Metabolic engineering of E. coli for pyocyanin production

Silva, A. J. da; Cunha, Josivan de Souza; Hreha, Teri N.; Micocci, Kelli Cristina; Araújo, Heloísa Sobreiro Selistre de; Barquera, Blanca; Koffas, Mattheos

doi:10.1016/j.ymben.2021.01.002

Cited by 27 publications

(22 citation statements)

References 43 publications

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“…Interestingly, the induction of ClearColi® BL21(DE3) cultures by a pulse of lactose led to higher levels of PspA4Pro than obtained in auto-induction cultivations, even though the final concentrations of lactose were the same in these cultivations. The same behavior was already observed for conventional E. coli BL21(DE3) cells and attributed to the earlier induction in auto-induction strategy, which triggered the so called “metabolic burden” related to heterologous protein production (Li et al 2006 ; Carneiro et al 2013 ; da Silva et al 2021 ) and restricted the allocation of cellular resources for biomass formation and other metabolic activities (da Silva et al 2021 ). Nevertheless, these results did not preclude the use of auto-induction medium in ClearColi cultivations, especially considering that it simplifies the control, monitoring, and operation of cultures (Hunt et al 2019 ).…”

Section: Discussionsupporting

confidence: 63%

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Cardoso

Paredes

Campani

et al. 2022

Appl Microbiol Biotechnol

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Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β- d -thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. Key points • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11758-9.

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Section: Discussionsupporting

confidence: 63%

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Cardoso

Paredes

Campani

et al. 2022

Appl Microbiol Biotechnol

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“…However, pyocyanin remains a costly reagent (e.g., €118 for 5 mg on Merck website). Therefore, the production of pyocyanin is optimized by many research groups by adjusting process conditions, genetic engineering of the microorganisms, or the addition of various substances to the medium, including nanomaterials (Sismaet et al 2014;El-Fouly et al 2015;Ozdal 2019;Ozdal et al 2019;Bacame-Valenzuela et al 2020;da Silva et al 2021;Elbargisy 2021). The analysis of the literature shows that most of the data concerning the influence of nanomaterials on pyocyanin production indicate the inhibition of pigment production.…”

Section: Introductionmentioning

confidence: 99%

The influence of nanomaterials on pyocyanin production by Pseudomonas aeruginosa

et al. 2022

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Pseudomonas aeruginosa is a bacterium producing industrially utile metabolites, such as rhamnolipids, biopolymers, and pigments. Pyocyanin, the most studied example of pigments, is a virulence factor that also shows the potential for application in, e.g., agriculture, anticancer therapy, and energy production. Therefore, potential inhibitors and stimulants of pyocyanin production by P. aeruginosa should be studied, and nanomaterials may cause both effects. The study aimed to examine the influence of zinc oxide and multi-walled carbon nanotubes (pristine or dispersed with alginic acid) on pyocyanin production by P. aeruginosa. First, the influence of different concentrations of nanomaterials (500.00–0.06 µg/mL) on culture optical density and biofilm formation was studied. These results helped select concentrations for further tests, i.e., growth curves and fluorescence measurements. Pyocyanin production was assessed by the chloroform–hydrochloric acid method. SEM analysis was conducted to assess the influence of nanomaterials on the cell's integrity and biofilm structure. Pristine multi-walled carbon nanotubes exhibited a stimulative effect on pigment production when applied in high concentrations (500.00 µg/mL), while dispersed material enhanced the production in lowered dosages (125.00 µg/mL). On the other hand, high concentrations of zinc oxide inhibited pyocyanin production, while minor increased bioproduct production. The research indicates the potential to use nanomaterials as the modulators of pyocyanin production and other metabolites.

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“…PYO serves its native host, P. aeuroginosa , in several ways, including as a toxin and as a signaling molecule [ 23 ]. Here we show that phenazine producing messenger cells and phenazine responding receiver cells can be developed from common laboratory E. coli strains for signal propagation and consortium coordination, complementing previous development of E. coli strains engineered to produce high levels of PYO [ 24 ]. Also, like other ported signaling transduction motifs, should there be a desire to minimize off-target effects stemming from PYO and PCA signaling, hosts can be generated as SoxRS deletion mutants or use multiplexed CRISPR-mediated downregulation [ 18 , 20 ].…”

Section: Resultsmentioning

confidence: 99%

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

et al. 2021

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Background Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. Results We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Conclusions Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

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Metabolic engineering of E. coli for pyocyanin production

Cited by 27 publications

References 43 publications

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

The influence of nanomaterials on pyocyanin production by Pseudomonas aeruginosa

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

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