Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

Kim, Ju-Hyun; Salvador, Manuel; Saunders, Elizabeth; González, J.M. Salicio; Avignone–Rossa, Claudio; Jiménez, José I.

doi:10.1042/ebc20160015

Cited by 52 publications

(27 citation statements)

References 74 publications

(68 reference statements)

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“…While most synthetic biology efforts make use of only one host chassis to develop and characterise genetic constructs, potential applications may require the same genetic devices to work with different cell types 19 .…”

Section: Introductionmentioning

confidence: 99%

Contextual dependencies expand the re-usability of genetic inverters

Tas

Grozinger

Stoof

et al. 2020

Preprint

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The design and implementation of Boolean logic functions in living cells has become a very active field within synthetic biology. By controlling networks of regulatory proteins, novel genetic circuits are engineered to generate predefined output responses. Although many current implementations focus solely on the genetic components of the circuit, the host context in which the circuit performs is crucial for its outcome. Here, we characterise 20 genetic NOT logic gates (inverters) in up to 7 bacterial-based contexts each, to finally generate 135 different functions. The contexts we focus on are particular combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each NOT logic gate shows seven different logic behaviours, depending on the context. That is, gates can be reconfigured to fit response requirements by changing only contextual parameters. Computational analysis shows that this range of behaviours improves the compatibility between gates, because there are considerably more possibilities for combination than when considering a unique function per genetic construct. Finally, we address the issue of interoperability and portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expand the scope of the functions that can be engineered in complex cellular environments, and advocate for considering context as a fundamental design parameter for synthetic biology.

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Section: Introductionmentioning

confidence: 99%

Contextual dependencies expand the re-usability of genetic inverters

Tas

Grozinger

Stoof

et al. 2020

Preprint

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“…Species of the bacterial genus Pseudomonas are of exceptional importance, not only as an infectious agent for a broad range of organisms including humans and plants (1–4), but also for the valuable role species of this genus play in biotechnology and basic science (5, 6). Important tools in both biotechnology and basic science are inducible gene expression systems that enable induced expression or repressed expression in the presence of an effector molecule.…”

Section: Introductionmentioning

confidence: 99%

Generation of Xylose-inducible promoter tools forPseudomonasspecies and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

Callaghan

Stella

Lehner

et al. 2020

Preprint

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ABSTRACTTunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and P. fluorescens. The Pxut promoter derived from the P. flurorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was expressed at a lower inducer concentration than PBAD in P. fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the P. aeruginosa type II secretion system gene from xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.ImportancePseudomonas species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in P. aeruginosa and P. fluorescens and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type 2 secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

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“…To address these problems and achieve broad commercial applications, scholars have developed a series of biological manufacturing methods with the advantages of self-assembly, proliferation, mild reaction condition requirements, and environment-friendly features to produce valuable monoterpenes, especially in this rapid development era of synthetic biology and bioengineering [25,26]. Several microorganisms, such as Escherichia coli and Saccharomyces cerevisiae, are considered as perfect chassis and have been designed as microbial cell factories for the industrialized production of significant monoterpenoids [27,28]. Both MEV and MEP pathways and their downstream enzyme systems are available to be incorporated and engineered in these chassis with different strategies to produce various valuable monoprenoids with high yield.…”

Section: Introductionmentioning

confidence: 99%

Advances in the Metabolic Engineering of Escherichia coli for the Manufacture of Monoterpenes

Xie

Zhu

et al. 2019

Catalysts

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Monoterpenes are commonly applied as pharmaceuticals and valuable chemicals in various areas. The bioproduction of valuable monoterpenes in prokaryotic microbial hosts, such as E. coli, has progressed considerably thanks to the development of different outstanding approaches. However, the large-scale production of monoterpenes still presents considerable limitations. Thus, process development warrants further investigations. This review discusses the endogenous methylerythritol-4-phosphate-dependent pathway engineering and the exogenous mevalonate-dependent isoprenoid pathway introduction, as well as the accompanied optimization of rate-limiting enzymes, metabolic flux, and product toxicity tolerance. We suggest further studies to focus on the development of systematical, integrational, and synthetic biological strategies in light of the inter disciplines at the cutting edge. Our review provides insights into the current advances of monoterpene bioengineering and serves as a reference for future studies to promote the industrial production of valuable monoterpenes.

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Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

Cited by 52 publications

References 74 publications

Contextual dependencies expand the re-usability of genetic inverters

Contextual dependencies expand the re-usability of genetic inverters

Generation of Xylose-inducible promoter tools forPseudomonasspecies and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

Advances in the Metabolic Engineering of Escherichia coli for the Manufacture of Monoterpenes

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