Design of an improved host platform for the over expression of recombinant proteins in Escherichia coli
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE29440 | GEO | 2011/05/24
SECONDARY ACCESSION(S): PRJNA141517
REPOSITORIES: GEO
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Project description:The objective of this work was to design an improved host platform for recombinant protein expression in E. coli. The approach involves first to create a library of the E. coli genomic DNA in different expression vectors and screen for probable transcripts which may lead to slow growing colonies and also simultaneously over-expression of recombinant proteins. To observe its effect on host performance, these genes were knocked out from the E. coli genome. A CG2 strain has been created by knocking in vhb gene gene downstream of the acetate promoter and knocking down ribB gene in DH5α and transformed with Recombinant GFP cloned in pBAD33. E. coli DH5α (control strain), and strain CG2 were transformed with pBAD33-GFP and cultured in well controlled fed batch fermentations. The effect of three parameters was sought to be studied. The first was the effect of time post induction and thus samples were collected 0, 2, 4 and 6 hours post induction. The second parameter was the effect of host modification and hence both the control and the modified host (CG2) were studied at μ = 0.3 h-1. The third parameter was the effect of specific growth rate and thus the host (CG2) was run at μ = 0.3 h-1 and 0.6 h-1.
2011-05-23 | E-GEOD-29440 | biostudies-arrayexpress
Project description:The ability to control the synthesis of products in the absence of cell division remains an attractive alternative for the optimization of microbial processes. If this could be achieved, it would dramatically expand the productivity of many microbial processes by increasing product synthesis in the absence of an increase in cell mass. In the present work we have looked at the factors involved in the design of a host where the fluxes would be channeled towards product formation rather than biomass synthesis. To identify the genes responsible for diverting the metabolic flux specifically towards product formation we have used for this study is quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabolically active cells. These cells channel the metabolic flux towards recombinant protein production and therefore the specific product yield per unit biomass is significantly higher. Indole which is previously known to be a signalling molecule is here used as an inducer of Quiescence. E.coli L-Asparaginase is used as a model protein in this work.
2011-06-01 | GSE29486 | GEO
Project description:Amino acid starvation during recombinant protein production (RPP) induces metabolic stress to cellular host which results in reduced productivity of the recombinant bioprocess. In present study, supplementation of amino acids in a chemically defined medium showed several folds increase in recombinant product titers in E. coli BL21 DE3 strain. To understand, the effect of amino acid supplementation in alleviating cellular stress during RPP a deep insight of cellular physiology must be studied. Here, we performed transcriptomic analysis of E. coli expressing a recombinant protein in two different conditions: with (5 mM of all 20 amino acids) as test and without supplementation of amino acids as control. RNA-seq data revealed downregulation of several genes associated with stress in test culture confirming the critical role of amino acids supplementation in improving RPP.
2020-05-18 | GSE138044 | GEO
Project description:Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. The objective of the present work is to gain a better understanding of the heat-shock response in recombinant cultures and how this response might impact recombinant protein production. To accomplish this objective, the transcriptome response of recombinant cultures subjected to a heat-shock and a dual heat-shock recombinant protein induction were analyzed. The transcriptome levels were determined using Affymetrix E. coli Antisense DNA microarrays, such that the entire genome was evaluated. These two transcriptome responses were also compared to recombinant cultures at normal growth temperature that were not over-expressing the recombinant protein and a set of literature recombinant culture data that were chemically induced to over-express the recombinant protein. Additionally, the heat-shock response of the recombinant cultures was compared to the literature report of the heat-shock response in wild-type cultures. The results of the global transcriptome analysis demonstrated that recombinant cultures respond differently to a heat-shock stress than wild-type cultures, where the transcriptome response of the recombinant cultures is further modified by production of a recombinant protein.
2009-08-07 | GSE17507 | GEO
Project description:The ability to control the synthesis of products in the absence of cell division remains an attractive alternative for the optimization of microbial processes. If this could be achieved, it would dramatically expand the productivity of many microbial processes by increasing product synthesis in the absence of an increase in cell mass. In the present work we have looked at the factors involved in the design of a host where the fluxes would be channeled towards product formation rather than biomass synthesis. To identify the genes responsible for diverting the metabolic flux specifically towards product formation we have used for this study is quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabolically active cells. These cells channel the metabolic flux towards recombinant protein production and therefore the specific product yield per unit biomass is significantly higher. Indole which is previously known to be a signalling molecule is here used as an inducer of Quiescence. E.coli L-Asparaginase is used as a model protein in this work. Fed batch cultures were carried out in Sartorius BIOSTAT B plus 5liter bioreactor. The data acquisition was done with the MFCS Shell software (version) provided with the bioreactor. The Sartorius BIOSTAT B plus had proportional integral derivative-based control loops for temperature, pH, antifoam, and dissolved-oxygen (DO) regulation. The culture was grown in a batch mode till 10-12 OD (µ=0.5) and then the feed was attached. In the next hour the culture was induced for product formation with 1M IPTG. In case of Q culture quiescence was induced with 0.5M Indole just 5min after induction with IPTG. Post induction every hour samples were collected and frozen with liquid nitrogen and stored at -80ºC for further analytical use. Total RNA extraction, RNA quality control (Agilent BioAnalyser), total RNA labeling, microarray hybridization and scanning were performed according to the Affymetrix GeneChip Expression analysis .
2011-05-31 | E-GEOD-29486 | biostudies-arrayexpress
Project description:For over 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease up-regulation. In this study, the effects of a serine hydroxamate addition on high cell density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas, recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas, recombinant protein production regulated numerous classical stringent response genes; however, not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses; however, are far from identical. It was hypothesized that recombinant protein production leads to a stringent response due to the high amino acid synthesis demands related to recombinant protein synthesis. A comparison of the transcriptomes during recombinant protein production and a chemical imposed stringent response would assist with determining what portion of the “metabolic burden” associated with recombinant protein production is due to amino acid shortages. In this study, the transcriptome profiles of recombinant E. coli were examined and compared for the three culture conditions: 1) Normal growth, no external stress; 2) L-serine hydroxamate addition (to mediate a stringent response); and 3) IPTG-induction to produce the recombinant protein chloramphenicol acetyltransferase (CAT). The transcriptome profiles from these three conditions were analyzed using Affymetrix Anti-sense E. coli GeneChip® microarrays.
2009-08-07 | GSE17506 | GEO
Project description:The transcriptome profiles for wild-type (plasmid-free) and recombinant (plasmid-bearing) Escherichia coli during well-controlled synchronized high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was observed that the growth phase significantly affected the transcriptome profiles, and the transcriptome profiles were significantly different for the recombinant and wild-type cultures. The response of the wild-type and recombinant cultures to an isopropyl-1-thio-β-D-galactopyranoside- (IPTG-) addition was examined, where IPTG induced recombinant protein production in the plasmid-bearing cultures. The IPTG-addition significantly altered the transcriptome response of the wild-type cultures entering the stationary phase. The IPTG-induced recombinant protein production resulted in a significant down-regulation of many energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage (psp, hfl) and transposon-related genes (tra, ins) were significantly regulated in the recombinant cultures due to the IPTG-induction. These results indicate that the signaling mechanism, associated with the recombinant protein production, may induce a metabolic burden in the form of a phage defense mechanism. Taken together, these results indicated that recombinant protein production initiated a cascade of transcriptome responses that down-regulated the very genes needed to sustain productivity. In this study, the transcriptome profiles for recombinant and wild-type E. coli cultures were compared. The recombinant and wild-type cultures were monitored during the exponential growth phase and as the cultures entered the stationary phase. Well-controlled fed-batch fermenters were used to synchronize the high-cell density cultures. E. coli MG1655 [pPROEx-CAT] and E. coli MG1655 (plasmid-free) were exposed to IPTG in the exponential phase, where chloramphenicol acetyltransferase (CAT) expression was induced in the plasmid-bearing cultures. Control cultures, not exposed to IPTG, were also examined. The gene expression profiles were determined using DNA microarrays. Recombinant protein production was shown to result in a metabolic burden due to significant down-regulation of the transcription, translation, and energy synthesis genes. This was the first comprehensive study that has examined the transcriptome of wild-type and recombinant cultures under the same set of conditions. This data indicated that one cannot merely extrapolate the behavior of recombinant cultures from wild-type culture data.
2009-08-07 | GSE17505 | GEO
Project description:In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional–translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.
2017-11-16 | MTBLS393 | MetaboLights
Project description:Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. The objective of the present work is to gain a better understanding of the heat-shock response in recombinant cultures and how this response might impact recombinant protein production. To accomplish this objective, the transcriptome response of recombinant cultures subjected to a heat-shock and a dual heat-shock recombinant protein induction were analyzed. The transcriptome levels were determined using Affymetrix E. coli Antisense DNA microarrays, such that the entire genome was evaluated. These two transcriptome responses were also compared to recombinant cultures at normal growth temperature that were not over-expressing the recombinant protein and a set of literature recombinant culture data that were chemically induced to over-express the recombinant protein. Additionally, the heat-shock response of the recombinant cultures was compared to the literature report of the heat-shock response in wild-type cultures. The results of the global transcriptome analysis demonstrated that recombinant cultures respond differently to a heat-shock stress than wild-type cultures, where the transcriptome response of the recombinant cultures is further modified by production of a recombinant protein. Experiment Overall Design: The heat-shock and recombinant protein production phases were synchronized to the cell density of 11.5 OD, which is referred to as Sample Time 0. For the heat-shocked cultures, the temperature was increased from 37°C to 50°C over 8 minutes beginning at Sample Time 0. The temperature and duration used in this study are the same conditions used to evaluate the heat-shock in wild-type cultures. The temperature was then decreased from 50°C to 37°C over 4 minutes. For the dual heat-shocked recombinant protein production cultures, 5 mM IPTG was added 8 minutes after Sample Time 0. The unstressed recombinant cultures were conducted similarly, except without the heat-shock or IPTG-addition. Each sample condition was obtained from at least two separate fermentations (two biological replicates). RNA from each biological replicate was purified and processed independently. Prior to hybridization, where only two biological replicates existed, one of the processed samples was divided (two technical replicates), resulting in three separate hybridized chips. The heat-shocked and dual stressed culture samples all consisted of three technical replicates from two biological duplicates. For the unstressed culture samples, triplicate samples were obtained for the 11.5 OD and duplicates for the 14 OD conditions. There were no statistical differences between the 11.5 and 14 OD unstressed samples (p ≤ 0.001). Thus, the unstressed culture transcriptome profile consisted of six technical replicates from five biological replicates and four independent fermentations.
2009-08-25 | E-GEOD-17507 | biostudies-arrayexpress
Project description:This study aimed to model formamide-based melting for the optimization of the sensitivity and specifcity of oligonucleotide probes in dignostic high-density microarrays. Formamide melting profiles of DNA oligonucleotides were obtained with a high-density microarray targeting 16S rRNA genes of Escherichia coli and Rhodobacter sphaeroides. One or two mismatched versions of perfect match probes were included on the array to systematically analyze the effect of formamide on mismatch stability and mismatch discrimination. A thermodynamics-based mathematical model of formamide denaturation was developed to predict the formamide melting profiles with sufficient accuracy to help with oligonucleotide design in microbial ecology applications. 16S rRNA sequences with GenBank accession codes U00006 ( E. coli ) and X53853 (R. sphaeroides) were used for probe design. The following oligonucleotide probe sets were used for the systematic analysis of the effect of formamide on probe-target hybrids (parenthetic information gives set name followed by the number of probes): 22-mer perfect match probes tiling the 16S rRNA gene of E. coli (TileE, n=1521), perfect match E.coli probes of variable length between 18 and 26 mers (Length, n=1045), E. coli probes with central single mismatches (OneM, n=1563), E. coli probes with single positional mismatches (PosM, n=4092), E. coli probes with single deletion mismatches (Gap, n=248), E. coli probes with single insertion mismatches (Insertion, n=248), E. coli probes with two separate mismatches (TwoM, n=1674), E. coli probes with central tandem mismatches (Tandem, n=558), and 22-mer perfect match probes tiling the 16S rRNA gene of R. sphaeroides. Also, a probe with no match to 16S rRNA genes was used as a background control. On the array, regular probes were replicated three times and the Nonsense probe ten times. See the manuscript of Yilmaz et al. for details.
2012-06-29 | E-GEOD-33021 | biostudies-arrayexpress