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1571 KiB

Open AccessArticle

Construction and Experimental Validation of a Quantitative Kinetic Model of Nitric Oxide Stress in Enterohemorrhagic Escherichia coli O157:H7

by Jonathan L. Robinson and Mark P. Brynildsen

Bioengineering 2016, 3(1), 9; https://doi.org/10.3390/bioengineering3010009 - 6 Feb 2016

Cited by 10 | Viewed by 6799

Abstract

Enterohemorrhagic Escherichia coli (EHEC) are responsible for large outbreaks of hemorrhagic colitis, which can progress to life-threatening hemolytic uremic syndrome (HUS) due to the release of Shiga-like toxins (Stx). The presence of a functional nitric oxide (NO·) reductase (NorV), which protects EHEC from [...] Read more.

Enterohemorrhagic Escherichia coli (EHEC) are responsible for large outbreaks of hemorrhagic colitis, which can progress to life-threatening hemolytic uremic syndrome (HUS) due to the release of Shiga-like toxins (Stx). The presence of a functional nitric oxide (NO·) reductase (NorV), which protects EHEC from NO· produced by immune cells, was previously found to correlate with high HUS incidence, and it was shown that NorV activity enabled prolonged EHEC survival and increased Stx production within macrophages. To enable quantitative study of EHEC NO· defenses and facilitate the development of NO·-potentiating therapeutics, we translated an existing kinetic model of the E. coli K-12 NO· response to an EHEC O157:H7 strain. To do this, we trained uncertain model parameters on measurements of [NO·] and [O₂] in EHEC cultures, assessed parametric and prediction uncertainty with the use of a Markov chain Monte Carlo approach, and confirmed the predictive accuracy of the model with experimental data from genetic mutants lacking NorV or Hmp (NO· dioxygenase). Collectively, these results establish a methodology for the translation of quantitative models of NO· stress in model organisms to pathogenic sub-species, which is a critical step toward the application of these models for the study of infectious disease. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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3965 KiB

Open AccessArticle

Metabolic Control in Mammalian Fed-Batch Cell Cultures for Reduced Lactic Acid Accumulation and Improved Process Robustness

by Viktor Konakovsky, Christoph Clemens, Markus Michael Müller, Jan Bechmann, Martina Berger, Stefan Schlatter and Christoph Herwig

Bioengineering 2016, 3(1), 5; https://doi.org/10.3390/bioengineering3010005 - 11 Jan 2016

Cited by 36 | Viewed by 16074

Abstract

Biomass and cell-specific metabolic rates usually change dynamically over time, making the “feed according to need” strategy difficult to realize in a commercial fed-batch process. We here demonstrate a novel feeding strategy which is designed to hold a particular metabolic state in a [...] Read more.

Biomass and cell-specific metabolic rates usually change dynamically over time, making the “feed according to need” strategy difficult to realize in a commercial fed-batch process. We here demonstrate a novel feeding strategy which is designed to hold a particular metabolic state in a fed-batch process by adaptive feeding in real time. The feed rate is calculated with a transferable biomass model based on capacitance, which changes the nutrient flow stoichiometrically in real time. A limited glucose environment was used to confine the cell in a particular metabolic state. In order to cope with uncertainty, two strategies were tested to change the adaptive feed rate and prevent starvation while in limitation: (i) inline pH and online glucose concentration measurement or (ii) inline pH alone, which was shown to be sufficient for the problem statement. In this contribution, we achieved metabolic control within a defined target range. The direct benefit was two-fold: the lactic acid profile was improved and pH could be kept stable. Multivariate Data Analysis (MVDA) has shown that pH influenced lactic acid production or consumption in historical data sets. We demonstrate that a low pH (around 6.8) is not required for our strategy, as glucose availability is already limiting the flux. On the contrary, we boosted glycolytic flux in glucose limitation by setting the pH to 7.4. This new approach led to a yield of lactic acid/glucose (Y L/G) around zero for the whole process time and high titers in our labs. We hypothesize that a higher carbon flux, resulting from a higher pH, may lead to more cells which produce more product. The relevance of this work aims at feeding mammalian cell cultures safely in limitation with a desired metabolic flux range. This resulted in extremely stable, low glucose levels, very robust pH profiles without acid/base interventions and a metabolic state in which lactic acid was consumed instead of being produced from day 1. With this contribution, we wish to extend the basic repertoire of available process control strategies, which will open up new avenues in automation technology and radically improve process robustness in both process development and manufacturing. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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2514 KiB

Open AccessArticle

Rebalancing Redox to Improve Biobutanol Production by Clostridium tyrobutyricum

by Chao Ma, Jianfa Ou, Ningning Xu, Janna L. Fierst, Shang-Tian Yang and Xiaoguang Liu

Bioengineering 2016, 3(1), 2; https://doi.org/10.3390/bioengineering3010002 - 24 Dec 2015

Cited by 13 | Viewed by 8309

Abstract

Biobutanol is a sustainable green biofuel that can substitute for gasoline. Carbon flux has been redistributed in Clostridium tyrobutyricum via metabolic cell engineering to produce biobutanol. However, the lack of reducing power hampered the further improvement of butanol production. The objective of this [...] Read more.

Biobutanol is a sustainable green biofuel that can substitute for gasoline. Carbon flux has been redistributed in Clostridium tyrobutyricum via metabolic cell engineering to produce biobutanol. However, the lack of reducing power hampered the further improvement of butanol production. The objective of this study was to improve butanol production by rebalancing redox. Firstly, a metabolically-engineered mutant CTC-fdh-adhE2 was constructed by introducing heterologous formate dehydrogenase (fdh) and bifunctional aldehyde/alcohol dehydrogenase (adhE2) simultaneously into wild-type C. tyrobutyricum. The mutant evaluation indicated that the fdh-catalyzed NADH-producing pathway improved butanol titer by 2.15-fold in the serum bottle and 2.72-fold in the bioreactor. Secondly, the medium supplements that could shift metabolic flux to improve the production of butyrate or butanol were identified, including vanadate, acetamide, sodium formate, vitamin B12 and methyl viologen hydrate. Finally, the free-cell fermentation produced 12.34 g/L of butanol from glucose using the mutant CTC-fdh-adhE2, which was 3.88-fold higher than that produced by the control mutant CTC-adhE2. This study demonstrated that the redox engineering in C. tyrobutyricum could greatly increase butanol production. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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2081 KiB

Open AccessArticle

Applying Acylated Fucose Analogues to Metabolic Glycoengineering

by Julia Rosenlöcher, Verena Böhrsch, Michael Sacharjat, Véronique Blanchard, Christoph Giese, Volker Sandig, Christian P. R. Hackenberger and Stephan Hinderlich

Bioengineering 2015, 2(4), 213-234; https://doi.org/10.3390/bioengineering2040213 - 30 Nov 2015

Cited by 4 | Viewed by 7933

Abstract

Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, [...] Read more.

Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, which so far limited the large-scale adaption of this technology, can be addressed by the introduction of acylated monosaccharides. In this work, we investigated tetra-O-acetylated, -propanoylated and -polyethylene glycol (PEG)ylated fucoses. Concentrations of up to 500 µM had no substantial effects on viability and recombinant glycoprotein production of human embryonic kidney (HEK)-293T cells. Analogues applied to an engineered Chinese hamster ovary (CHO) cell line with blocked fucose de novo synthesis revealed an increase in cell surface and recombinant antibody fucosylation as proved by lectin blotting, mass spectrometry and monosaccharide analysis. Significant fucose incorporation was achieved for tetra-O-acetylated and -propanoylated fucoses already at 20 µM. Sequential fucosylation of the recombinant glycoprotein, achieved by the application of increasing concentrations of PEGylated fucose up to 70 µM, correlated with a reduced antibody’s binding activity in a Fcγ receptor IIIa (FcγRIIIa) binding assay. Our results provide further insights to modulate fucosylation by exploiting the salvage pathway via metabolic glycoengineering. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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Review

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680 KiB

Open AccessReview

Metabolic Engineering Strategies for Co-Utilization of Carbon Sources in Microbes

by Yifei Wu, Xiaolin Shen, Qipeng Yuan and Yajun Yan

Bioengineering 2016, 3(1), 10; https://doi.org/10.3390/bioengineering3010010 - 6 Feb 2016

Cited by 38 | Viewed by 10065

Abstract

Co-utilization of carbon sources in microbes is an important topic in metabolic engineering research. It is not only a way to reduce microbial production costs but also an attempt for either improving the yields of target products or decreasing the formation of byproducts. [...] Read more.

Co-utilization of carbon sources in microbes is an important topic in metabolic engineering research. It is not only a way to reduce microbial production costs but also an attempt for either improving the yields of target products or decreasing the formation of byproducts. However, there are barriers in co-utilization of carbon sources in microbes, such as carbon catabolite repression. To overcome the barriers, different metabolic engineering strategies have been developed, such as inactivation of the phosphotransferase system and rewiring carbon assimilation pathways. This review summarizes the most recent developments of different strategies that support microbes to utilize two or more carbon sources simultaneously. The main content focuses on the co-utilization of glucose and pentoses, major sugars in lignocellulose. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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4124 KiB

Open AccessReview

¹³C-Metabolic Flux Analysis: An Accurate Approach to Demystify Microbial Metabolism for Biochemical Production

by Weihua Guo, Jiayuan Sheng and Xueyang Feng

Bioengineering 2016, 3(1), 3; https://doi.org/10.3390/bioengineering3010003 - 25 Dec 2015

Cited by 18 | Viewed by 13798

Abstract

Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to [...] Read more.

Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to achieve a satisfactory yield, titer or productivity of the target chemicals for industrial commercialization. In order to overcome this challenge, ¹³C Metabolic Flux Analysis (¹³C-MFA) has been continuously developed and widely applied to rigorously investigate cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, many ¹³C-MFA studies have been performed in academic labs and biotechnology industries to pinpoint key issues related to microbe-based chemical production. Insightful information about the metabolic rewiring has been provided to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this review, we will introduce the basics of ¹³C-MFA and illustrate how ¹³C-MFA has been applied via integration with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production for various host microorganisms Full article

(This article belongs to the Special Issue Metabolic Engineering)

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1702 KiB

Open AccessReview

Metabolic Engineering of the Phenylpropanoid and Its Primary, Precursor Pathway to Enhance the Flavor of Fruits and the Aroma of Flowers

by Hadas Peled-Zehavi, Moran Oliva, Qingjun Xie, Vered Tzin, Michal Oren-Shamir, Asaph Aharoni and Gad Galili

Bioengineering 2015, 2(4), 204-212; https://doi.org/10.3390/bioengineering2040204 - 27 Nov 2015

Cited by 29 | Viewed by 9100

Abstract

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life [...] Read more.

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life of crops have generally been associated with reduced levels of volatile specialized metabolites and hence with decreased aroma and flavor. Thus, there is a need for the development of new varieties that will retain their desired traits while gaining enhanced scent and flavor. Metabolic engineering holds great promise as a tool for improving the profile of emitted volatiles of domesticated crops. This mini review discusses recent attempts to utilize metabolic engineering of the phenylpropanoid and its primary precursor pathway to enhance the aroma and flavor of flowers and fruits. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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1348 KiB

Open AccessReview

Converting Sugars to Biofuels: Ethanol and Beyond

by Aram Kang and Taek Soon Lee

Bioengineering 2015, 2(4), 184-203; https://doi.org/10.3390/bioengineering2040184 - 27 Oct 2015

Cited by 57 | Viewed by 14371

Abstract

To date, the most significant sources of biofuels are starch- or sugarcane-based ethanol, which have been industrially produced in large quantities in the USA and Brazil, respectively. However, the ultimate goal of biofuel production is to produce fuels from lignocellulosic biomass-derived sugars with [...] Read more.

To date, the most significant sources of biofuels are starch- or sugarcane-based ethanol, which have been industrially produced in large quantities in the USA and Brazil, respectively. However, the ultimate goal of biofuel production is to produce fuels from lignocellulosic biomass-derived sugars with optimal fuel properties and compatibility with the existing fuel distribution infrastructure. To achieve this goal, metabolic pathways have been constructed to produce various fuel molecules that are categorized into fermentative alcohols (butanol and isobutanol), non-fermentative alcohols from 2-keto acid pathways, fatty acids-derived fuels and isoprenoid-derived fuels. This review will focus on current metabolic engineering efforts to improve the productivity and the yield of several key biofuel molecules. Strategies used in these metabolic engineering efforts can be summarized as follows: (1) identification of better enzymes; (2) flux control of intermediates and precursors; (3) elimination of competing pathways; (4) redox balance and cofactor regeneration; and (5) bypassing regulatory mechanisms. In addition to metabolic engineering approaches, host strains are optimized by improving sugar uptake and utilization, and increasing tolerance to toxic hydrolysates, metabolic intermediates and/or biofuel products. Full article

(This article belongs to the Special Issue Metabolic Engineering)

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