<i>Vitreoscilla</i> hemoglobin expression in engineered <i>Escherichia coli</i>: Improved performance in high cell‐density batch cultivations

Pablos, Tania E.; Mora, Eugenio Meza; Borgne, Sylvie Le; Ramı́rez, Octavio T.; Gosset, Guillermo; Lara, Alvaro R.

doi:10.1002/biot.201000405

Cited by 38 publications

(12 citation statements)

References 25 publications

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“…To better understand the effect of VHb on the engineered strain, the metabolic fluxes were estimated by flux balance analysis during the exponential growth of strains W3110 and VAL24. The simulations estimated values of μ close to the experimental values (data not shown) and the specific oxygen uptake rates estimated were in agreement with previously published values . The results are depicted in Fig.…”

Section: Resultssupporting

confidence: 91%

Toward efficient microaerobic processes using engineered Escherichia coli W3110 strains

Pablos

Olivares²,

Sigala³

et al. 2016

Engineering in Life Sciences

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Operational and economic constraints in large-scale bioreactors often result in local or global microaerobic conditions, which lead to less efficient bioprocesses. Escherichia coli adapts to microaerobicity by activating fermentation pathways that accumulate acidic by-products, in detriment of growth rate (μ) and biomass yield on glucose (Y X/S ). In this study, the metabolism of E. coli was modified to better cope with microaerobicity. For that purpose, genes coding for global regulators like carbon source responsive B protein and aerobic respiratory control A protein, or for fermentative pathways were inactivated. The performance of a wild-type (W3110) and engineered E. coli strains was evaluated in batch cultures at constant low dissolved oxygen tension (3% air sat.). By combining the partial elimination of fermentation pathways and the expression of the Vitreoscilla hemoglobin (VHb), a 32% decrease on carbon waste as by-products, 24 % increase on Y X/S and 13% increase of μ were obtained. Flux balance analysis of the best strain estimated major differences in the fluxes through the pentose phosphate pathway and tricarboxylic acid cycle as consequence of VHb presence. Overall, our results show that E. coli can be genetically modified to overcome some of the disadvantages of microaerobic growth, which is potentially useful for better bioreactor scale-up and operation.

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Section: Resultssupporting

confidence: 91%

Toward efficient microaerobic processes using engineered Escherichia coli W3110 strains

Pablos

Olivares²,

Sigala³

et al. 2016

Engineering in Life Sciences

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“…The expression vector was previously designed in order to increase VHb synthesis under fully aerobic conditions. It was shown that, in this case, overflow metabolism was reduced and plasmid DNA yield was increased . VHb facilitates oxygen uptake from the media and delivers it to the cytochromes more efficiently, thus enhancing the electron transport chain activity.…”

Section: Resultsmentioning

confidence: 90%

Microbial population heterogeneity versus bioreactor heterogeneity: Evaluation of Redox Sensor Green as an exogenous metabolic biosensor

Baert

Delepierre

Telek

et al. 2016

Engineering in Life Sciences

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Practical applicationIt is known that isogenic microbial population is able to display phenotypic diversification upon environmental changes. In this study, we developed a high throughput methodology based on the use of flow cytometry and the preliminary staining of the cells with Redox Sensor Green (RSG). We demonstrated that RSG is related to the activity of the electron transport chain and www.els-journal.com Page 2 Engineering in Life SciencesThis article is protected by copyright. All rights reserved.2 can be used as an efficient tracker of metabolic activity at the single cell level in different bioreactor configurations. We showed that RSG can be efficiently used to detect carbon limitation and dissolved oxygen limitation in bioreactor. More specifically, our methodology was used in order to assess a potential relationship between bioreactor heterogeneity and microbial population heterogeneity. Unexpectedly, no such correlation was found based on scale-down analysis. AbstractMicrobial heterogeneity in metabolic performances has attracted a lot of attention, considering its potential impact on industrial bioprocesses. However, little is known about the impact of extracellular perturbations (i.e. bioreactor heterogeneity) on cell-to-cell variability in metabolic performances (i.e. microbial population heterogeneity). In this work, we have evaluated the relevance of Redox Sensor Green (RSG) as an exogenous biosensor of metabolic activity at the single cell level. RSG signal is proportional to the activity of the electron transport chain and its signal is strongly affected by metabolic burden, availability of electron final acceptor and side metabolisms (i.e. overflow and mixed acid fermentation). RSG can also be used for the estimation of the impact of scale-down conditions on microbial metabolic robustness. The relationship linking averaged RSG activity and its cell-to-cell variability (noise) has been highlighted but seems unaffected by environmental perturbations.

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“…To alleviate the oxygen‐limiting conditions, Vitreoscilla haemoglobin (VHb) has also been used to improve the production of metabolites in high cell‐density fermentation (Pablos et al . ). To the best of our knowledge, there are a few researches for investigating the effect of VHb gene on cell growth and lipid metabolism in oleaginous micro‐organisms (Liu et al .…”

Section: Introductionmentioning

confidence: 97%

“…Overexpression of VHb also increased alpha-amylase production and total protein secretion in Schwanniomyces occidentalis (Suthar and Chattoo 2006). To alleviate the oxygen-limiting conditions, Vitreoscilla haemoglobin (VHb) has also been used to improve the production of metabolites in high cell-density fermentation (Pablos et al 2011). To the best of our knowledge, there are a few researches for investigating the effect of VHb gene on cell growth and lipid metabolism in oleaginous micro-organisms (Liu et al 2017;Zhang et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Intracellular expression ofVitreoscillahaemoglobin improves lipid production inYarrowia lipolytica

Zhang

Kang

Xiao

et al. 2019

Lett Appl Microbiol

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Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in oleaginous yeast, Yarrowia lipolytica, and its effect on lipid accumulation was analysed. During fermentation, the dissolved oxygen (DO) concentration was controlled at 5, 10, 20 and 30 of full O2 saturation and also uncontrolled by varying the stirring speed and aeration rate. Yarrowia lipolytica harbouring the VHb gene (VHb+ strain) displayed more pseudomycelium than the control strain (VHb‐ strain), and VHb expression also enhanced the cell density. When DO level was controlled at 30%, the biomass of VHb+ strains reached about 19 g l−1 and increased by 27% compared to VHb− strains. Total fatty acid contents, although, were higher in VHb+ strains than in VHb− strains under all DO levels, and maximally increased by c. 40% (from 10·5 to 14·5% of the biomass) when the DO concentration was controlled at 30%. VHb overexpression, however, markedly suppressed citrate secretion. In addition, expression of VHb also induced significant changes in fatty acid composition and increased the oleic acid content. Significance and Impact of the Study Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in Yarrowia lipolytica, which is a most attractive model for microbial oil production because of its excellent lipid accumulation capacity and convenient genetic tools. Expression of VHb resulted in lipid accumulation increased and cell morphology shift, however, markedly suppressed citrate secretion. This study provided the new strategy for fermentation technology to improve lipid production in oleaginous micro‐organisms.

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Vitreoscilla hemoglobin expression in engineered Escherichia coli: Improved performance in high cell‐density batch cultivations

Cited by 38 publications

References 25 publications

Toward efficient microaerobic processes using engineered Escherichia coli W3110 strains

Toward efficient microaerobic processes using engineered Escherichia coli W3110 strains

Microbial population heterogeneity versus bioreactor heterogeneity: Evaluation of Redox Sensor Green as an exogenous metabolic biosensor

Intracellular expression ofVitreoscillahaemoglobin improves lipid production inYarrowia lipolytica

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