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 Optimization of Phosphate Limited Autoinduction Broth for 2-Stage Heterologous Protein Expression in E. coli

2021 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Jennifer N Hennigan ◽  
Michael D Lynch
Keyword(s):  
Protein Expression ◽  
Small Scale ◽  
Heterologous Protein Expression ◽  
Scale Production ◽  
Inducible Promoters ◽  
E Coli ◽  
Media Formulation ◽  
Microtiter Plates ◽  
Shake Flasks ◽  
Consistent Performance

Autoinducible, 2-stage protein expression leveraging phosphate inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date small scale production using microtiter plates and shake flasks rely on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, we develop a simpler media formulation (AB-2) with just a few autoclavable components. We show that AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate adoption of phosphate limited 2-stage protein expression protocols.

scholarly journals Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in Escherichia coli

BioTechniques ◽  
2021 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Jennifer N Hennigan ◽  
Michael D Lynch
Keyword(s):  
Protein Expression ◽  
Small Scale ◽  
Heterologous Protein Expression ◽  
Scale Production ◽  
Two Stage ◽  
Inducible Promoters ◽  
Media Formulation ◽  
Microtiter Plates ◽  
Shake Flasks ◽  
Consistent Performance

Autoinducible, two-stage protein expression leveraging phosphate-inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date, small-scale production using microtiter plates and shake flasks relies on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, the authors develop a simpler media formulation (AB-2) with just a few autoclavable components. AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate the adoption of phosphate-limited two-stage protein expression protocols.

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

scholarly journals Automation assisted anaerobic phenotyping for metabolic engineering

2021 ◽  
Author(s):  
Kaushik Raj ◽  
Naveen Venayak ◽  
Patrick Diep ◽  
Sai Akhil Golla ◽  
Alexander F. Yakunin ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Throughput ◽  
Large Scale ◽  
Small Scale ◽  
Chemical Production ◽  
E Coli ◽  
Strain Design ◽  
Phenotypic Screen ◽  
Wide Range ◽  
Scale Down

Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for a high-throughput, laboratory scale methods to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes. Several small-scale fermentation techniques, in conjunction with laboratory automation have enhanced the throughput of enzyme and strain phenotyping experiments. However, such high throughput experimentation typically entails large operational costs and generate massive amounts of laboratory plastic waste. In this work, we develop an eco-friendly automation workflow that effectively calibrates and decontaminates fixed-tip liquid handling systems to reduce tip waste. We also investigate inexpensive methods to establish anaerobic conditions in microplates for high-throughput anaerobic phenotyping. To validate our phenotyping platform, we assess its performance in two case studies - an anaerobic enzyme screen, and a microbial phenotypic screen. We used our automation platform to investigate conditions under which several strains of E. coli exhibit the same phenotypes in 0.5 L bioreactors and in our scaled-down fermentation platform. Further, we propose the use of dimensionality reduction through t-distributed stochastic neighbours embedding, in conjunction with our phenotyping platform to serve as an effective scale-down model for bioreactor phenotypes. By integrating an in-house data-analysis pipeline, we were able to accelerate the 'test' phase of the design-build-test-learn cycle of metabolic engineering.

scholarly journals Large-scale Production of Novel Porcine Circovirus Type 2d (PCV2d) Subunit Vaccine Using E. coli Platform

2021 ◽  
Author(s):  
Sarawuth Noppiboon ◽  
Thunyaluk Kerdboon ◽  
Neeracha Lapanusorn ◽  
Pisit Ekkpongpaisit ◽  
Sarah Slack ◽  
...  
Keyword(s):  
Large Scale ◽  
Pilot Scale ◽  
Porcine Circovirus Type ◽  
Exchange Chromatography ◽  
Porcine Circovirus ◽  
Scale Production ◽  
E Coli ◽  
Large Scale Production ◽  
Production Platform ◽  
Cost Efficient

Porcine Circovirus type 2d (PCV2d) is becoming the predominant PCV genotype and considerably affects the global pig industry. Nevertheless, currently no commercial PCV2d vaccine is available. Preventing and controlling the disease caused by PCV2d is therefore based on other genotype-based vaccines. However, their production platforms are laborious, limited in expression level and relatively expensive for veterinary applications. To address these challenges, we have developed a simple and cost-efficient platform for a novel PCV2d vaccine production platform, using fed-batch E. coli fermentation followed by cell disruption and filtration, and a single purification step via cation exchange chromatography. The process was developed at bench scale and then pilot scale where the PCV2d subunit protein yield was approximately 0.8 g/L fermentation volume in a short production time. Moreover, we have successfully implemented this production process at two different sites, in Southeast Asia and Europe. This demonstrates transferability and the high potential for successful industrial production.

scholarly journals Heterotrophy as a tool to overcome the long and costly autotrophic scale-up process for large scale production of microalgae

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
A. Barros ◽  
H. Pereira ◽  
J. Campos ◽  
A. Marques ◽  
J. Varela ◽  
...  
Keyword(s):  
Large Scale ◽  
Scale Up ◽  
Biomass Concentration ◽  
Growth Conditions ◽  
Pilot Scale ◽  
Industrial Scale ◽  
Scale Production ◽  
Two Stage ◽  
Chlorophyll Contents ◽  
Large Scale Production

Abstract Industrial scale-up of microalgal cultures is often a protracted step prone to culture collapse and the occurrence of unwanted contaminants. To solve this problem, a two-stage scale-up process was developed – heterotrophically Chlorella vulgaris cells grown in fermenters (1st stage) were used to directly inoculate an outdoor industrial autotrophic microalgal production unit (2nd stage). A preliminary pilot-scale trial revealed that C. vulgaris cells grown heterotrophically adapted readily to outdoor autotrophic growth conditions (1-m3 photobioreactors) without any measurable difference as compared to conventional autotrophic inocula. Biomass concentration of 174.5 g L−1, the highest value ever reported for this microalga, was achieved in a 5-L fermenter during scale-up using the heterotrophic route. Inocula grown in 0.2- and 5-m3 industrial fermenters with mean productivity of 27.54 ± 5.07 and 31.86 ± 2.87 g L−1 d−1, respectively, were later used to seed several outdoor 100-m3 tubular photobioreactors. Overall, all photobioreactor cultures seeded from the heterotrophic route reached standard protein and chlorophyll contents of 52.18 ± 1.30% of DW and 23.98 ± 1.57 mg g−1 DW, respectively. In addition to providing reproducible, high-quality inocula, this two-stage approach led to a 5-fold and 12-fold decrease in scale-up time and occupancy area used for industrial scale-up, respectively.

scholarly journals Pilot-scale production of a highly thermostable α-amylase enzyme from Thermotoga petrophila cloned into E. coli and its application as a desizer in textile industry

RSC Advances ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 984-992 ◽  
Author(s):  
Asma Zafar ◽  
Muhammad Nauman Aftab ◽  
Irfana Iqbal ◽  
Zia ud Din ◽  
Mushtaq Ahmad Saleem
Keyword(s):  
Textile Industry ◽  
Large Scale ◽  
Pilot Scale ◽  
Cotton Cloth ◽  
Scale Production ◽  
E Coli ◽  
Thermotoga Petrophila ◽  
Pilot Scale Production

Thermostable recombinant α-amylase was produced in a large scale for the desizing of cotton cloth in the textile industry.

Laboratory and Pilot-Scale Dead-End Ultrafiltration Concentration of Sanitizer-Free and Chlorinated Lettuce Wash Water for Improved Detection of Escherichia coli O157:H7

2014 ◽  
Vol 77 (8) ◽  
pp. 1260-1268 ◽  
Author(s):  
SONIA MAGAÑA ◽  
SARAH M. SCHLEMMER ◽  
GORDON R. DAVIDSON ◽  
ELLIOT T. RYSER ◽  
DANIEL V. LIM
Keyword(s):  
Escherichia Coli ◽  
Pilot Scale ◽  
Wash Water ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Scale Production ◽  
E Coli ◽  
Concentration Method ◽  
Dead End ◽  
Electrochemiluminescence Immunoassay

An automated dead-end (single pass, no recirculation) ultrafiltration device, the Portable Multi-use Automated Concentration System (PMACS), was evaluated as a means to concentrate Escherichia coli O157:H7 from 40 liters of simulated commercial lettuce wash water. The assessment included generating, sieving, and concentrating sanitizer-free lettuce wash water, either uninoculated or inoculated with green fluorescent protein–transformed E. coli O157:H7 at a high (1.00 log CFU/ml) or low (−1.00 log CFU/ml) concentration. Cells collected within the filters were recovered in approximately 400 ml of buffer to create lettuce wash retentates. The extent of concentration was determined by viable plate counts using a medium selective for the transformed E. coli O157:H7. The samples were qualitatively analyzed for E. coli O157:H7 according to the U.S. Food and Drug Administration Bacteriological Analytical Manual enrichment method and with an electrochemiluminescence immunoassay. This concentration method was then evaluated in a pilot-scale production line at Michigan State University using chlorinated (100, 30, and 10 ppm of available chlorine) lettuce wash water. The total PMACS processing times were 82 ±6 and 65 ±5 min for sanitizer-free and chlorinated washes, respectively. Overall, E. coli O157:H7 populations were approximately 2 log higher in retentates than in unconcentrated lettuce wash samples. The higher E. coli O157:H7 levels in the retentates enabled cultural and electrochemiluminescence immunoassay detection in some samples when the corresponding lettuce wash samples were negative. When combined with standard and rapid detection methods, the PMACS concentration method may provide a means to enhance pathogen monitoring of produce wash water.

scholarly journals Sensory quality and shelf-life of locally produced British butters compared to large-scale, industrially produced butters

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Agnieszka Dudkiewicz ◽  
William Hayes ◽  
Bukola Onarinde
Keyword(s):  
Shelf Life ◽  
Large Scale ◽  
Sensory Quality ◽  
Pilot Scale ◽  
Small Scale ◽  
Scale Production ◽  
Content Type ◽  
Life Study ◽  
Quality And Shelf Life

PurposeThe purpose of this pilot-scale study was to compare the quality of traditionally manufactured butters from local, small British producers with the quality of butters that are produced industrially.Design/methodology/approachButter samples were obtained after supervised site inspections of three traditional-butter manufacturers and one large-scale butter producer. The samples were subject to initial microbiological, chemical and sensory testing, followed by a refrigerated shelf-life study over 24 weeks.FindingsTraditional butters matched or exceeded the sensory quality of industrial butters, but spoilage microorganisms tended to grow faster on traditional butters. This seemed to be related to poorer water droplet dispersion in the manufacture of some of the traditionally made butters. Visible mould appeared on two of the traditional butters after eight weeks, but this occurred well after the nominal “best before” date.Originality/valueProlonged lockdowns due to the current coronavirus disease (COVID-19) pandemic pose a threat to the food supply chain, and food produced by local manufacturers may become increasingly important. However, are foods produced by local small-scale manufacturers of a quality comparable to that produced using large-scale production facilities? To the best of the authors' knowledge, there is no comparative study of the quality and shelf-life of traditionally-produced and industrially-produced butters. The current work presents such a comparison together with an outline of how the process of traditional butter-making differs from commercial production in Britain.

scholarly journals Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

2020 ◽  
Vol 14 (1) ◽  
Author(s):  
Timm Keil ◽  
Barbara Dittrich ◽  
Clemens Lattermann ◽  
Jochen Büchs
Keyword(s):  
Release Rate ◽  
Oxygen Transfer ◽  
Oxygen Transfer Rate ◽  
Small Scale ◽  
Fed Batch ◽  
Release Rates ◽  
Glucose Release ◽  
E Coli ◽  
Batch Phase ◽  
Microtiter Plates

Abstract Background Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24–0.90 mg/h per well. Conclusions The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

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