Combinatorial InVitroFlow-assisted mutagenesis (CombIMut) yields a 41-fold improved CelA2 cellulase.

The combination of diversity generation methods and ultrahigh-throughput screening (uHTS) technologies is key to efficiently explore nature's sequence space and elucidate structure-function relationships of enzymes. Beneficial substitutions often cluster in a few regions and simultaneous amino acid substitutions at multiple positions (e.g., by OmniChange) will likely lead to further improved enzyme variants. An extensive screening effort is required to identify such variants, as the simultaneous randomization of four codons can easily yield over 105 potential enzyme variants. The combination of flow cytometer-based uHTS with cell-free compartmentalization technology using (w/o/w) double emulsions (InVitroFlow), provides analysis capabilities of up to 107 events per hour, thus enabling efficient screening. InVitroFlow is an elegant solution since diversity loss through a transformation of host cells is omitted and emulsion compartments provide a genotype-phenotype linkage through a fluorescence readout. In this study, a multisite saturation mutagenesis and an OmniChange library with four simultaneously saturated positions in the active site of CelA2 cellulase were screened using InVitroFlow. Screening of over 36 million events, yielded a significantly improved cellulase variant CelA2-M3 (H288F/H524Q) with an 8-fold increase in specific activity compared to the parent CelA2-H288F (83.9 U/mg) and a 41-fold increased specific activity (674.5 U/mg) compared to wildtype CelA2 (16.6 U/mg) for the substrate 4-MUC (4-methylumbelliferyl-ß d-cellobioside).

Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH.

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced Khalf at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).

Enzyme-Compatible Dynamic Nanoreactors from Electrostatically Bridged Like-Charged Surfactants and Polyelectrolytes.

Reported is an unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid (PAA) with like-charged surfactants. Amphiphilic polymer-surfactant complexes with high interfacial activity and a solubilization capacity exceeding that of conventional micelles are formed by bridging with Ca2+ ions. Incorporation of a protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance because of the improved solubilization of poorly water-soluble immobilized proteins. Competitive interfacial and intermolecular interactions on different time- and length-scales have been resolved using colorimetric analysis, dynamic tensiometry, light scattering, and molecular dynamic simulations. The discovered bridging association mechanism suggests reengineering of surfactant/polymer/enzyme formulations of modern detergents and opens new opportunities in advancing labile delivery systems.

Recombinant RNA Polymerase from Geobacillus sp. GHH01 as tool for rapid generation of metagenomic RNAs using in vitro technologies.

The exciting promises of functional metagenomics for the efficient discovery of novel biomolecules from nature are often hindered by factors associated with expression hosts. Aiming to shift functional metagenomics to a host independent innovative system, we here report on the cloning, heterologous expression, and reconstitution of an RNA polymerase (RNAP) from the thermophilic Geobacillus sp. GHH01 and in vitro transcription thereafter. The five genes coding for RNAP subunits, a house keeping sigma factor and two transcription elongation factors were cloned and over expressed as His6 -tagged and/ or tag-free proteins. Purified subunits were reconstituted into a functional polymerase through either the classical method of denaturation and subsequent renaturation or through a new resource and time efficient thermo-reconstitution method which takes advantage of the subunits' temperature stability. Additionally, all subunits were cloned into a single vector system for a co-expression and in vivo reconstitution to the RNAP core enzyme. Both the core and holoenzyme form of the RNAP exhibited a robust transcription activity and were stable up to a temperature of 55°C close to their fullest activity. The Geobacillus RNAP showed a remarkable in vitro transcription profile recognizing DNA template sequences of diverse bacteria and archaea as well as metagenomic samples. Coupled with a subsequent in vitro translation step, this recombinant transcription system could allow a new, clone-free, and functional metagenomic screening approach.

In vitro flow cytometry-based screening platform for cellulase engineering.

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-ß-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg).

Correction: A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels.

Correction for 'A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels' by Nina Lülsdorf et al., Chem. Commun., 2015, 51, 8679-8682.

A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution.

Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, especially for complex aromatic substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing phenyl benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compound 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5 %), linear detection range (15 to 250 µM), lower detection limit (15 µM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temperatures. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temperature (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).

A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels.

A high throughput whole cell flow cytometer screening toolbox was developed and validated by identifying improved variants (1.3-7-fold) for three hydrolases (esterase, lipase, cellulase). The screening principle is based on coupled enzymatic reaction using glucose derivatives which yield upon hydrolysis a fluorescent-hydrogel-layer on the surface of E. coli cells.

Advances in protease engineering for laundry detergents.

Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents.

A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes.

Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton's reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10(7) phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials.

Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg.

Enzyme promiscuity describes the ability of biocatalysts to catalyze conversions beyond their natural reactions. Enzyme engineering to promote side reactions is attractive for synthetic and industrial applications. For instance, a subtilisin Carlsberg protease variant (T58A/L216W) catalyzes in addition to its proteolytic activity the generation of peroxycarboxylic acids from corresponding esters in the presence of hydrogen peroxide. In the current study we used a semi-rational design approach to shift the specificity of subtilisin Carlsberg towards production of peroxycarboxylic acid. Among other identified amino acid substitutions, position Gly165 in the S1 binding pocket provided insights in subtilisin Carlsberg's promiscuity by promoting ester perhydrolysis. Catalytic constants of subtilisin Carlsberg for perhydrolysis of methyl-propionate, methyl-butyrate and methyl-pentanoate were increased up to 3.5-, 5.4- and 5.5-fold, respectively, while proteolysis was decreased up to 100-fold for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate (suc-AAPF-pNA).

Reengineering of subtilisin Carlsberg for oxidative resistance.

Mild bleaching conditions by in situ production of hydrogen peroxide or peroxycarboxylic acid is attractive for pulp, textile, and cosmetics industries. The enzymatic generation of chemical oxidants is often limited by enzyme stability. The subtilisin Carlsberg variant T58A/L216W/M221 is a promiscuous protease that was improved in producing peroxycarboxylic acids. In the current article, we identified two amino acid positions (Trp216 and Met221) that are important for the oxidative resistance of subtilisin Carlsberg T58A/L216W/M221. Site-saturation mutagenesis at positions Trp216 and Met221, which are located close to the active site, resulted in variants M4 (T58/W216M/M221) and M6 (T58A/W216L/M221C). Variants M4 (T58/W216M/M221) and M6 (T58A/W216L/M221C) have a 2.6-fold (M4) and 1.5-fold (M6) increased oxidative resistance and 1.4-fold increased kcat values for peroxycarboxylic acid formation, compared with wild-type subtilisin Carlsberg.

An efficient transformation method for Bacillus subtilis DB104.

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10(3) transformants/µg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is "easy" in handling and allows for the first time to generate large libraries (1.5 × 10(5) transformants/µg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.

Fluorescent assay for directed evolution of perhydrolases.

Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the µM range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.