scholarly journals Two-stage dynamic deregulation of metabolism improves process robustness & scalability in engineered E. coli.

2021 ◽  
Author(s):  
Zhixia Ye ◽  
Shuai Li ◽  
Jennifer N. Hennigan ◽  
Juliana Lebeau ◽  
Eirik A. Moreb ◽  
...  
Keyword(s):  
Two Stage ◽  
E Coli ◽  
Process Robustness

scholarly journals Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in Engineered E. coli

2020 ◽  
Author(s):  
Zhixia Ye ◽  
Shuai Li ◽  
Jennifer N. Hennigan ◽  
Juliana Lebeau ◽  
Eirik A. Moreb ◽  
...  
Keyword(s):  
High Throughput ◽  
Dynamic Control ◽  
Pilot Scale ◽  
Small Scale ◽  
Scale Production ◽  
Industrial Chemicals ◽  
Two Stage ◽  
E Coli ◽  
Process Robustness ◽  
Microtiter Plates

AbstractWe report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

scholarly journals Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in E. coli

2020 ◽  
Author(s):  
Eirik A Moreb ◽  
Zhixia Ye ◽  
John P Efromson ◽  
Jennifer N Hennigan ◽  
Romel Menacho-Melgar ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Heterologous Protein ◽  
Scale Up ◽  
Mineral Salts ◽  
Two Stage ◽  
Robustness Testing ◽  
E Coli ◽  
Media Formulation ◽  
The Impact ◽  
Regulated Promoters

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the robustness testing of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Media dependent differences were promoter dependent. 4 out of 16 promoters tested were identified to have tightly controlled expression which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlight the impact of the environment on the performance of biological parts, and the importance of robustness testing in synthetic biology.

scholarly journals High-throughput enrichment of temperature-sensitive argininosuccinate synthetase for two-stage citrulline production in E. coli

2020 ◽  
Vol 60 ◽  
pp. 14-24 ◽  
Author(s):  
Thorben Schramm ◽  
Martin Lempp ◽  
Dominik Beuter ◽  
Silvia González Sierra ◽  
Timo Glatter ◽  
...  
Keyword(s):  
High Throughput ◽  
Temperature Sensitive ◽  
Argininosuccinate Synthetase ◽  
Two Stage ◽  
E Coli

PRODUKSI REKOMBINAN SEFALOSPORIN ASILASE SEBAGAI BIOKATALIS UNTUK PRODUKSI ASAM 7-AMINOSEFALOSPORANAT

2017 ◽  
Vol 4 (1) ◽  
pp. 28 ◽  
Author(s):  
Bima Wedana Isdiyono ◽  
Dudi Hardianto ◽  
Fransiskus Xaverius Ivan
Keyword(s):  
Escherichia Coli ◽  
Induction Time ◽  
Acid Oxidase ◽  
Enzymatic Method ◽  
Amino Acid Oxidase ◽  
Two Stage ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Cephalosporin Acylase ◽  
The One

Production of Cephalosporin Acylase Recombinant as Biocatalyst for 7-Aminocephalosporanic Acid Production7-aminocephalosporanic acid (7-ACA) is a precursor for the production of semisynthetic cephalosporin derivatives. The enzymatic 7-ACA production can use two-stage and one-step enzymatic methods. Two-stage enzymatic method uses D-amino acid oxidase (DAAO) enzyme to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA) in the first stage and glutaryl-7-aminocephalosporanic acid acylase to produce 7-ACA in the second stage. The one-stage enzymatic method using cephalosporin acylase (CPC acylase) converts the CPC to 7-ACA directly. The aim of this research was to produce recombinant CPC acylase in Escherichia coli BL21(DE3). Transformantion culture E. coli BL21(DE3) was induced with concentrations of IPTG 0; 0.25; 0.5; 0.75; 1; 2 mM for 5 hours. The induction time of IPTG was determined at 0, 1, 2, 3, 4, and 5 hours. The results showed that CPC acylase produced by E. coli BL21(DE3) with optimum condition of CPC acylase production was 0.5 mM IPTG and optimal induction time of IPTG was 5 hours.Keywords: Cephalosporin, cephalosporin acylase, 7-ACA, protein expression, Escherichia coli BL21(DE3) ABSTRAKAsam 7-aminosefalosporanat (7-ACA) merupakan prekursor untuk produksi turunan sefalosporin semisintetik. Produksi 7-ACA secara enzimatik dapat menggunakan metode dua tahap dan satu tahap enzimatik. Metode enzimatik secara dua tahap menggunakan enzim asam D-amino oksidase (DAAO) untuk menghasilkan asam glutaril-7-aminosefalosporinat (GL-7-ACA) pada tahap pertama dan menggunakan asam glutaril-7-aminosefalosporinat asilase untuk menghasilkan 7-ACA pada tahap kedua. Metode enzimatik satu tahap dengan sefalosporin asilase (CPC asilase) mengubah CPC menjadi 7-ACA secara langsung. Tujuan penelitian adalah memproduksi rekombinan CPC asilase di dalam sel Escherichia coli BL21(DE3). Kultur Transforman E. coli BL21(DE3) diinduksi dengan konsentrasi IPTG 0; 0,25; 0,5; 0,75; 1; 2 mM selama 5 jam. Waktu induksi IPTG ditentukan pada 0, 1, 2, 3, 4 dan 5 jam. Hasil penelitian menunjukan bahwa CPC asilase diproduksi oleh E. coli BL21(DE3) dengan kondisi optimal produksi CPC asilase adalah konsentrasi IPTG 0,5 mM dan waktu induksi IPTG optimal adalah 5 jam.

scholarly journals Evolution of bidirectional costly mutualism from byproduct consumption

2018 ◽  
Vol 115 (47) ◽  
pp. 12000-12004 ◽  
Author(s):  
William R. Harcombe ◽  
Jeremy M. Chacón ◽  
Elizabeth M. Adamowicz ◽  
Lon M. Chubiz ◽  
Christopher J. Marx
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Species Interactions ◽  
De Novo ◽  
Two Stage ◽  
E Coli ◽  
Stage Process ◽  
Life On Earth

Mutualisms are essential for life, yet it is unclear how they arise. A two-stage process has been proposed for the evolution of mutualisms that involve exchanges of two costly resources. First, costly provisioning by one species may be selected for if that species gains a benefit from costless byproducts generated by a second species, and cooperators get disproportionate access to byproducts. Selection could then drive the second species to provide costly resources in return. Previously, a synthetic consortium evolved the first stage of this scenario: Salmonella enterica evolved costly production of methionine in exchange for costless carbon byproducts generated by an auxotrophic Escherichia coli. Growth on agar plates localized the benefits of cooperation around methionine-secreting S. enterica. Here, we report that further evolution of these partners on plates led to hypercooperative E. coli that secrete the sugar galactose. Sugar secretion arose repeatedly across replicate communities and is costly to E. coli producers, but enhances the growth of S. enterica. The tradeoff between individual costs and group benefits led to maintenance of both cooperative and efficient E. coli genotypes in this spatially structured environment. This study provides an experimental example of de novo, bidirectional costly mutualism evolving from byproduct consumption. The results validate the plausibility of costly cooperation emerging from initially costless exchange, a scenario widely used to explain the origin of the mutualistic species interactions that are central to life on Earth.

scholarly journals Does the Use of a Closed-Suction Drain Reduce the Effectiveness of an Antibiotic-loaded Spacer in Two-Stage Exchange Arthroplasty for Periprosthetic Hip Infection? A Prospective, Randomized, Controlled Study

2019 ◽  
Author(s):  
Chi Xu ◽  
Cheng-Qi Jia ◽  
Feng-Chih Kuo ◽  
Wei Chai ◽  
Ming-Hua Zhang ◽  
...  
Keyword(s):  
Controlled Study ◽  
Cement Spacer ◽  
Two Stage ◽  
E Coli ◽  
Closed Suction ◽  
Randomized Controlled ◽  
Suction Drain ◽  
Exchange Arthroplasty ◽  
Closed Suction Drain ◽  
Hip Infection

Abstract Objectives There is a concern regarding the use of a closed-suction drain (CSD) in two-stage exchange arthroplasty for periprosthetic joint infection as it may decrease the antibiotic concentrations in the joint fluids. The purpose of this study was to identify whether the use of a CSD could reduce local antibiotic concentrations following spacer implantation. Methods A prospective, randomized, controlled trial was conducted at our institution between January 2018 and November 2018. We enrolled 32 patients undergoing two-stage exchange arthroplasty for periprosthetic hip infection with an interim cement spacer containing 4-g vancomycin and 2-g meropenem per 40-g methyl-methacrylate cement polymer. Patients were randomized and evenly divided into the clamped CSD group and open CSD group after surgery. Drainage samples of joint fluids (n=160) were collected every 24 hours for the first five days following spacer implantation. The antibiotic concentrations of drainage samples were measured by high-performance liquid chromatography, and the bioactivities of the drainage samples against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) and E. coli were assessed. Results There was no significant difference in the decrease of vancomycin (p=0.917) and meropenem concentration (P=0.548) between the two groups during the first five days following spacer implantation. All joint drainage samples in each group exhibited antibacterial activity against MSSA, MRSA and E. Coli. Conclusions The use of CSD following the implantation of an antibiotic-loaded cement spacer does not reduce the effectiveness of such a spacer in two-stage exchange arthroplasty.

scholarly journals Does the use of a closed-suction drain reduce the effectiveness of an antibiotic-loaded spacer in two-stage exchange Arthroplasty for Periprosthetic hip infection? A prospective, randomized, controlled study

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Chi Xu ◽  
Cheng-Qi Jia ◽  
Feng-Chih Kuo ◽  
Wei Chai ◽  
Ming-Hua Zhang ◽  
...  
Keyword(s):  
Control Group ◽  
Cement Spacer ◽  
Two Stage ◽  
E Coli ◽  
Closed Suction ◽  
Randomized Controlled ◽  
Suction Drain ◽  
Exchange Arthroplasty ◽  
Closed Suction Drain ◽  
Hip Infection

Abstract Background There is a concern regarding the use of a closed-suction drain (CSD) in two-stage exchange arthroplasty for periprosthetic joint infection as it may decrease the antibiotic concentrations in the joint fluids. The purpose of this study was to identify whether the use of a CSD could reduce local antibiotic concentrations following spacer implantation. Methods A prospective, randomized, controlled trial was conducted at our institution between January 2018 and November 2018. We enrolled 32 patients undergoing two-stage exchange arthroplasty for periprosthetic hip infection with an interim cement spacer containing 4-g vancomycin and 2-g meropenem per 40-g methyl-methacrylate cement polymer. Patients were randomized and evenly divided into the study group (non-CSD) and control group (CSD group) by sealed envelopes. Drainage samples of joint fluids (n = 160) were collected every 24 h for the first five days following spacer implantation. The antibiotic concentrations of drainage samples were measured by high-performance liquid chromatography, and the bioactivities of the drainage samples against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) and E. coli were assessed. Results There was no significant difference in the decrease of vancomycin (study group vs. control group: 163.20 ± 77.05 vs. 162.39 ± 36.31; p = 0.917) and meropenem concentration (123.78 ± 21.04 vs. 117.27 ± 19.38; P = 0.548) between the two groups during the first five days following spacer implantation. All joint drainage samples in each group exhibited antibacterial activity against MSSA, MRSA and E. coli. Conclusions The use of CSD following the implantation of an antibiotic-loaded cement spacer does not reduce the effectiveness of such a spacer in two-stage exchange arthroplasty. (Chinese Clinical Trial Registry, ChiCTR-INR-17014162. Registered 26 December 2017.)

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