Efficient Metabolic Pathways Research Articles

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as "vitamin E") as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules

BackgroundThe depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach.ResultsWe developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and α-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and α-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design.ConclusionsUsing directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and α-pinene. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms.

Abstract 9643: Effect of Partial Fatty Acids Oxidation Inhibition With Trimetazidine on Survival in Heart Failure: Results From an International Multicentre Retrospective Cohort Study

Background: Trimetazidine (TMZ) shifts myocardial energy production from free fatty acids to glucose oxidation, a more efficient metabolic pathway. Previous small studies in patients with chronic heart failure (CHF) have shown that TMZ can improve contractile left ventricular function, exercise capacity and NYHA class compared to placebo. However, no data on the effects of TMZ on survival in patients with CHF have ever been produced. Methods: In this international multicentre retrospective cohort study 669 patients (521 post-ischaemic) from the CHF clinics of our institutions were recruited. 362 patients (288 post-ischaemic) were on TMZ (20 mg tid or 35 mg bid) due to symptoms persistence (dyspnoea on exertion, fatigue, orthopnoea, oedema, alone or in combination) despite up titration of optimal CHF medical therapy, while the remaining patients continued conventional CHF therapy alone. Effects of TMZ administration were evaluated by Kaplan-Meier survival analysis and Cox-Regression survival analysis. Obtained results were corrected with propensity score (PS) in order to minimize selection bias. The protocol has been registered in the Italian Pharmaceutical Agency (AIFA) - Italian Ministry of Health, on the observational studies registry, ID number 458. Results: Kaplan-Meier analysis for global mortality showed 11•3% improved global survival (p=0•015) and 8•5% improved survival for death from cardiovascular disease (CVD) causes (p=0•050) in the TMZ group. Cox regression analysis for global mortality showed a significant risk reduction for TMZ treated patients with a PS-corrected hazard ratio (HR)= 0•189 (confidence interval - CI 95%: 0•079-0•454; p=0•0002). TMZ also showed a good risk reduction profile for CVD death causes (PS-adjusted HR=0•072, CI 95%: 0•019-0•268, p=0•0001). The rate of hospitalization for cardiovascular causes was reduced by 10.4% at 5 years (p < 0•0005) with increased hospitalization-free survival of •8 months. Conclusions: TMZ is effective in reducing both global and cardiovascular mortality and event-free survival in patients with CHF. The addition of TMZ on top of optimal medical therapy improves long-term survival in CHF patients.

Metabolic engineering for improved microbial pentose fermentation

Global concern over the depletion of fossil fuel reserves, and the detrimental impact that combustion of these materials has on the environment, is focusing attention on initiatives to create sustainable approaches for the production and use of biofuels from various biomass substrates. The development of a low-cost, safe and eco-friendly process for the utilisation of renewable resources to generate value-added products with biotechnological potential as well as robust microorganisms capable of efficient fermentation of all types of sugars are essential to underpin the economic production of biofuels from biomass feedstocks. Saccharomyces cerevisiae, the most established fermentation yeast used in large scale bioconversion strategies, does not however metabolise the pentose sugars, xylose and arabinose and bioengineering is required for introduction of efficient pentose metabolic pathways and pentose sugar transport proteins for bioconversion of these substrates. Our approach provided a basis for future experiments that may ultimately lead to the development of industrial S. cerevisiae strains engineered to express pentose metabolising proteins from thermophilic fungi living on decaying plant material and here we expand our original article and discuss the strategies implemented to improve pentose fermentation.

Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic fungus Botrytis cinerea: from plant hexoses to mannitol

The main steps for carbon acquisition and conversion by Botrytis cinerea during pathogenesis of sunflower cotyledon were investigated here. A sequential view of soluble carbon metabolites detected by NMR spectroscopy during infection is presented. Disappearance of plant hexoses and their conversion to fungal metabolites were investigated by expression analysis of an extended gene family of hexose transporters (Bchxts) and of the mannitol pathway, using quantitative PCR. In order to analyse the main fungal metabolic routes used by B. cinerea in real time, we performed, for the first time, in vivo NMR analyses during plant infection. During infection, B. cinerea converts plant hexoses into mannitol. Expression analysis of the sugar porter gene family suggested predominance for transcription induced upon low glucose conditions and regulated according to the developmental phase. Allocation of plant hexoses by the pathogen revealed a conversion to mannitol, trehalose and glycogen for glucose and a preponderant transformation of fructose to mannitol by a more efficient metabolic pathway. Uptake of plant hexoses by B. cinerea is based on a multigenic flexible hexose uptake system. Their conversion into mannitol, enabled by two simultaneously expressed pathways, generates a dynamic intracellular carbon pool.

The tragedy of the commons in microbial populations: insights from theoretical, comparative and experimental studies

First principles of thermodynamics imply that metabolic pathways are faced with a trade-off between the rate and yield of ATP production. Simple evolutionary models argue that this trade-off generates a fundamental social conflict in microbial populations: average fitness in a population is highest if all individuals exploit common resources efficiently, but individual reproductive rate is maximized by consuming common resources at the highest possible rate, a scenario known as the tragedy of the commons. In this paper, I review studies that have addressed two key questions: What is the evidence that the rate-yield trade-off is an evolutionary constraint on metabolic pathways? And, if so, what determines evolutionary outcome of the conflicts generated by this trade-off? Comparative studies and microbial experiments provide evidence that the rate-yield trade-off is an evolutionary constraint that is driven by thermodynamic constraints that are common to all metabolic pathways and pathway-specific constraints that reflect the evolutionary history of populations. Microbial selection experiments show that the evolutionary consequences of this trade-off depend on both kin selection and biochemical constraints. In well-mixed populations with low relatedness, genotypes with rapid and efficient metabolism can coexist as a result of negative frequency-dependent selection generated by density-dependent biochemical costs of rapid metabolism. Kin selection can promote the maintenance of efficient metabolism in structured populations with high relatedness by ensuring that genotypes with efficient metabolic pathways gain an indirect fitness benefit from their competitive restraint. I conclude by suggesting avenues for future research and by discussing the broader implications of this work for microbial social evolution.

Solved upscaling problems for implementing deammonification of rejection water

So far, extremely efficient metabolic pathways for nitrogen removal exclusively by autotrophic organisms are well established in scientific literature but not in practice. This paper presents results from the successful implementation of rejection water deammonification in a full-scale single sludge system at the WWTP Strass, Austria. Anaerobic ammonia oxidising biomass has been accumulated during a 2.5 year start-up period when the reactor size was gradually scaled up in the steps. The pH-controlled deammonification system (DEMON) has reached a design capacity of eliminating approximately 300 kg of nitrogen per day. Energy savings outperform expectations, decreasing the mean specific demand for compressed air from 109 m3(kg N)(-1) to 29 m3(kg N)(-1). Dominance of autotrophic metabolism is confirmed by organic effluent loads topping influent loads.

Regulation of ubiquinone metabolism

Interest in ubiquinone (UQ) has increased during recent years, mainly because of its antioxidant function and its use as a dietary supplement. However, our knowledge of the biosynthesis, catabolism, and regulation of this lipid in mammalian tissues is quite limited. UQ exhibits a high rate of turnover in all tissues indicating that cells possess efficient metabolic pathways for handling this compound and controlling its tissue levels. Besides reviewing the generally accepted metabolic pathway, alternative synthetic mechanisms are described. The lack of data concerning catabolism and regulation of this compound is emphasized. Reasons for the rather limited uptake of dietary UQ are discussed and alternative mechanisms for its beneficial effects on organ function are suggested. Since appropriate tissue uptake of dietary UQ probably only occurs in deficient states, the definition of partial UQ deficiency and its consequences is urgently needed. The possibility of raising tissue UQ levels by drug treatment or natural metabolites is raised as a choice of preference for the future.

Metabolically optimised BHK cell fed-batch cultures

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h −1) and cell viability, higher maximum cell concentration (2.5 vs. 1.1×10 6 cell ml −1), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.

Mitochondrial disorders.

Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.