The crystal structure analysis of group B Streptococcus sortase C1: a model for the "lid" movement upon substrate binding

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible “lid” anchored in the active site. However, the mechanistic details of the “lid” displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its “lid” mutant and proposed a role of the “lid” as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.     

Cell surface display of minor pilin adhesins in the form of a simple heterodimeric assembly in Corynebacterium diphtheriae

Pilus assembly in Gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, the pilin-specific sortase SrtA catalyses polymerization of the SpaA-type pilus, consisting of the shaft pilin SpaA, tip pilin SpaC and minor pilin SpaB. Cell wall anchoring of the SpaA polymers is triggered when SrtA incorporates SpaB into the pilus base via lysine-mediated transpeptidation; anchoring to the cell wall peptidoglycan is subsequently catalysed by the housekeeping sortase SrtF. Here we show that SpaB and SpaC formed a heterodimer independent of SpaA polymerization. SrtA was absolutely required for the formation of the SpaBC heterodimer, while SrtF facilitated the optimal cell wall anchoring of this heterodimer. Alanine substitution of the SpaB lysine residue K139 or truncation of the SpaB cell wall-sorting signal (CWSS) abolished assembly of the SpaBC heterodimer, hence underscoring SpaB function in transpeptidation and cell wall linkage. Importantly, sortase specificity for the cell wall-anchoring step was found to be dependent on the LAFTG motif within the SpaB CWSS. Thus, C. diphtheriae employs a common sortase-catalysed mechanism involving lysine-mediated transpeptidation to generate both adhesive pilus and simple heterodimeric structures on the bacterial the cell wall.     

Using Magnetically Responsive Tea Waste to Remove Lead in Waters under Environmentally Relevant Conditions

We report the use of a simple yet highly effective magnetite-waste tea composite to remove lead(II) (Pb²⁺) ions from water. Magnetite-waste tea composites were dispersed in four different types of water–deionized (DI), artificial rainwater, artificial groundwater and artificial freshwater–that mimic actual environmental conditions. The water samples had varying initial concentrations (0.16–5.55 ppm) of Pb²⁺ ions and were mixed with the magnetite-waste tea composite for at least 24 hours to allow adsorption of the Pb²⁺ ions to reach equilibrium. The magnetite-waste tea composites were stable in all the water samples for at least 3 months and could be easily removed from the aqueous media via the use of permanent magnets. We detected no significant leaching of iron (Fe) ions into the water from the magnetite-waste tea composites. The percentage of Pb adsorbed onto the magnetite-waste tea composite ranged from ∼70% to 100%; the composites were as effective as activated carbon (AC) in removing the Pb²⁺ ions from water, depending on the initial Pb concentration. Our prepared magnetite-waste tea composites show promise as a green, inexpensive and highly effective sorbent for removal of Pb in water under environmentally realistic conditions.     

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.     

Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production

Lactobacillus plantarum ferments glucose through the Embden–Meyerhof–Parnas pathway: the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A PDC gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into an LDH-deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent TF103 and produced 90–130 mM ethanol. Although slightly more ethanol was observed, carbon flow was not significantly improved toward ethanol, suggesting that a further understanding of this organism’s metabolism is necessary.     

Full-scale On-farm Pretreatment of Perennial Grasses with Dilute Acid for Fuel Ethanol Production

Biorefineries that rely on lignocellulosic feedstocks require dependable and safe methods for storing biomass. Storing biomass wet in the presence of sulfuric acid and the absence of oxygen has been shown to preserve carbohydrates and enhance cellulose conversion but has not been demonstrated at farm-scale. To that end, switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.) were pretreated with 18?N sulfuric acid with two methods: during bagging (on-line) and thoroughly mixed in a commercial feed mixer (mixed) and both stored for 90 days. The two methods, applied at rates from 28 to 54 g(kg DM)^?1 not only helped to preserve biomass substrates under on-farm conditions (anaerobic, ambient temperature and pressure) through inhibition of microbial activity but also enhanced conversion of cellulose to ethanol by simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae. Acid-pretreated substrate yielded 19 and 7 percentage points higher ethanol conversion efficiencies than fresh reed canarygrass and switchgrass, respectively. The on-line method of pretreatment out-yielded the mixed method both as a preservative and as an agent for enhanced cell wall degradation. This result was thought to be an outcome of more uniform acid application as indicated by the on-line method’s more consistent pH profile and decreased fermentation products, as compared to the mixed method. Although significant levels of acetate and lactate were present in the biomass following storage, concentrations were not sufficient to inhibit S. cerevisiae in SSFs with a 10% solids loading.     

Protective effect of wogonin on endotoxin-induced lethal shock in D-galactosamine-sensitized mice.

The effects of wogonin, a major flavonoid from Scutellaria baicalensis Georgi, on lipopolysaccharide (LPS)-induced lethal shock in mice was investigated. Wogonin pretreatment prevented the lethal shock in mice injected with D-galactosamine (D-GalN) and LPS, but not in mice injected with a high dose of LPS. Wogonin definitely inhibited the hepatic injury in mice injected with D-GalN, and LPS and reduced the level of circulating tumor necrosis factor (TNF)-alpha. The reduction was more marked in mice injected with D-GalN and LPS compared with that in mice injected with a high dose of LPS. Wogonin pretreatment did not inhibit the lipid peroxidation in mice receiving either D-GalN and LPS or a high dose of LPS. Wogonin inhibited the in vitro production of TNF-alpha and nitric oxide in LPS-stimulated RAW 264.7 cells. The mechanism of the protective effect of wogonin on the lethal shock in mice injected with D-GalN and LPS is discussed.     

Applications of metabolic modeling to drive bioprocess development for the production of value-added chemicals

Increasing numbers of value added chemicals are being produced using microbial fermentation strategies. Computational modeling and simulation of microbial metabolism is rapidly becoming an enabling technology that is driving a new paradigm to accelerate the bioprocess development cycle. In particular, constraint-based modeling and the development of genome-scale models of industrial microbes are finding increasing utility across many phases of the bioprocess development workflow. Herein, we review and discuss the requirements and trends in the industrial application of this technology as we build toward integrated computational/experimental platforms for bioprocess engineering. Specifically we cover the following topics: (1) genome-scale models as genetically and biochemically consistent representations of metabolic networks; (2) the ability of these models to predict, assess, and interpret metabolic physiology and flux states of metabolism; (3) the model-guided integrative analysis of high throughput ‘omics’ data; (4) the reconciliation and analysis of on- and off-line fermentation data as well as flux tracing data; (5) model-aided strain design strategies and the integration of calculated biotransformation routes; and (6) control and optimization of the fermentation processes. Collectively, constraint-based modeling strategies are impacting the iterative characterization of metabolic flux states throughout the bioprocess development cycle, while also driving metabolic engineering strategies and fermentation optimization.     

Impact of Harvest Time and Switchgrass Cultivar on Sugar Release Through Enzymatic Hydrolysis

Switchgrass (Panicum virgatum L.) is a native North American prairie grass being developed for bioenergy production in the central and eastern USA. The objective of this study was to identify the impacts of harvest time and switchgrass cultivar had on sugar release variables determined through enzymatic hydrolysis. Previously, we reported that delaying harvest of switchgrass until after frost and until after winter resulted in decreased yields of switchgrass but it reduced the amount of ash and nutrients in the biomass. The current study used near-infrared reflectance spectroscopy (NIRS) to broaden an existing set of calibration equations designed to predict composition and sugar release variables of switchgrass. These updated calibrations were then applied to the full set of samples from a multi-year and multi-location switchgrass harvest-management study. Composition and processor sugar yields were significantly affected by location, year, cultivar, and harvest time, of which the time of harvest was the most important. Delaying the time of harvest until after frost or post-winter increased the concentration of structural carbohydrates from 500 to over 570 g kg^?1 in the biomass and lignin content from 160 to over 200 g kg^?1. Conversely, delaying harvest time lowered the amounts of ash and soluble sugars. The later harvest times also yielded more sugars following processing with yields increasing over 20% from the first harvest. Increased sugar yields are attributable to both increased concentration of sugars in the biomass upon harvest and reduced biomass recalcitrance. Based upon processed sugar yields, it is estimated that a biorefinery producing 76 million liters of ethanol per year would require 229–373 km² of land cultivated with switchgrass.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates

Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH(2)-Gly(3), sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH(2)-Gly(3). In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate.