scholarly journals Local genic base composition impacts protein production and cellular fitness

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4286 ◽  
Author(s):  
Erik M. Quandt ◽  
Charles C. Traverse ◽  
Howard Ochman
Keyword(s):  
Escherichia Coli ◽  
Base Composition ◽  
Protein Production ◽  
Purifying Selection ◽  
Specific Protein ◽  
Compositional Variation ◽  
Terminal Portion ◽  
A Genome ◽  
Selection For ◽  
Protein Feature

The maintenance of a G + C content that is higher than the mutational input to a genome provides support for the view that selection serves to increase G + C contents in bacteria. Recent experimental evidence fromEscherichia colidemonstrated that selection for increasing G + C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G + C content acts across all sites within a gene or is confined to particular genic regions or nucleotide positions. We systematically altered the G + C contents of the GFP gene and assayed its effects on the fitness of strains harboring each variant. Fitness differences were attributable to the base compositional variation in the terminal portion of the gene, suggesting a connection to the folding of a specific protein feature. Variants containing sequence features that are thought to result in rapid translation, such as low G + C content and high levels of codon adaptation, displayed highly reduced growth rates. Taken together, our results show that purifying selection acting against A and T mutations most likely results from their tendency to increase the rate of translation, which can perturb the dynamics of protein folding.

scholarly journals Selection on translation rate impacts genic base composition

2017 ◽  
Author(s):  
Erik M Quandt ◽  
Charles Traverse ◽  
Howard Ochman
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Experimental Evidence ◽  
Base Composition ◽  
Purifying Selection ◽  
Compositional Variation ◽  
Terminal Portion ◽  
Translation Rate ◽  
A Genome ◽  
Selection For

The maintenance of a G+C content that is higher than the mutational input to a genome supports the view that selection serves to increase G+C contents in many bacteria. Recent experimental evidence from Escherichia coli has demonstrated that selection for increasing G+C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G+C content acts across all sites within a gene or was confined to particular nucleotide positions or genic regions. We systematically altered the G+C contents of the GFP gene and assayed the effects of each variant on cellular fitness. The fitness differences were attributable to the base compositional variation in the terminal portion of the gene: increasing G+C content produced more stable mRNA secondary structures, which, in turn, slowed translation rate and allowed proper protein folding. We show that purifying selection against A and T mutations results from their tendency to increase the rate of translation and perturb the dynamics of protein folding.

scholarly journals Selection on translation rate impacts genic base composition

2017 ◽  
Author(s):  
Erik M Quandt ◽  
Charles Traverse ◽  
Howard Ochman
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Experimental Evidence ◽  
Base Composition ◽  
Purifying Selection ◽  
Compositional Variation ◽  
Terminal Portion ◽  
Translation Rate ◽  
A Genome ◽  
Selection For

The maintenance of a G+C content that is higher than the mutational input to a genome supports the view that selection serves to increase G+C contents in many bacteria. Recent experimental evidence from Escherichia coli has demonstrated that selection for increasing G+C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G+C content acts across all sites within a gene or was confined to particular nucleotide positions or genic regions. We systematically altered the G+C contents of the GFP gene and assayed the effects of each variant on cellular fitness. The fitness differences were attributable to the base compositional variation in the terminal portion of the gene: increasing G+C content produced more stable mRNA secondary structures, which, in turn, slowed translation rate and allowed proper protein folding. We show that purifying selection against A and T mutations results from their tendency to increase the rate of translation and perturb the dynamics of protein folding.

scholarly journals Spontaneous Deletion of a 209-Kilobase-Pair Fragment from the Escherichia coli Genome Occurs with Acquisition of Resistance to an Assortment of Infectious Phages

2008 ◽  
Vol 74 (14) ◽  
pp. 4256-4263 ◽  
Author(s):  
Yasunori Tanji ◽  
Kenji Hattori ◽  
Kohichi Suzuki ◽  
Kazuhiko Miyanaga
Keyword(s):  
Escherichia Coli ◽  
Parent Strain ◽  
Protein C ◽  
Protein Production ◽  
Growth Characteristics ◽  
Phage Resistance ◽  
Continuous Cultures ◽  
A Genome ◽  
Lipopolysaccharide Biosynthesis ◽  
Pair Fragment

ABSTRACT To breed resistance to an assortment of infectious phages, continuous cultures of Escherichia coli JM109 grown in a chemostat were exposed to phage mixtures prepared from sewage influent. Four sequential chemostat-grown cultures were each infected with a different phage mixture. At the end of a chemostat run, one phage-resistant colony was isolated and used to inoculate the subsequent culture. This process was repeated, and increased phage resistance of the input bacterial strain resulted from the successive challenges with different phage cocktails. Multiple mutations apparently accumulated progressively. A mutant isolated at the end of the four runs, designated D198, showed resistance to 38 of 40 phages that infect the parent strain, JM109. D198 produced less outer membrane protein C (OmpC) than JM109. However, restoration of the OmpC protein by plasmid-mediated complementation did not completely restore the susceptibility of D198 to the 38 phages. Therefore, alterations beyond the level of OmpC protein production contribute to the phage resistance of D198. PCR-based genetic analysis revealed that D198 has a genome that is 209 kbp (about 200 genes) smaller than JM109. The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.

scholarly journals A constraint-based modeling approach to reach an improved chemically defined minimal medium for recombinant antiEpEX-scFv production by Escherichia coli

2021 ◽  
Author(s):  
Aidin Behravan ◽  
atieh hashemi ◽  
Sayed-Amir Marashi
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Cell Growth ◽  
Recombinant Protein ◽  
Minimal Medium ◽  
Protein Production ◽  
Feeding Strategy ◽  
E Coli ◽  
A Genome ◽  
Constraint Based Modeling

Increasing demand for recombinant therapeutic proteins highlights the necessity of their yield improvement. Culture medium formulation is a popular approach for bioprocess optimization to improve therapeutic protein production. Constraint-based modeling can empower high-precision optimization through information on how media compounds affect metabolism and cell growth. In the current study, a genome-scale metabolic model (GEMM) of Escherichia coli cells was employed to design strategies of minimal medium supplementation for higher antiEpEX-scFv production. Dynamic flux balance analysis of the recombinant E. coli cell model predicted that ammonium was depleted during the process. Based on the simulations, three amino acids (Asn, Gln and Arg) were chosen to be added to the medium to compensate for ammonium depletion. Experimental validation suggested that the addition of these amino acids (one-by-one, or in combinations) can indeed improve cell growth and recombinant protein production. Then, design of experiment was used to optimize the concentrations of amino acids in the growth medium. About two-fold increase in the growth rate and total scFv expression level was observed using this strategy. We conclude that the GEMM-based approach can provide insights into an effective feeding strategy to improve the production of recombinant protein in E. coli.

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

scholarly journals Genetic basis and identification of candidate genes for wooden breast and white striping in commercial broiler chickens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Juniper A. Lake ◽  
Jack C. M. Dekkers ◽  
Behnam Abasht
Keyword(s):  
Candidate Genes ◽  
Broiler Chickens ◽  
Genetic Basis ◽  
Genome Wide Association Study ◽  
Specific Protein ◽  
Chromosome 11 ◽  
A Genome ◽  
White Striping ◽  
Commercial Broiler ◽  
Wooden Breast

AbstractWooden breast (WB) and white striping (WS) are highly prevalent and economically damaging muscle disorders of modern commercial broiler chickens characterized respectively by palpable firmness and fatty white striations running parallel to the muscle fiber. High feed efficiency and rapid growth, especially of the breast muscle, are believed to contribute to development of such muscle defects; however, their etiology remains poorly understood. To gain insight into the genetic basis of these myopathies, a genome-wide association study was conducted using a commercial crossbred broiler population (n = 1193). Heritability was estimated at 0.5 for WB and WS with high genetic correlation between them (0.88). GWAS revealed 28 quantitative trait loci (QTL) on five chromosomes for WB and 6 QTL on one chromosome for WS, with the majority of QTL for both myopathies located in a ~ 8 Mb region of chromosome 5. This region has highly conserved synteny with a portion of human chromosome 11 containing a cluster of imprinted genes associated with growth and metabolic disorders such as type 2 diabetes and Beckwith-Wiedemann syndrome. Candidate genes include potassium voltage-gated channel subfamily Q member 1 (KCNQ1), involved in insulin secretion and cardiac electrical activity, lymphocyte-specific protein 1 (LSP1), involved in inflammation and immune response.

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