Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing

AbstractPyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.

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Improved pyrrolysine biosynthesis through continuous directed evolution of the complete pathway

10.21203/rs.3.rs-78440/v1 ◽

2020 ◽

Author(s):

Joanne Ho ◽

Corwin Miller ◽

Jacob Mattia ◽

Matthew Bennett

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Directed Evolution ◽

Biosynthetic Pathway ◽

Fluorescent Protein ◽

Proteinogenic Amino Acid ◽

E Coli ◽

Rational Engineering ◽

Fundamental Building Block ◽

Green Fluorescent

Abstract Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improved Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we devised an approach termed Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway exhibited a 32-fold improved yield of Pyl-containing super- folder green fluorescent protein (sfGFP) compared to the rationally engineered ancestor, whereas the WT pathway produced no detectable quantities of Pyl-containing sfGFP. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl- proteins in E. coli.

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The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability

Viruses ◽

10.3390/v13061101 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1101

Author(s):

Michael J. Love ◽

David Coombes ◽

Sarah H. Manners ◽

Gayan S. Abeysekera ◽

Craig Billington ◽

...

Keyword(s):

Escherichia Coli ◽

Thermal Stability ◽

Antibacterial Agents ◽

Catalytic Mechanism ◽

Bioinformatic Analysis ◽

Catalytic Triad ◽

Escherichia Coli O157 ◽

Hydrophobic Core ◽

Rational Engineering ◽

Enzymatic Function

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

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Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit DefectiveN-Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

Applied and Environmental Microbiology ◽

10.1128/aem.00499-14 ◽

2014 ◽

Vol 80 (12) ◽

pp. 3729-3740 ◽

Cited By ~ 43

Author(s):

James D. Winkler ◽

Carlos Garcia ◽

Michelle Olson ◽

Emily Callaway ◽

Katy C. Kao

Keyword(s):

Escherichia Coli ◽

Osmotic Stress ◽

Cell Shape ◽

Current Knowledge ◽

Point Mutations ◽

Nacl Stress ◽

Transcriptional Analysis ◽

Content Type ◽

Rational Engineering ◽

Osmotic Stress Tolerance

ABSTRACTBiocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable ofin situDNA exchange) and asexualEscherichia colistrains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controllingN-acetylglucosamine catabolism (nagC,nagA), cell shape (mrdA,mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions betweennagC,nagA,fimA, andproVdeletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes inompACGLporin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future.

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Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesis

Metabolic Engineering Communications ◽

10.1016/j.mec.2021.e00167 ◽

2021 ◽

Vol 12 ◽

pp. e00167

Author(s):

Chen Minliang ◽

Ma Chengwei ◽

Chen Lin ◽

An-Ping Zeng

Keyword(s):

Escherichia Coli ◽

Tryptophan Biosynthesis ◽

Laboratory Evolution ◽

Rational Engineering

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An Engineered Synthetic Pathway for Discovering Nonnatural Nonribosomal Peptides in Escherichia coli

mBio ◽

10.1128/mbio.01474-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 3

Author(s):

Sara Cleto ◽

Timothy K. Lu

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Chemical Diversity ◽

Bioactive Molecules ◽

Nonribosomal Peptides ◽

Biosynthetic Pathways ◽

Biosynthetic Genes ◽

Synthetic Pathway ◽

Therapeutic Applications ◽

Rational Engineering

ABSTRACT Peptides that are synthesized independently of the ribosome in plants, fungi, and bacteria can have clinically relevant anticancer, antihemochromatosis, and antiviral activities, among many other. Despite their natural origin, discovering new natural products is challenging, and there is a need to expand the chemical diversity that is accessible. In this work, we created a novel, compressed synthetic pathway for the heterologous expression and diversification of nonribosomal peptides (NRPs) based on homologs of siderophore pathways from Escherichia coli and Vibrio cholerae. To enhance the likelihood of successful molecule production, we established a selective pressure via the iron-chelating properties of siderophores. By supplementing cells containing our synthetic pathway with different precursors that are incorporated into the pathway independently of NRP enzymes, we generated over 20 predesigned, novel, and structurally diverse NRPs. This engineering approach, where phylogenetically related genes from different organisms are integrated and supplemented with novel precursors, should enable heterologous expression and molecular diversification of NRPs. IMPORTANCE Nonribosomal peptides (NRPs) constitute a source of bioactive molecules with potential therapeutic applications. However, discovering novel NRPs by rational engineering of biosynthetic pathways remains challenging. Here, we show that a synthetic compressed pathway in which we replaced biosynthetic genes with their ancestral homologs and orthologs enabled successful heterologous NRP expression. Polyamines added exogenously were incorporated into nascent NRPs, and molecular production was pressured by growing the host under conditions that make such NRPs beneficial for survival. This multilayered approach resulted in the assembly of over 20 distinct and novel molecules. We envision this strategy being used to enable the production of NRPs from heterologous pathways. IMPORTANCE Nonribosomal peptides (NRPs) constitute a source of bioactive molecules with potential therapeutic applications. However, discovering novel NRPs by rational engineering of biosynthetic pathways remains challenging. Here, we show that a synthetic compressed pathway in which we replaced biosynthetic genes with their ancestral homologs and orthologs enabled successful heterologous NRP expression. Polyamines added exogenously were incorporated into nascent NRPs, and molecular production was pressured by growing the host under conditions that make such NRPs beneficial for survival. This multilayered approach resulted in the assembly of over 20 distinct and novel molecules. We envision this strategy being used to enable the production of NRPs from heterologous pathways.

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Characterization of the Effects ofn-butanol on the cell envelope ofE. coli

10.1101/062547 ◽

2016 ◽

Author(s):

Eugene Fletcher ◽

Teuta Pilizota ◽

Philip R. Davies ◽

Alexander McVey ◽

Chris E. French

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cell Envelope ◽

Membrane Protrusion ◽

Bacterial Strains ◽

External Concentration ◽

Inner Membrane ◽

E Coli ◽

Rational Engineering

ABSTRACTBiofuel alcohols have severe consequences on the microbial hosts used in their biosynthesis, which limits the productivity of the bioconversion. The cell envelope is one of the most strongly affected structures, in particular, as the external concentration of biofuels rises during biosynthesis. Damage to the cell envelope can have severe consequences, such as impairment of transport into and out of the cell; however the nature of butanol-induced envelope damage has not been well characterized. In the present study, the effects ofn-butanol on the cell envelope ofEscherichia coliwere investigated. Using enzyme and fluorescence-based assays, we observed that 1% v/v n-butanol resulted in release of lipopolysaccharides from the outer membrane ofE. coliand caused ‘leakiness’ in both outer and inner membranes. Higher concentrations ofn-butanol, within the range of 2% – 10% (v/v), resulted in inner membrane protrusion through the peptidoglycan observed by characteristic blebs. The findings suggest that strategies for rational engineering of butanol-tolerant bacterial strains should take into account all components of the cell envelope.

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Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081693 ◽

1978 ◽

Vol 36 (2) ◽

pp. 166-167 ◽

Cited By ~ 1

Author(s):

G. Stöffler ◽

R.W. Bald ◽

J. Dieckhoff ◽

H. Eckhard ◽

R. Lührmann ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Spatial Arrangement ◽

Small Subunit ◽

Antibody Binding ◽

Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

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