Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations

<p>Abstract</p> <p>Background</p> <p>Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, <it>Miscanthus</it>, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.</p> <p>Results</p> <p>When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and <it>Miscanthus </it>gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.</p> <p>Conclusion</p> <p>The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.</p

Gene family encoding the major toxins of lethal \u3ci\u3eAmanita\u3c/i\u3e mushrooms

Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode α-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. α-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable ‘‘toxin’’ region capable of encoding a wide variety of peptides of 7–10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes

Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose

<p>Abstract</p> <p>Background</p> <p>Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide (AHP)) was shown by Gould and coworkers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here, we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production.</p> <p>Results</p> <p>The effects of biomass loading, hydrogen peroxide loading, residence time, and pH control were tested in combination with subsequent digestion with a commercial enzyme preparation, optimized mixtures of four commercial enzymes, or optimized synthetic mixtures of pure enzymes. AHP pretreatment was performed at room temperature (23°C) and atmospheric pressure, and after AHP pretreatment the biomass was neutralized with HCl but not washed before enzyme digestion. Standard enzyme digestion conditions were 0.2% glucan loading, 15 mg protein/g glucan, and 48 h digestion at 50°C. Higher pretreatment biomass loadings (10% to 20%) gave higher monomeric glucose (Glc) and xylose (Xyl) yields than the 2% loading used in earlier studies. An H₂O₂loading of 0.25 g/g biomass was almost as effective as 0.5 g/g, but 0.125 g/g was significantly less effective. Optimized mixtures of four commercial enzymes substantially increased post-AHP-pretreatment enzymatic hydrolysis yields at all H₂O₂concentrations compared to any single commercial enzyme. At a pretreatment biomass loading of 10% and an H₂O₂loading of 0.5 g/g biomass, an optimized commercial mixture at total protein loadings of 8 or 15 mg/g glucan gave monomeric Glc yields of 83% or 95%, respectively. Yields of Glc and Xyl after pretreatment at a low hydrogen peroxide loading (0.125 g H₂O₂/g biomass) could be improved by extending the pretreatment residence time to 48 h and readjusting the pH to 11.5 every 6 h during the pretreatment. A Glc yield of 77% was obtained using a pretreatment of 15% biomass loading, 0.125 g H₂O₂/g biomass, and 48 h with pH adjustment, followed by digestion with an optimized commercial enzyme mixture at an enzyme loading of 15 mg protein/g glucan.</p> <p>Conclusions</p> <p>Alkaline peroxide is an effective pretreatment for corn stover. Particular advantages are the use of reagents with low environmental impact and avoidance of special reaction chambers. Reasonable yields of monomeric Glc can be obtained at an H₂O₂concentration one-quarter of that used in previous AHP research. Additional improvements in the AHP process, such as peroxide stabilization, peroxide recycling, and improved pH control, could lead to further improvements in AHP pretreatment.</p

Influence of carbon source on the expression of Cochliobolus carbonum xylan-degrading enzyme genes

The expression of four Cochliobolus carbonum endo-1,4-b-xylanase genes (XYL1, XYL2, XYL3, XYL4), and an exo-1,4-b-xylosidase gene (XYP1) was studied following the growth of the fungus in minimal medium containing glucose, sucrose, xylose, xylan, pectin, or cellulose. The XYL1 and XYL2 genes were expressed only when the culture medium contained xylan or cellulose. Both XYL3 and XYL4 are induced by xylose and xylan, and XYP1 expression is induced by xylose, xylan, pectin and cellulose. None of these genes is expressed in glucose or sucrose media. The differential expression of these enzymes may provide means for the fungus to adapt to different conditions

Submaximal Oxygen Uptake Kinetics, Functional Mobility, and Physical Activity in Older Adults with Heart Failure and Reduced Ejection Fraction

Background: Submaximal oxygen uptake measures are more feasible and may better predict clinical cardiac outcomes than maximal tests in older adults with heart failure (HF). We examined relationships between maximal oxygen uptake, submaximal oxygen kinetics, functional mobility, and physical activity in older adults with HF and reduced ejection fraction. Methods: Older adults with HF and reduced ejection fraction (n = 25, age 75 ± 7 years) were compared to 25 healthy age- and gender-matched controls. Assessments included a maximal treadmill test for peak oxygen uptake (VO2peak), oxygen uptake kinetics at onset of and on recovery from a submaximal treadmill test, functional mobility testing [Get Up and Go (GUG), Comfortable Gait Speed (CGS), Unipedal Stance (US)], and self-reported physical activity (PA). Results: Compared to controls, HF had worse performance on GUG, CGS, and US, greater delays in submaximal oxygen uptake kinetics, and lower PA. In controls, VO2peak was more strongly associated with functional mobility and PA than submaximal oxygen uptake kinetics. In HF patients, submaximal oxygen uptake kinetics were similarly associated with GUG and CGS as VO2peak, but weakly associated with PA. Conclusions: Based on their mobility performance, older HF patients with reduced ejection fraction are at risk for adverse functional outcomes. In this population, submaximal oxygen uptake measures may be equivalent to VO2 peak in predicting functional mobility, and in addition to being more feasible, may provide better insight into how aerobic function relates to mobility in older adults with HF

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF

The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris

Outcomes and effect of treatment according to etiology in HFrEF: an analysis of PARADIGM-HF

Objectives: The purpose of this study was to compare outcomes (and the effect of sacubitril/valsartan) according to etiology in the PARADIGM-HF (Prospective comparison of angiotensin-receptor-neprilysin inhibitor [ARNI] with angiotensin-converting-enzyme inhibitor [ACEI] to Determine Impact on Global Mortality and morbidity in Heart Failure) trial. Background: Etiology of heart failure (HF) has changed over time in more developed countries and is also evolving in non-Western societies. Outcomes may vary according to etiology, as may the effects of therapy. Methods: We examined outcomes and the effect of sacubtril/valsartan according to investigator-reported etiology in PARADIGM-HF. The outcomes analyzed were the primary composite of cardiovascular death or HF hospitalization, and components, and death from any cause. Outcomes were adjusted for known prognostic variables including N terminal pro-B type natriuretic peptide. Results: Among the 8,399 patients randomized, 5,036 patients (60.0%) had an ischemic etiology. Among the 3,363 patients (40.0%) with a nonischemic etiology, 1,595 (19.0% of all patients; 47% of nonischemic patients) had idiopathic dilated cardiomyopathy, 968 (11.5% of all patients; 28.8% of nonischemic patients) had a hypertensive cause, and 800 (9.5% of all patients, 23.8% of nonischemic patients) another cause (185 infective/viral, 158 alcoholic, 110 valvular, 66 diabetes, 30 drug-related, 14 peripartum–related, and 237 other). Whereas the unadjusted rates of all outcomes were highest in patients with an ischemic etiology, the adjusted hazard ratios (HRs) were not different from patients in the 2 major nonischemic etiology categories; for example, for the primary outcome, compared with ischemic (HR: 1.00), hypertensive 0.87 (95% confidence interval [CI]: 0.75 to 1.02), idiopathic 0.92 (95% CI: 0.82 to 1.04) and other 1.00 (95% CI: 0.85 to 1.17). The benefit of sacubitril/valsartan over enalapril was consistent across etiologic categories (interaction for primary outcome; p = 0.11). Conclusions: Just under one-half of patients in this global trial had nonischemic HF with reduced ejection fraction, with idiopathic and hypertensive the most commonly ascribed etiologies. Adjusted outcomes were similar across etiologic categories, as was the benefit of sacubitril/valsartan over enalapril. (Efficacy and Safety of LCZ696 Compared to Enalapril on Morbidity and Mortality of Patients With Chronic Heart Failure; NCT01035255

The XMM Cluster Survey: Evidence for energy injection at high redshift from evolution of the X-ray luminosity-temperature relation

We measure the evolution of the X-ray luminosity-temperature (L_X-T) relation since z~1.5 using a sample of 211 serendipitously detected galaxy clusters with spectroscopic redshifts drawn from the XMM Cluster Survey first data release (XCS-DR1). This is the first study spanning this redshift range using a single, large, homogeneous cluster sample. Using an orthogonal regression technique, we find no evidence for evolution in the slope or intrinsic scatter of the relation since z~1.5, finding both to be consistent with previous measurements at z~0.1. However, the normalisation is seen to evolve negatively with respect to the self-similar expectation: we find E(z)^{-1} L_X = 10^{44.67 +/- 0.09} (T/5)^{3.04 +/- 0.16} (1+z)^{-1.5 +/- 0.5}, which is within 2 sigma of the zero evolution case. We see milder, but still negative, evolution with respect to self-similar when using a bisector regression technique. We compare our results to numerical simulations, where we fit simulated cluster samples using the same methods used on the XCS data. Our data favour models in which the majority of the excess entropy required to explain the slope of the L_X-T relation is injected at high redshift. Simulations in which AGN feedback is implemented using prescriptions from current semi-analytic galaxy formation models predict positive evolution of the normalisation, and differ from our data at more than 5 sigma. This suggests that more efficient feedback at high redshift may be needed in these models.Comment: Accepted for publication in MNRAS; 12 pages, 6 figures; added references to match published versio

Administrative Managers – A Critical Link

Institutional responses to changes in the higher education environment have caused movements in the roles and identities of administrative managers in UK universities. These shifts have highlighted the problem for individuals of balancing traditional public service considerations of administration with institutional innovation and development. Administrative managers find themselves not only acting as independent arbiters, giving impartial advice on the basis of professional expertise, but also becoming involved in political judgements about institutional futures. They increasingly undertake an interpretive function between the various communities of the university and its external partners. As the boundaries of the university have become more permeable, administrative and academic management have inter-digitated, and hybrid roles have developed. In undertaking increasingly complex functions, therefore, administrative managers play a critical role in linking the academic and executive arms of governance in the university