Turbo‐charging proximity labeling: directed evolution of promiscuous biotin ligase for efficient proteomic mapping in vivo

AbstractEngineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

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Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

Journal of Virology ◽

10.1128/jvi.00593-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 4

Author(s):

Hui Cai ◽

Meisui Liu ◽

Charles J. Russell

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Virus Infection ◽

Directed Evolution ◽

Influenza Virus Infection ◽

Foreign Gene ◽

Reporter Virus ◽

Wild Type ◽

Bioluminescence Signal

ABSTRACTReporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus withNanoLuc luciferaseinserted into the 5′ end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we comparedin vivoreplication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations andPB2noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-likein vivofitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCEInfluenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

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Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms19102989 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2989 ◽

Cited By ~ 3

Author(s):

Ji Zhang ◽

Fuying Ma ◽

Xiaoyu Zhang ◽

Anli Geng

Keyword(s):

Directed Evolution ◽

Laccase Activity ◽

Catalytic Properties ◽

Random Mutagenesis ◽

Catalytic Performance ◽

Industrial Applications ◽

Subunit Interaction ◽

Cerrena Unicolor ◽

Protein Model

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.

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Creating Hybrid Genes by Homologous Recombination

Disease Markers ◽

10.1155/2000/596468 ◽

2000 ◽

Vol 16 (1-2) ◽

pp. 3-13 ◽

Cited By ~ 5

Author(s):

Peter L. Wang

Keyword(s):

Directed Evolution ◽

High Efficiency ◽

Genomic Sequence ◽

Sequence Data ◽

Strand Breaks ◽

E Coli ◽

Hybrid Genes ◽

Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

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Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase

Biochemical Journal ◽

10.1042/bj3020881 ◽

1994 ◽

Vol 302 (3) ◽

pp. 881-887 ◽

Cited By ~ 97

Author(s):

A Chapman-Smith ◽

D L Turner ◽

J E Cronan ◽

T W Morris ◽

J C Wallace

Keyword(s):

Escherichia Coli ◽

Exchange Chromatography ◽

Carrier Protein ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

E Coli ◽

Biotin Ligase ◽

Domain Peptide

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

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Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants

Protein Engineering Design and Selection ◽

10.1093/protein/gzr030 ◽

2011 ◽

Vol 24 (8) ◽

pp. 607-616 ◽

Cited By ~ 22

Author(s):

Yasuhisa Asano ◽

Mohammad Dadashipour ◽

Mizue Yamazaki ◽

Nobutaka Doi ◽

Hidenobu Komeda

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Functional Expression ◽

Hydroxynitrile Lyase

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In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

10.26434/chemrxiv.12485993.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shreyans Chordia ◽

Siddarth Narasimhan ◽

Alessandra Lucini Paioni ◽

Marc Baldus ◽

Gerard Roelfes

Keyword(s):

Directed Evolution ◽

Cell Fractionation ◽

Whole Cell ◽

Whole Cells ◽

E Coli ◽

Whole Cell Biocatalysis ◽

Catalytically Active ◽

Active Transition ◽

Artificial Metalloenzymes

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

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Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting

The Analyst ◽

10.1039/c8an00851e ◽

2018 ◽

Vol 143 (19) ◽

pp. 4747-4755 ◽

Cited By ~ 8

Author(s):

Joanna C. Sadler ◽

Andrew Currin ◽

Douglas B. Kell

Keyword(s):

Monoamine Oxidase ◽

Directed Evolution ◽

High Throughput ◽

Cell Sorting ◽

Oxidase Activity ◽

Functional Enrichment ◽

High Throughput Screen ◽

Fluorescence Activated Cell Sorting ◽

E Coli

A novel ultra-high throughput screen forin vivodetection of oxidase activity inE. colicells and its application to directed evolution.

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Characterisation, Manipulation and Directed Evolution of Non-Ribosomal Peptide Synthetase Enzymes

10.26686/wgtn.17009951 ◽

2021 ◽

Author(s):

◽

Jeremy George Owen

Keyword(s):

Amino Acid ◽

Directed Evolution ◽

Pseudomonas Syringae ◽

Gene Knockout ◽

Iron Starvation ◽

Biochemical Characterisation ◽

T Domain ◽

Stereo Selectivity

Non-ribosomal peptide synthetases (NRPS) are large, modular enzymes that synthesisebiologically active secondary metabolites from amino acid precursors without the need for anucleic acid template. NRPS play an integral role in microbial physiology and also havepotential applications in the synthesis of novel peptide molecules. Both of these aspects areexamined in this thesis. Under conditions of iron starvation Pseudomonas syringae synthesises siderophores for activeuptake of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent moleculethat is assembled from amino acid (aa) precursors by NRPS. Five putative pyoverdine NRPSgenes in P. syringae pv. phaseolicola 1448a (Ps1448a) were identified and characterised insilico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Creation ofpyoverdine null Ps1448a enabled identification of a previously uncharacterised temperatureregulatedsecondary siderophore, achromobactin, which is NRPS independent and has loweraffinity for iron. Pyoverdine and achromobactin null mutants were characterised in regard toiron uptake, virulence and growth in iron-limited conditions. Determination of the substratespecificity for the seven adenylation (A) domains of the Ps1448a pyoverdine sidechain NRPSwas also attempted. Although ultimately unsuccessful, these attempts provided a rigorousassessment of methods for the expression, purification and biochemical characterisation of Adomains. The Ps1448a NRPS were subsequently employed in domain swapping experiments to testcondensation (C) domain specificity for aa substrates during peptide formation in vivo. Experiments in which the terminal C- and/or A-domain of the Pseudomonas aeruginosa(PAO1) pyoverdine NRPS system were replaced with alternative domains from Ps1448a andPAO1 were consistent with previous in vitro observations that C-domains exhibit strongsidechain and stereo-selectivity at the downstream aa position, but only stereo-selectivity atthe upstream aa position. These results prompted investigation into the role of inter-domain communication in NRPSfunction, to test the hypothesis that the thiolation (T) domain enters into specific interactionswith other domains, which might provide an alternative explanation for the diminished activityof recombinant NRPS enzymes. A recently characterised single-module NRPS, bpsA, waschosen as a reporter gene for these experiments based on its ability to generate blue pigment inEscherichia coli. Substitution of the native bpsA T-domain consistently impaired function,consistent with the hypothesis. It was shown that directed evolution could be applied to restorefunction in substituted T-domains. Mutations that restored function were mapped in silico, anda structural model for interaction between the thioester (TE) and T-domain of BpsA wasderived. The utility of bpsA for discovery and characterisation of phosphopantetheinyl transferase(PPTase) enzymes was also investigated. In vivo and in vitro assays for determination ofPPTase activity were developed and a high-throughput screen for discovery of new PPTases inenvironmental DNA libraries was successfully implemented.

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Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Life ◽

10.3390/life10090179 ◽

2020 ◽

Vol 10 (9) ◽

pp. 179

Author(s):

Jorge D. García-García ◽

Jaya Joshi ◽

Jenelle A. Patterson ◽

Lidimarie Trujillo-Rodriguez ◽

Christopher R. Reisch ◽

...

Keyword(s):

Directed Evolution ◽

Target Gene ◽

Enzyme Engineering ◽

Plant Evolution ◽

Kinetic Properties ◽

Continuous Systems ◽

Yield And Quality ◽

Plant Enzyme

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

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