Into new territory: improved microbial synthesis through engineering of the essential metabolic network

Lynch, Michael

doi:10.1016/j.copbio.2016.01.009

Cited by 36 publications

(20 citation statements)

References 48 publications

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“…While the expression of heterologous proteins during stationary phase may seem counterintuitive and at odds with maximal production, stationary phase cells can maintain significant metabolic activity and produce high levels of protein. [25][26][27][28][29] Specifically, phosphate depletion has been used routinely for heterologous protein expression. [30][31][32] In addition, it has been shown that phosphate depletion can be used to amplify the expression of heterologous proteins using the pET based T7 promoters in E. coli.…”

Section: Introductionmentioning

confidence: 99%

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

Menacho‐Melgar

Moreb

et al. 2019

Preprint

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We report the improved production of recombinant proteins in E. coli , reliant on tightly controlled autoinduction, triggered by phosphate depletion in 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 total cellular protein.Initial use of the method in instrumented fed batch fermentations enables cell densities of 10 grams dry cell weight (gCDW) per liter and protein titers up to 2.7+/-0.2 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 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach~5-7 gCDW per liter, 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 Highlights :• Stationary phase protein expression results in high titers.• Autoinduction by phosphate depletion enables protein titers from 2-3 g/L.• Autoinduction has been validated from 384 well plates to instrumented bioreactors.

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Section: Introductionmentioning

confidence: 99%

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

Menacho‐Melgar

Moreb

et al. 2019

Preprint

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“…These promoters (as well as insulated version) have utility in two-stage expression and production. 22,23 None of these 4 promoters were found to be robust to oxygen conditions, and optimal expression requires adequate culture aeration, limiting use in oxygen limited or anaerobic cultures.…”

Section: Resultsmentioning

confidence: 99%

Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction inE. coli

Moreb¹,

Ye²,

Efromson³

et al. 2020

Preprint

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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.

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“…To our knowledge this is the first combination of these two mechanisms to improve cellular lysis and DNA removal, and an example of the potential benefits of two stage production. [43][44][45] This system enables > 95% lysis and hydrolysis. Due to tightly controlled expression these strains are stable to shear forces in stirred tank bioreactors and even when subjected to freeze thaw cycles in deionized water, with 10% glycerol.…”

Section: Discussionmentioning

confidence: 99%

Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

Menacho‐Melgar

Moreb

Efromson

et al. 2020

Preprint

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We report improved release of recombinant proteins in E. coli , which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. 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. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). 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. Highlights :• Autoinduction of both cell lysis and nucleotide hydrolysis • >90 % lysis and DNA degradation • Strains are stable to lysis in the absence of phosphate depletion.

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Into new territory: improved microbial synthesis through engineering of the essential metabolic network

Cited by 36 publications

References 48 publications

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction inE. coli

Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

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