Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting

<p>Abstract</p> <p>Background</p> <p>Growth substrates, aerobic/anaerobic conditions, specific growth rate (μ) etc. strongly influence <it>Escherichia coli </it>cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA.</p> <p>Results</p> <p>We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor <it>E. coli </it>K-12 MG1655 flux and energy metabolism dynamic responses to change of μ and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing μ and carbon wasting to non-CO₂by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of <it>E. coli </it>pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on μ, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway.</p> <p>Conclusions</p> <p>It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.</p

Clostridium ljungdahlii as a biocatalyst in microbial electrosynthesis – Effect of culture conditions on product formation

Microbial electrosynthesis enables the production of value-added chemicals from CO2 and electrons provided by an electrode. Clostridium ljungdahlii is an electroactive acetogen that potentially could be used in microbial electrosynthesis systems. However, the optimal operational parameters for microbial electrosynthesis using C. ljungdahlii are not known. Here, we explored the effects of yeast extract, pH, and cathode potential. A low initial pH increased the rate of acetate production from CO2 and H2 in serum bottle cultures. When cultivated in bioelectrochemical systems, the optimal coulombic efficiency (i.e. close to 100 %) was observed at a cathode potential between −0.8 V and −1.0 V, while the highest productivity was reached at −1.0 V. Addition of yeast extract to the medium was needed to ensure reproducible results. Using cyclic voltammetry, we detected hydrogen-mediated extracellular electron transfer of C. ljungdahlii during growth on CO2 in a bioelectrochemical system. These results show that operational parameters should be chosen carefully to maximise the efficiency of microbial electrosynthesis

Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase

<p>Abstract</p> <p>Background</p> <p>The biotechnology industry has extensively exploited <it>Escherichia coli </it>for producing recombinant proteins, biofuels etc. However, high growth rate aerobic <it>E. coli </it>cultivations are accompanied by acetate excretion <it>i.e</it>. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.</p> <p>Results</p> <p>In the current work, growth rate dependent acetate overflow metabolism of <it>E. coli </it>was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h^-1) is the repression of acetyl-CoA synthethase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h^-1with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for <it>E. coli </it>chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of <it>E. coli </it>decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h^-1leading to total Acs inhibition and fast accumulation of acetate.</p> <p>Conclusion</p> <p>This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in <it>E. coli </it>shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in <it>E. coli </it>is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.</p

Systems-level engineering and characterization of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO + H as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H) and the other steel mill off-gas (2% H). Results were characterized using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimization of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens

Unraveling acetogen gas fermentation using quantitative systems biology

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Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Advanced continuous cultivation methods for systems microbiology

Increasing the throughput of systems biology-based experimental characterization of in silico designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state – growth space analysis (GSA) – can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories