Strain engineering for improved expression of recombinant proteins in bacteria

Makino, Tomohiro; Σκρέτας, Γεώργιος; Georgiou, George

doi:10.1186/1475-2859-10-32

Cited by 176 publications

(114 citation statements)

References 94 publications

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“…Glycosyltransferase and glycosidases are enzymes responsible for glycosylation of many proteins. Glycoproteins, which are commonly distributed in eukaryotic cells, are rarely presented in prokaryotic organisms because cellular organelles essential for glycosylation are missing in these organisms [4,[6][7][8] .…”

Section: Protein Phosphorylation and Glycosylationmentioning

confidence: 99%

“…Degradation of mRNA by RNases can be protected through RNA folding, ribosomes and stability modulation by polyadenylation. Recombinant expression systems with mRNA stability enhancement is commercially available, for example, Invitrogen BL21 star strain, containing a mutation in the gene encoding RNaseE [1,8,9] .…”

Section: Stability Of Mrnamentioning

confidence: 99%

“…Expression of soluble proteins can be regulated through many factors that the host cell normally use in controlling of toxic protein expression [6,8,[12][13][14] .…”

Section: Tight Control Of the E Coli Cellular Milieumentioning

confidence: 99%

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Strategies for production of active eukaryotic proteins in bacterial expression system

Khow

Suntrarachun

2012

Asian Pacific Journal of Tropical Biomedicine

113

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Section: Protein Phosphorylation and Glycosylationmentioning

confidence: 99%

Section: Stability Of Mrnamentioning

confidence: 99%

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Strategies for production of active eukaryotic proteins in bacterial expression system

Khow

Suntrarachun

2012

Asian Pacific Journal of Tropical Biomedicine

113

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“…The implicit assumption is that DNA acts as a sort of software which, if entered in a reading machine already in place (the host), will result in the expression of the genes at stake at the user's will [2,3]. This somewhat naïve concept has proven, however, very successful, and the number of genes and pathways that have been functionally expressed in archetypal hosts such as Escherichia coli just by knocking-in the DNA sequences of interest is very large [4][5][6]. This view has been exacerbated in the recent times with the inception of Synthetic Biology, which entertains the performance of a biological chassis (i.e., the basic, complete genetic, and biochemical scaffold needed for the gene expression flow [7]) in which different engineered DNA constructs are plugged-in and out for specific purposes.…”

Section: Introductionmentioning

confidence: 99%

Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression

et al. 2014

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Background: Because of its adaptability to sites polluted with toxic chemicals, the model soil bacterium Pseudomonas putida is naturally endowed with a number of metabolic and stress-endurance qualities which have considerable value for hosting energy-demanding and redox reactions thereof. The growing body of knowledge on P. putida strain KT2440 has been exploited for the rational design of a derivative strain in which the genome has been heavily edited in order to construct a robust microbial cell factory. Results: Eleven non-adjacent genomic deletions, which span 300 genes (i.e., 4.3% of the entire P. putida KT2440 genome), were eliminated; thereby enhancing desirable traits and eliminating attributes which are detrimental in an expression host. Since ATP and NAD(P)H availability -as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery. Four prophages, two transposons, and three components of DNA restriction-modification systems were eliminated as well. The resulting strain (P. putida EM383) displayed growth properties (i.e., lag times, biomass yield, and specific growth rates) clearly superior to the precursor wild-type strain KT2440. Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase. The performance of a bi-cistronic GFP-LuxCDABE reporter system as a proxy of combined metabolic vitality, revealed that the deletions in P. putida strain EM383 brought about an increase of >50% in the overall physiological vigour.

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“…We found a clear positive relationship between the number of genes added and the length of the cell cycle which was consistent for all four genes examined. Externally added genes commonly affect the growth rate of the host 20,32 although the magnitude of the effect varies between organisms. 33 The relationship between the number of lacI genes added and the length of the cell cycle was approximately linear.…”

Section: Discussionmentioning

confidence: 99%

Towards a whole-cell modeling approach for synthetic biology

Purcell

Jain

Karr

et al. 2013

Chaos: An Interdisciplinary Journal of Nonlinear Science

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Despite rapid advances over the last decade, synthetic biology lacks the predictive tools needed to enable rational design. Unlike established engineering disciplines, the engineering of synthetic gene circuits still relies heavily on experimental trial-and-error, a time-consuming and inefficient process that slows down the biological design cycle. This reliance on experimental tuning is because current modeling approaches are unable to make reliable predictions about the in vivo behavior of synthetic circuits. A major reason for this lack of predictability is that current models view circuits in isolation, ignoring the vast number of complex cellular processes that impinge on the dynamics of the synthetic circuit and vice versa. To address this problem, we present a modeling approach for the design of synthetic circuits in the context of cellular networks. Using the recently published whole-cell model of Mycoplasma genitalium, we examined the effect of adding genes into the host genome. We also investigated how codon usage correlates with gene expression and find agreement with existing experimental results. Finally, we successfully implemented a synthetic Goodwin oscillator in the whole-cell model. We provide an updated software framework for the whole-cell model that lays the foundation for the integration of wholecell models with synthetic gene circuit models. This software framework is made freely available to the community to enable future extensions. We envision that this approach will be critical to transforming the field of synthetic biology into a rational and predictive engineering discipline. Synthetic biology aims to engineer synthetic genetic circuits to endow cells and organisms with the ability to address new applications, including the production of drugs and industrial products, the design of new therapeutics for human diseases, and the study of basic biological processes. Despite significant advances in the field over the last decade, the synthetic biology design cycle has been hampered by the lack of predictive models. In contrast, more established engineering disciplines, such as civil and electrical engineering can rely on rational design by using models that can capture the behavior of real-world systems with a high degree of accuracy. This is currently not the case for synthetic biology, as models are generally only able to make qualitative predictions about system behavior. As a result, producing a functional engineered biological system usually requires extensive manual tuning by trial-and-error, a timeconsuming process that significantly slows the biological design process. The lack of predictive power in current models is partly because these models account for only the synthetic circuits themselves, but not other ongoing processes in the cells in which they reside. However, the complex dynamics of synthetic circuits may affect the host cell and vice versa, leading to divergence between current models and experimental results. Recently, a whole-cell model of a small and simple bacteri...

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Strain engineering for improved expression of recombinant proteins in bacteria

Cited by 176 publications

References 94 publications

Strategies for production of active eukaryotic proteins in bacterial expression system

Strategies for production of active eukaryotic proteins in bacterial expression system

Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression

Towards a whole-cell modeling approach for synthetic biology

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