Cross-Species Synthetic Promoter Library: Finding Common Ground between Pseudomonas taiwanensis VLB120 and Escherichia coli.

The potential of nonmodel organisms for industrial biotechnology is increasingly becoming evident since advances in systems and synthetic biology have made it possible to explore their unique traits. However, the lack of adequately characterized genetic elements that drive gene expression impedes benchmarking nonmodel with model organisms. Promoters are one of the genetic elements that contribute significantly to gene expression, but information about their performance in different organisms is limited. This work addresses this bottleneck by characterizing libraries of synthetic σ70-dependent promoters controlling the expression of msfGFP, a monomeric, superfolder green fluorescent protein, in both Escherichia coli TOP10 and Pseudomonas taiwanensis VLB120, a less explored microbe with industrially attractive attributes. We adopted a standardized method for comparing gene promoter strength across species and laboratories. Our approach uses fluorescein calibration and adjusts for cell growth variation, enabling accurate cross-species comparisons. The quantitative description of promoter strength is a valuable expansion of P. taiwanensis VLB120's genetic toolbox, while the comparison with the performance in E. coli facilitates the evaluation of P. taiwanensis VLB120's potential as a chassis for biotechnology applications.

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

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 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.

A Competitive Flow Cytometry Screening System for Directed Evolution of Therapeutic Enzyme.

A ligand-mediated eGFP-expression system (LiMEx) was developed as a novel flow cytometry based screening platform that relies on a competitive conversion/binding of arginine between arginine deiminase and arginine repressor. Unlike product-driven detection systems, the competitive screening platform allows to evolve enzymes toward efficient operation at low substrate concentrations under physiological conditions. The principle of LiMEx was validated by evolving arginine deiminase (ADI, an anticancer therapeutic) for stronger inhibition of tumor growth. After screening of ∼8.2 × 10(6) clones in three iterative rounds of epPCR libraries, PpADI (ADI from Pseudomonas plecoglossicida) variant M31 with reduced S0.5 value (0.17 mM compared to 1.23 mM (WT)) and, importantly, increased activity at physiological arginine concentration (M31:6.14 s(-1); WT: not detectable) was identified. Moreover, M31 showed a significant inhibitory effect against SK-MEL-28 and G361 melanoma cell lines. (IC50 = 0.02 µg/mL for SK-MEL-28 and G361).

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.