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 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 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 HreP, an In Vivo-Expressed Protease of Yersinia enterocolitica, Is a New Member of the Family of Subtilisin/Kexin-Like Proteases

2001 ◽  
Vol 183 (12) ◽  
pp. 3556-3563 ◽  
Author(s):  
Gerhard Heusipp ◽  
Glenn M. Young ◽  
Virginia L. Miller
Keyword(s):  
Yersinia Enterocolitica ◽  
Gene Encoding ◽  
Significant Similarity ◽  
E Coli ◽  
Calcium Dependent ◽  
Autocatalytic Processing ◽  
The Family ◽  
Flagellum Biosynthesis

ABSTRACT The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene,hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocoliticathat were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica,hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in Escherichia coli, Salmonella, andYersinia pestis than in Y. enterocolitica. Analysis of the distribution ofhreP between different Yersinia isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of Anabaena variabilis, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His6 tag fusion protein in E. coli revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein.

scholarly journals Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evgeniya Schastnaya ◽  
Zrinka Raguz Nakic ◽  
Christoph H. Gruber ◽  
Peter Francis Doubleday ◽  
Aarti Krishnan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate ◽  
Acetate Metabolism ◽  
Post Translational Modification ◽  
E Coli ◽  
In Vivo Experiments ◽  
Functional Relevance ◽  
Regulation Of Enzyme Activity

AbstractProtein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

Nitrofurazone-reducing enzymes in E. coli and their role in drug activation in vivo

1975 ◽  
Vol 21 (10) ◽  
pp. 1484-1491 ◽  
Author(s):  
D. R. McCalla ◽  
P. Olive ◽  
Yu Tu ◽  
M. L. Fan
Keyword(s):  
Resistant Mutant ◽  
Reductive Activation ◽  
E Coli ◽  
In Vivo Experiments ◽  
Nitrofuran Derivatives ◽  
Oxygen Reductase ◽  
Resistant Strains ◽  
Drug Activation

Earlier work showed that Escherichia coli contains at least two enzymes which reduce nitrofurazone and other nitrofuran derivatives. One of these enzymes is lacking in some nitrofurazone-resistant mutant strains. We now report that there are three separable nitrofuran reductases in this organism: reductase I (mol. wt. ~50 000, insensitive to O2), reductase IIa (mol. wt. ~120 000, inhibited by oxygen), reductase IIb (mol. wt. ~700 000, inhibited by O2).Unstable metabolites formed during the reduction of nitrofurazone by preparations containing reductases IIa and IIb produce breaks in DNA in vitro. In vivo experiments with nitrofurazone-resistant strains, which lack reductase II but contain reductases IIa and IIb, demonstrated that lethality, mutation, and DNA breakage are all greatly increased when cultures are incubated under anaerobic conditions, i.e., conditions such that reductase II is active. These results provide further evidence for the importance of reductive activation of nitrofurazone.

scholarly journals The p50 Subunit of NF-κB Is Critical for In Vivo Clearance of the Noninvasive Enteric Pathogen Citrobacter rodentium

2008 ◽  
Vol 76 (11) ◽  
pp. 4978-4988 ◽  
Author(s):  
Alison Dennis ◽  
Takahiro Kudo ◽  
Laurens Kruidenier ◽  
Francis Girard ◽  
Valerie F. Crepin ◽  
...  
Keyword(s):  
Immune Response ◽  
Citrobacter Rodentium ◽  
Necrosis Factor Alpha ◽  
E Coli ◽  
The Family ◽  
Factor Alpha ◽  
Mucosal Thickening ◽  
Transcription Factor Nuclear Factor ◽  
Necrosis Factor

ABSTRACT Citrobacter rodentium, a natural mouse pathogen, belongs to the family of extracellular enteric pathogens that includes enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). C. rodentium shares many virulence factors with EPEC and EHEC and relies on attaching-and-effacing lesion formation for colonization and infection of the gut. In vivo, C. rodentium infection is characterized by increased epithelial cell proliferation, mucosal thickening, and a TH1-type immune response, but with protective immunity believed to be mediated by serum immunoglobulin G (IgG). In this work, we characterize the immune response and pathology of mice lacking the p50 subunit of the transcription factor nuclear factor kappa B (NF-κB) during C. rodentium infection. We show that p50−/− mice are unable to clear C. rodentium infection. Furthermore, these animals show a reduced influx of immune cells into infected colonic tissue and greater levels of mucosal hyperplasia and the cytokines tumor necrosis factor alpha and gamma interferon. Surprisingly, despite being unable to eliminate infection, p50−/− mice showed markedly higher levels of anti-Citrobacter IgG and IgM, suggesting that antibody alone is not responsible for bacterial clearance. These data also demonstrate that non-NF-κB-dependent defenses are insufficient to control C. rodentium infection, and hence, the NF-κB p50 subunit is critical for defense against this noninvasive pathogen.

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