Modification of the Genome of Rhodobacter sphaeroides and Construction of Synthetic Operons

Jaschke, Paul R.; Saer, Rafael G.; Noll, Stephan; Beatty, J. Thomas

doi:10.1016/b978-0-12-385075-1.00023-8

Cited by 32 publications

(29 citation statements)

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“…In obtaining such integrated views, genome-scale metabolic network models can serve both as databases for storage and organization and as tools for the combination and analysis of heterogeneous data sets [6]. A particular interest of our laboratory is developing an integrated understanding of metabolic networks in photosynthetic microbes, because of their abundance in nature, the unique aspects of a solar-driven lifestyle and their growing importance in biotechnological applications [7-9]. …”

Section: Introductionmentioning

confidence: 99%

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

2013

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BackgroundImproving our understanding of processes at the core of cellular lifestyles can be aided by combining information from genetic analyses, high-throughput experiments and computational predictions.ResultsWe combined data and predictions derived from phenotypic, physiological, genetic and computational analyses to dissect the metabolic and energetic networks of the facultative photosynthetic bacterium Rhodobacter sphaeroides. We focused our analysis on pathways crucial to the production and recycling of pyridine nucleotides during aerobic respiratory and anaerobic photosynthetic growth in the presence of an organic electron donor. In particular, we assessed the requirement for NADH/NADPH transhydrogenase enzyme, PntAB during respiratory and photosynthetic growth. Using high-throughput phenotype microarrays (PMs), we found that PntAB is essential for photosynthetic growth in the presence of many organic electron donors, particularly those predicted to require its activity to produce NADPH. Utilizing the genome-scale metabolic model iRsp1095, we predicted alternative routes of NADPH synthesis and used gene expression analyses to show that transcripts from a subset of the corresponding genes were conditionally increased in a ΔpntAB mutant. We then used a combination of metabolic flux predictions and mutational analysis to identify flux redistribution patterns utilized in the ΔpntAB mutant to compensate for the loss of this enzyme. Data generated from metabolic and phenotypic analyses of wild type and mutant cells were used to develop iRsp1140, an expanded genome-scale metabolic reconstruction for R. sphaeroides with improved ability to analyze and predict pathways associated with photosynthesis and other metabolic processes.ConclusionsThese analyses increased our understanding of key aspects of the photosynthetic lifestyle, highlighting the added importance of NADPH production under these conditions. It also led to a significant improvement in the predictive capabilities of a metabolic model for the different energetic lifestyles of a facultative organism.

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Section: Introductionmentioning

confidence: 99%

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

2013

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“…Alternatively, these anoxygenic photosynthetic microorganisms themselves may be employed as chassis for engineering photo-electro-autotrophic production. Some basic tools for genetic and metabolic engineering are available for some of these organisms, such as for the green bacterium Chlorobaculum tepidum [ 67 ] and purple bacterium Rhodobacter sphaeroides [ 68 , 69 ].…”

Section: Resultsmentioning

confidence: 99%

Integrated In Silico Analysis of Pathway Designs for Synthetic Photo-Electro-Autotrophy

et al. 2016

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The strong advances in synthetic biology enable the engineering of novel functions and complex biological features in unprecedented ways, such as implementing synthetic autotrophic metabolism into heterotrophic hosts. A key challenge for the sustainable production of fuels and chemicals entails the engineering of synthetic autotrophic organisms that can effectively and efficiently fix carbon dioxide by using sustainable energy sources. This challenge involves the integration of carbon fixation and energy uptake systems. A variety of carbon fixation pathways and several types of photosystems and other energy uptake systems can be chosen and, potentially, modularly combined to design synthetic autotrophic metabolism. Prior to implementation, these designs can be evaluated by the combination of several computational pathway analysis techniques. Here we present a systematic, integrated in silico analysis of photo-electro-autotrophic pathway designs, consisting of natural and synthetic carbon fixation pathways, a proton-pumping rhodopsin photosystem for ATP regeneration and an electron uptake pathway. We integrated Flux Balance Analysis of the heterotrophic chassis Escherichia coli with kinetic pathway analysis and thermodynamic pathway analysis (Max-min Driving Force). The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the proton pumping rhodopsin photosystems and the high ATP maintenance of E. coli. If these factors can be tackled, our analysis indicates the highest growth potential for the natural reductive tricarboxylic acid cycle and the synthetic pyruvate synthase–pyruvate carboxylate -glyoxylate bicycle. Both carbon fixation cycles are very ATP efficient, while maintaining fast kinetics, which also results in relatively low estimated protein costs for these pathways. Furthermore, the synthetic bicycles are highly thermodynamic favorable under conditions analysed. However, the most important challenge identified for improving photo-electro-autotrophic growth is increasing the proton-pumping rate of the rhodopsin photosystems, allowing for higher ATP regeneration. Alternatively, other designs of autotrophy may be considered, therefore the herein presented integrated modeling approach allows synthetic biologists to evaluate and compare complex pathway designs before experimental implementation.

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“…Bacteria revert to the acetate metabolism, which allows the low energy production because of the finite space within their inner bacterial membrane [17]. The metabolites flood the membrane and the cost of holding them outweighs the advantage of producing energy because it impedes the electron transport function [18]. When supplemented with environmental resources, R. sphaeroides adapts well to its expanded metabolic capabilities, and becomes permeable to metabolites.…”

Section: Bacterial Growth In Minimal Media With Additional Carbon Sourcementioning

confidence: 99%

The Effects of Different Carbon Sources on the Growth of <i>Rhodobacter sphaeroides</i>

Zavala¹,

Baeza²,

González³

et al. 2019

AiM

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Rhodobacter sphaeroides is a purple non-sulfur bacterium that belongs to the α-3 subdivision of Proteobacteria. R. sphaeroides is a model bacterial species because of its complex genome structure and expanded metabolic capabilities.The genome of R. sphaeroides consists of two circular chromosomes and five endogenous plasmids. It has the ability to grow under a wide variety of environmental conditions. It grows aerobically (~20% O 2 ), semi-aerobically (~2% O 2 ), and photosynthetically (under anaerobic condition plus light). It has been previously shown that many bacterial species utilize a number of alternate carbon sources for their optimal growth under a variety of growth conditions. We hypothesize that different or an additional carbon source in the minimal medium differentially affects the bacterial growth under dark-aerobic conditions. The bacterial growth kinetics and the number of cells in the bacterial culture were analyzed by measuring the optical density (OD at 600 nm) and the colony forming units (CFUs) at regular intervals of bacterial cultures. Results reveal that sodium succinate is the preferred sole carbon source for the optimal growth of R. sphaeroides. The results of growth kinetics and CFUs together concluded that from the tested carbon sources, sodium succinate is the best single carbon source in the minimal media for the optimal growth of R. sphaeroides. Interestingly, cell culture grown in SIS supplemented with sodium acetate exhibits a prolonged lag phase with the lowest ODs and CFUs that later switches to the growth-burst phase support previously discovered similar phenomenon of the growth-rate switch in the presence of acetate metabolism. Future work will utilize the aerobically grown R. sphaeroides' cells as a biocatalyst to deplete the oxygen levels from natural gas streams and industrial gas pipelines.

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Modification of the Genome of Rhodobacter sphaeroides and Construction of Synthetic Operons

Cited by 32 publications

References 75 publications

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

Integrated In Silico Analysis of Pathway Designs for Synthetic Photo-Electro-Autotrophy

The Effects of Different Carbon Sources on the Growth of <i>Rhodobacter sphaeroides</i>

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Modification of the Genome of Rhodobacter sphaeroides and Construction of Synthetic Operons

Cited by 32 publications

References 75 publications

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

Integrated In Silico Analysis of Pathway Designs for Synthetic Photo-Electro-Autotrophy

The Effects of Different Carbon Sources on the Growth of &lt;i&gt;Rhodobacter sphaeroides&lt;/i&gt;

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The Effects of Different Carbon Sources on the Growth of <i>Rhodobacter sphaeroides</i>