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Romel Menacho-Melgar Zhixia Ye Eirik A. Moreb Tian Yang John P. Efromson John S. Decker Ruixin Wang Michael D. Lynch 《Biotechnology and bioengineering》2020,117(9):2715-2727
We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed-batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384-well plates), 100 μl (96-well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5–7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation. 相似文献
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Romel Menacho-Melgar Eirik A. Moreb John P. Efromson Tian Yang Jennifer N. Hennigan Ruixin Wang Michael D. Lynch 《Biotechnology and bioengineering》2020,117(9):2852-2860
We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion. 相似文献
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As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on-line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on-line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively. 相似文献
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Kristina Stephens Amin Zargar Milad Emamian Nadia Abutaleb Erica Choi David N. Quan Gregory Payne William E. Bentley 《Biotechnology progress》2019,35(6):e2881
The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell–cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an “enhanced” strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 μM AI-2 to growing cell cultures, we determine that this “enhanced” AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications. 相似文献