting and facilitating circular economy. However, engineering industrially relevant model organisms to use food wastes as their sole carbon source has remained an outstanding challenge so far. Here, we reprogrammed Escherichia coli metabolism using modular pathway engineering followed by laboratory adaptive evolution to establish a strain that can efficiently utilize waste cooking oil (WCO) as the sole carbon source to produce monomers of bioplastics, namely, medium-chain α,ω-dicarboxylic acids (MCDCAs). First, the biosynthetic pathway of MCDCAs was designed and rewired by modifying the ß-oxidation pathway and introducing an ω-oxidation pathway. Then, metabolic engineering and laboratory adaptive evolution were applied for improving the pathway efficiency of fatty acids utilization. Finally, the engineered strain E. coli AA0306 was able to produce 15.26 g/L MCDCAs with WCO as the sole carbon source. This study provides an economically attractive strategy for biomanufacturing bioplastics from food wastes, which has a great potentiality to be developed as a wide range of enabling biotechnologies for achieving green revolution.

19.
Biotechnol Bioeng ; 118(11): 4503-4515, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34406648

RESUMO

The compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of adenosine triphosphate (ATP) (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided "ADP expulsion" strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme's flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1 ) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor, and achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. Then, the PAPS catalytic system was combined with the chondroitin 4-sulfotransferase using a one-pot method. Finally, chondroitin sulfate was transformed from chondroitin at a conversion rate of 98.75%. This strategy has great potential for scale biosynthesis of PAPS and chondroitin sulfate.

20.
PLoS Genet ; 17(8): e1009724, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34398892

RESUMO

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. Here, we investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. We found that feeding increases the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, we found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, we have identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. Our findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but our experiments cannot exclude further parallel pathways. Importantly, we show that the underlying mechanisms are conserved in the two distantly related insect species.

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