Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

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.

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Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

Author(s):

Sviatlana Smolskaya ◽

Yaroslav Andreev

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Nonsense Suppression ◽

Expression Vectors ◽

Site Specific ◽

Suppressor Trna ◽

E Coli ◽

Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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Effect of E. Coli endotoxin on mammalian cell growth and recombinant protein production

In Vitro Cellular & Developmental Biology ◽

10.1007/bf02623686 ◽

1990 ◽

Vol 26 (12) ◽

pp. 1121-1122 ◽

Cited By ~ 10

Author(s):

J. Epstein ◽

C. E. Kelly ◽

M. M. Lee ◽

P. K. Donahoe

Keyword(s):

Cell Growth ◽

Recombinant Protein ◽

Mammalian Cell ◽

Recombinant Protein Production ◽

Protein Production ◽

E Coli ◽

Mammalian Cell Growth

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E. coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00402-21 ◽

2021 ◽

Author(s):

Chunhui Chen ◽

Yin Wang ◽

Hidemasa Nakaminami ◽

Eu Suk Kim ◽

George A. Jacoby ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Dna Gyrase ◽

Amino Acid Residues ◽

Site Specific ◽

E Coli ◽

Small Deletion ◽

Interaction Sites ◽

Repeat Proteins

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo crosslinking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286-298 on the Tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286-289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

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Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system

Microbial Cell Factories ◽

10.1186/s12934-021-01690-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Qin Fan ◽

Peter Neubauer ◽

Matthias Gimpel

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Protein Production ◽

Ralstonia Eutropha ◽

Process Time ◽

Synthesis Rate ◽

Heterologous Production ◽

Fed Batch ◽

Iptg Induction ◽

E Coli

Abstract Background Autoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate. Results We were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date. Conclusions The findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.

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Design of Orthogonal Pairs for Protein Translation: Selection Systems for Genetically Encoding Noncanonical Amino Acids in E. coli

Springer Protocols Handbooks - Hydrocarbon and Lipid Microbiology Protocols ◽

10.1007/8623_2015_105 ◽

2015 ◽

pp. 71-82 ◽

Cited By ~ 1

Author(s):

Jelena Jaric ◽

Nediljko Budisa

Keyword(s):

Amino Acids ◽

Protein Translation ◽

Noncanonical Amino Acids ◽

E Coli ◽

Selection Systems ◽

Orthogonal Pairs

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Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

Genes ◽

10.3390/genes9080414 ◽

2018 ◽

Vol 9 (8) ◽

pp. 414 ◽

Cited By ~ 3

Author(s):

Hao Chen ◽

Sumana Venkat ◽

Jessica Wilson ◽

Paige McGuire ◽

Abigail Chang ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Host Cell ◽

Protein Production ◽

Gene Overexpression ◽

Amino Acid Supplementation ◽

E Coli ◽

Biological Studies ◽

Associated Proteins ◽

K 12

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

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Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

Frontiers in Microbiology ◽

10.3389/fmicb.2021.744284 ◽

2021 ◽

Vol 12 ◽

Author(s):

Huawei Yi ◽

Jing Zhang ◽

Famin Ke ◽

Xiurong Guo ◽

Jian Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Expression ◽

Rna Polymerase ◽

T7 Rna Polymerase ◽

Noncanonical Amino Acids ◽

E Coli ◽

Growth Defects ◽

Genetic Code Expansion ◽

And Function

Incorporation of noncanonical amino acids (ncAAs) into proteins has been proven to be a powerful tool to manipulate protein structure and function, and to investigate many biological processes. Improving the yields of ncAA-containing proteins is of great significance in industrial-scale applications. Escherichia coli C321.ΔA was generated by the replacement of all known amber codons and the deletion of RF1 in the genome and has been proven to be an ideal host for ncAA-containing protein expression using genetic code expansion. In this study, we investigated the transcriptome and proteome profiles of this first codon reassignment strain and found that some functions and metabolic pathways were differentially expressed when compared with those of its parent strain. Genes involved in carbohydrate and energy metabolism were remarkably downregulated. Our results may provide important clues about the growth defects in E. coli C321.ΔA. Furthermore, we improved the yields of ncAA-containing proteins in E. coli C321.ΔA by integrating the T7 RNA polymerase system.

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Catalyst-Free and Site-Specific One-Pot Dual-Labeling of a Protein Directed by Two Genetically Incorporated Noncanonical Amino Acids

ChemBioChem ◽

10.1002/cbic.201200281 ◽

2012 ◽

Vol 13 (10) ◽

pp. 1405-1408 ◽

Cited By ~ 46

Author(s):

Bo Wu ◽

Zhiyong Wang ◽

Ying Huang ◽

Wenshe R. Liu

Keyword(s):

Amino Acids ◽

One Pot ◽

Noncanonical Amino Acids ◽

Site Specific ◽

Catalyst Free

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