scholarly journals A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

2020 ◽  
Author(s):  
yilan liu ◽  
jinjin chen ◽  
Anna N. Khusnutdinova ◽  
Kevin Correia ◽  
Patrick Diep ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Stability ◽  
Degradation Mechanism ◽  
Bioinformatic Analysis ◽  
Evolution System ◽  
Marine Cyanobacterium ◽  
E Coli ◽  
Renewable Feedstock ◽  
Promising Solution

Abstract Background: Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability.Results: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo. Conclusion: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

scholarly journals A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

2020 ◽  
Author(s):  
yilan liu ◽  
jinjin chen ◽  
Anna N. Khusnutdinova ◽  
Kevin Correia ◽  
Patrick Diep ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Degradation Mechanism ◽  
Bioinformatic Analysis ◽  
Evolution System ◽  
E Coli ◽  
In Vivo Experiments ◽  
Renewable Feedstock ◽  
The Family ◽  
Promising Solution

Abstract Background: Aldehyde decarbonylase (AD), which converts acyl aldehydes into alkanes, supplies promising solution for producing alkanes from renewable feedstock. However the instability of AD impeded its further application. Therefore, the current study aimed to investigate the degradation mechanism of AD and engineer it towards high stability. Results: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus via error-prone PCR based directed evolution system. Bioinformatic analysis revealed this C-terminal degron is common in the family of bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli , thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo . Conclusions: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

scholarly journals Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

2020 ◽  
Author(s):  
Ayushi Agrawal ◽  
Chandra Kanth Bandi ◽  
Tucker Burgin ◽  
Youngwoo Woo ◽  
Heather B. Mayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Click Chemistry ◽  
Directed Evolution ◽  
Cell Sorting ◽  
Screening Methods ◽  
Carbohydrate Active Enzymes ◽  
E Coli ◽  
Highly Sensitive ◽  
Increased Activity

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.

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
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.

scholarly journals Multiple Interaction Domains in FtsL, a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

2010 ◽  
Vol 192 (11) ◽  
pp. 2757-2768 ◽  
Author(s):  
Mark D. Gonzalez ◽  
Esra A. Akbay ◽  
Dana Boyd ◽  
Jon Beckwith
Keyword(s):  
Cell Division ◽  
Bioinformatic Analysis ◽  
The Other ◽  
Its Sequence ◽  
E Coli ◽  
Assembly Pathway ◽  
Enormous Amount ◽  
Interaction Domains ◽  
Multiple Interaction

ABSTRACT A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.

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

2020 ◽  
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.

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

The Analyst ◽  
2018 ◽  
Vol 143 (19) ◽  
pp. 4747-4755 ◽  
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.

scholarly journals In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

2008 ◽  
Vol 75 (2) ◽  
pp. 337-344 ◽  
Author(s):  
Daniel J. Koch ◽  
Mike M. Chen ◽  
Jan B. van Beilen ◽  
Frances H. Arnold
Keyword(s):  
Directed Evolution ◽  
Chain Length ◽  
Carbon Sources ◽  
Evolution System ◽  
Alkane Hydroxylase ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Selection For ◽  
Alkane Hydroxylases

ABSTRACT Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.

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

2020 ◽  
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.

Combinatorial Genetic Technology for the Development of New Anti-infectives

2004 ◽  
Vol 128 (12) ◽  
pp. 1351-1359
Author(s):  
Eleftheria Laios ◽  
Marny Waddington ◽  
Ashesh A. Saraiya ◽  
Kris Ann Baker ◽  
Elizabeth O'Connor ◽  
...  
Keyword(s):  
Drug Resistance ◽  
16S Rrna ◽  
High Throughput ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Evolution System ◽  
Genetic Technology ◽  
E Coli

Abstract Context.—We previously developed a novel technology known as instant evolution for high-throughput analysis of mutations in Escherichia coli ribosomal RNA. Objective.—To develop a genetic platform for the isolation of new classes of antiinfectives that are not susceptible to drug resistance based on the instant evolution system. Design.—Mutation libraries were constructed in the 16S rRNA gene of E coli and analyzed. In addition, the rRNA genes from a number of pathogenic bacteria were cloned and expressed in E coli. The 16S rRNA genes were incorporated into the instant-evolution system in E coli. Setting.—The Department of Biological Sciences, Wayne State University, Detroit, Mich. Main Outcome Measures.—Ribosome function was assayed by measuring the amount of green fluorescent protein produced by ribosomes containing mutant or foreign RNA in vivo. Results.—We have developed a new combinatorial genetic technology (CGT) platform that allows high-throughput in vivo isolation and analysis of rRNA mutations that might lead to drug resistance. This information is being used to develop anti-infectives that recognize the wild type and all viable mutants of the drug target. CGT also provides a novel mechanism for identifying new drug targets. Conclusions.—Antimicrobials produced using CGT will provide new therapies for the treatment of infections caused by human pathogens that are resistant to current antibiotics. The new therapeutics will be less susceptible to de novo resistance because CGT identifies all mutations of the target that might lead to resistance during the earliest stages of the drug discovery process.

scholarly journals Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences

2016 ◽  
Vol 113 (26) ◽  
pp. 7166-7170 ◽  
Author(s):  
Sharon Penias Navon ◽  
Guy Kornberg ◽  
Jin Chen ◽  
Tali Schwartzman ◽  
Albert Tsai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Bioinformatic Analysis ◽  
Single Amino Acid ◽  
E Coli ◽  
Naturally Occurring ◽  
Exit Tunnel ◽  
Amino Acid Triplet

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

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