High-throughput screening, next generation sequencing and machine learning: advanced methods in enzyme engineering

Enzyme engineering is an important biotechnological process capable of generating tailored biocatalysts for applications in industrial chemical conversion and biopharma. Typical enhancements sought in enzyme engineering and in vitro evolution campaigns include improved folding stability, catalytic activity, and/or substrate specificity. Despite significant progress in recent years in the areas of high-throughput screening and DNA sequencing, our ability to explore the vast space of functional enzyme sequences remains severely limited. Here, we review the currently available suite of modern methods for enzyme engineering, with a focus on novel readout systems based on enzyme cascades, and new approaches to reaction compartmentalization including single-cell hydrogel encapsulation techniques to achieve a genotype–phenotype link. We further summarize systematic scanning mutagenesis approaches and their merger with deep mutational scanning and massively parallel next-generation DNA sequencing technologies to generate mutability landscapes. Finally, we discuss the implementation of machine learning models for computational prediction of enzyme phenotypic fitness from sequence. This broad overview of current state-of-the-art approaches for enzyme engineering and evolution will aid newcomers and experienced researchers alike in identifying the important challenges that should be addressed to move the field forward

Risk of Recurrent Arterial Ischemic Stroke in Childhood: A Prospective International Study.

Background and purposePublished cohorts of children with arterial ischemic stroke (AIS) in the 1990s to early 2000s reported 5-year cumulative recurrence rates approaching 20%. Since then, utilization of antithrombotic agents for secondary stroke prevention in children has increased. We sought to determine rates and predictors of recurrent stroke in the current era.MethodsThe Vascular Effects of Infection in Pediatric Stroke (VIPS) study enrolled 355 children with AIS at 37 international centers from 2009 to 2014 and followed them prospectively for recurrent stroke. Index and recurrent strokes underwent central review and confirmation, as well as central classification of causes of stroke, including arteriopathies. Other predictors were measured via parental interview or chart review.ResultsOf the 355 children, 354 survived their acute index stroke, and 308 (87%) were treated with an antithrombotic medication. During a median follow-up of 2.0 years (interquartile range, 1.0-3.0), 40 children had a recurrent AIS, and none had a hemorrhagic stroke. The cumulative stroke recurrence rate was 6.8% (95% confidence interval, 4.6%-10%) at 1 month and 12% (8.5%-15%) at 1 year. The sole predictor of recurrence was the presence of an arteriopathy, which increased the risk of recurrence 5-fold when compared with an idiopathic AIS (hazard ratio, 5.0; 95% confidence interval, 1.8-14). The 1-year recurrence rate was 32% (95% confidence interval, 18%-51%) for moyamoya, 25% (12%-48%) for transient cerebral arteriopathy, and 19% (8.5%-40%) for arterial dissection.ConclusionsChildren with AIS, particularly those with arteriopathy, remain at high risk for recurrent AIS despite increased utilization of antithrombotic agents. Therapies directed at the arteriopathies themselves are needed

Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger

Glucose oxidase (GOx) is a promising candidate for construction of implantable miniature biofuel cells and biosensors for continuous glucose monitoring. The main drawback that limits current application of GOx in these devices is its low stability, especially sensitivity to reactive oxygen species. In order to address this problem, we performed saturation mutagenesis at all 11 methionine residues as their interaction with reactive oxygen species inactivates enzymes. For successful screening of these libraries a method based on yeast surface display (YSD) systems was developed. Mutations at methionine positions close to the GOx active site contributed the most to the oxidative stability, and combinations of the four best single mutations were tested. Combined mutants did not show higher stability or activity compared to the parental single mutants. To confirm oxidative stability of YSD expressed GOx mutants they were re-cloned in Pichia pastoris, purified and immobilized on macroporous copolymer. The additional kinetic analysis of immobilized GOx mutants confirmed that the best mutant with only one mutation close to the active site (M561S) has 2.5 times increased half-life in the presence of hydrogen peroxide compared to the wild-type variant

Dietary fructose-related adiposity and glucocorticoid receptor function in visceral adipose tissue of female rats

Excessive fructose intake coincides with the growing rate of obesity and metabolic syndrome, with women being more prone to these disorders than men. Findings that detrimental effects of fructose might be mediated by glucocorticoid regeneration in adipose tissue only indirectly implicated glucocorticoid receptor (GR) in the phenomenon. The aim of the present study was to elucidate whether fructose overconsumption induces derangements in GR expression and function that might be associated with fructose-induced adiposity in females. We examined effects of fructose-enriched diet on GR expression and function in visceral adipose tissue of female rats. Additionally, we analyzed the expression of genes involved in glucocorticoid prereceptor metabolism {[}11 beta-hydroxysteroid dehydrogenase type 1 (11 beta HSD1) and hexose-6-phosphate dehydrogenase], lipolysis (hormone-sensitive lipase) and lipogenesis (sterol regulatory element binding protein 1 and peroxisomal proliferator-activated receptor gamma). Fructose-fed rats had elevated energy intake that resulted in visceral adiposity, as indicated by increased visceral adipose tissue mass and its share in the whole-body weight. GR hormone binding capacity and affinity, as well as the expression of GR gene at both mRNA and protein levels were reduced in visceral adipose tissue of the rats on fructose diet. The glucocorticoid prereceptor metabolism was stimulated, as evidenced by elevated tissue corticosterone, while the key regulators of lipolysis and lipogenesis remained unaffected by fructose diet. The results suggest that the 11 beta HSD1-mediated elevation of intracellular corticosterone may induce GR downregulation, which may be associated with failure of GR to stimulate lipolysis in fructose-fed female rats.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}III41009

Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator

Yeast surface display is a valuable tool for protein engineering and directed evolution; however, significant variability in the copy number (i.e., avidity) of displayed variants on the yeast cell wall complicates screening and selection campaigns. Here, we report an engineered titratable display platform that modulates the avidity of Aga2-fusion proteins on the yeast cell wall dependent on the concentration of the anhydrotetracycline (aTc) inducer. Our design is based on a genomic Aga1 gene copy and an episomal Aga2-fusion construct both under the control of an aTc-dependent transcriptional regulator that enables stoichiometric and titratable expression, secretion, and display of Aga2-fusion proteins. We demonstrate tunable display levels over 2-3 orders of magnitude for various model proteins, including glucose oxidase enzyme variants, mechanostable dockerin-binding domains, and anti-PDL1 affibody domains. By regulating the copy number of displayed proteins, we demonstrate the effects of titratable avidity levels on several specific phenotypic activities, including enzyme activity and cell adhesion to surfaces under shear flow. Finally, we show that titrating down the display level allows yeast-based binding affinity measurements to be performed in a regime that avoids ligand depletion effects while maintaining small sample volumes, avoiding a well-known artifact in yeast-based binding assays. The ability to titrate the multivalency of proteins on the yeast cell wall through simple inducer control will benefit protein engineering and directed evolution methodology relying on yeast display for broad classes of therapeutic and diagnostic proteins of interest.ISSN:2161-506

Purification and functional analysis of the recombinant protein isolated from E. coli by employing three different methods of bacterial lysis

In this paper, the purification of the human recombinant protein expressed in E. coli using the GSTGene Fusion System, by applying various methods of bacterial lysis: sonication, freeze/thaw and beadbeating, is presented. The study was an attempt to compare the properties of the proteins obtained by the sonication method, recommended by manufacturers but inaccessible for many researchers, with those obtained using two other readily available lysis methods. The data show that all purified proteins were soluble and intact with the highest protein yield being obtained via the freeze/thaw method. The results of functional analysis indicate that the proteins purified using the sonication and freeze/thaw methods of lysis exhibited similar DNA binding affinity, while the protein purified by beadbeating was also functional but with a lower binding affinity. The conclusion of this study is that all three lysis methods could be successfully employed for protein purification