Current Metabolomics (v.4, #1)

Meet Our Editor by David G. Watson (1-1).

Preface by David G. Watson (3-3).

Background: Rising costs and a depleting supply of oil, as well as environmental concerns, have led to strong interest in renewable fuels and chemicals. In the last decade, there was considerable interest for microbial production of biofuels by metabolic engineering approach as an attractive alternative to transportation fuels. This review is aimed to provide details of different biosynthetic pathways for microbial production of biofuels.
Methods: Recent advancements of synthetic biology have led to the further development of genetic engineering of microorganism, which has been great motivation for developing strategy for microbial production of biofuels. Rational design of metabolism is very important for production of biofuels. In silico prediction of metabolic flux distribution of the metabolic pathways enabled us to decide the time consuming steps in metabolic engineering.
Results: In recent years significant efforts have been made to engineer microorganisms to produce bioethanol, higher chain alcohols, fatty acids and isoprenoid based biodiesel. Metabolic engineering involves improvement of biofuels formation through the modification of specific genes or addition new genes involved in biochemical reactions with the use of recombinant DNA technology. System-level approach to analyze and engineer metabolism based on flux distributions obtained from metabolomics and 13C metabolic flux analysis have been extensively used to produce biofuels in Escherichia coli and yeast.
Conclusion: The biofuels obtained from microorganisms can be used as renewable resource for gasoline, diesel and jet fuel. The development of renewable bioenergy will significantly reduce our dependence on fossil fuels for elegantly safer environment.

Bioenergy is one of the best options towards replacing fossil fuels with renewable, carbon-neutral alternatives. To generate bioenergy at an industrial scale, we must be able to design an optimized cell factory that will convert chemically complex residual biomass or lignocellulose to useful liquid fuels. Microorganisms have evolved a sophisticated enzymatic machinery to degrade these heterogeneous biomass substrates to soluble sugar. These enzyme cocktails are primarily composed of various glycoside hydrolases (GHs) and act synergistically to efficiently cleave the glycosidic linkages in the biomass. Here we review the current strategies to engineer and improve cellulases and the efforts to understand and create microbes with improved catalytic proficiencies.

Background: Xylanase (EC, endo-1,4--xylan 4-xylanohydrolase) is a naturally occurring enzyme that breaks down hemicellulose by converting -1,4-xylan into xylooligosaccharides, xylobiose and xylose. It is frequently found in microbes and fungi. Considering the depleting resources of natural petroleum products especially petrol, current status of xylanase especially with respect to structural aspects and its application in biofuel production has been reviewed to show evidences that describe the significance of xylanase in bio-processing industry.
Methods: The systematic investigation and web content related to xylanase and its industrial applications are reviewed.
Results: Diverse primary studies and structured literature reviews met the review criteria. Studies comprised involvement of xylanase in various commercial applications. It has been reported to play an indispensable role in bio-bleaching of paper pulp, human digestion, baking industry, clarification of fruit juices and beer, malting and in the production of biofuels. Besides, structural aspects indicated importance of carbohydrate binding module in catalysis.
Conclusion: The wide usage of xylanase from dough conditioning to biofuel production has garnered commercial importance to the enzyme. Many such explorations are anticipated in near future.

Strain Engineering for Improved Bio-Fuel Production by Supriya Ratnaparkhe, Milind B. Ratnaparkhe, Arun Kumar Jaiswal, Anil Kumar (38-48).
Background: Biofuel research has gained considerable interest in the recent past since these are renewable fuels and are considered environment friendly unlike their conventional counterparts. Microbes play important roles in most aspects of biofuel production particularly hydrolysis of biomass, lipid production, fermentation of sugars and production of advanced biofuels.
Methods: In the present review, we have discussed some interesting studies pertaining to strain engineering of microorganisms for improved uptake of multiple sugars, consolidated bioprocessing, production of advanced fuels, and biofuel production in photosynthetic microorganisms.
Result: Genetically manipulated microbes for co-consumption of multiple sugars, especially glucose and xylose which are abundantly present in hydrolysate of lignocellulosic biomass, have improved the overall efficiency of the whole process. Besides, some designer microbes have been reported for consolidated bioprocessing that aims at a single-step cellulosic biomass fermentation thus cutting down the cost involved in various processes. There are also interesting reports of engineered microbes capable of producing high titers of advanced fuels such as butanol and isopropanol. Engineered photosynthetic microbes such as microalgae and cyanobacteria are promising sources of enhanced lipid and alcohol production. The ability of photosynthetic microbes to grow in water, thus not competing for arable land, make them suitable for production of sustainable fuel.
Conclusion: Biofuels have the potential to completely replace fossil fuels in future but the current production of biofuels is unable to fulfil the global fuel requirement. Microorganisms play an important role in various aspects of biofuel production. Microbial strains have been developed to overcome some of the challenges in biofuel production such as lack of multiple sugar assimilation, intolerance to inhibitors and solvents, and low titer of the products. In spite of all the advancements, more improvement is needed in order to achieve the fuel titers high enough for economically sustainable production. The pace at which this field is advancing, there exist possibilities of synthetic microbes designed with engineered minimal genome to produce desired fuels and chemicals at industrially relevant levels.

Designer Plants for Biofuels: A Review by Ajay Badhan, Tim McAllister (49-55).
Injudicious over-consumption of fossil fuels over the last century has generated an imbalance in the carbon cycle, dramatically increasing atmospheric carbon dioxide and contributing to climate change. Much of the released carbon is ancient, accumulating in the earth's crust in the form of oil, coal or methane gas over millions of years. In contrast, the carbon in biofuels has only recently been sequestered from the atmosphere as a result of photosynthesis. From this perspective, the release of carbon dioxide from biofuels can be viewed as carbon neutral making biofuels particularly attractive as an energy source. First generation biofuels relied primarily on cereal starch as a source of glucose for biofuel production, a practice that faced scrutiny due to its competition for human food, its impact on biodiversity and reliance on chemical fertilizers and fossil fuels for harvest. Second generation biofuels focused on lignocellulosic waste as a carbon resource for bioenergy production. Cellulosic biomass is the most abundant of the renewable resources and represents an enormous storehouse of sugars. However, plant cell walls are chemically complex, heterogeneous structures that have evolved to counter the process of enzymatic digestion. The layered structure with cross-linkages among cellulose, xylan and lignin are examples of the recalcitrant moieties that resist enzymatic breakdown. The use of exogenous enzymes or chemical treatment to weaken these barriers is expensive and typically achieves only partial hydrolysis. Recent advancements in plant genetic engineering have demonstrated the potential to design plants for improved cell wall deconstruction as well as to produce cell wall carbohydrases within the plant biomass. These technologies offer significant promise to lower the cost of production of biofuels from lignocellulosic biomass. In this review, we provide summary of recent research efforts focused on improving the biomass characteristics of plants for biofuel production through alteration of the major cell wall biosynthesis genes or pathways.

Profiling of Extracellular Metabolites in CHO-K1 Cells Cultured in Medium with Different Levels of Serum and Glutamine by Yumi Zuhanis Has-Yun Hashim, Salfarina Ezrina Mohmad Saberi, Maizirwan Mel, Vasila Packeer Mohamed (56-62).
Background: Gas Chromatography Mass Spectrometry (GCMS)-based metabolomics has been shown to substantially contribute to understanding of the physiological state of mammalian producer cells leading to improved large-scale production of biopharmaceuticals. In this study, GCMSbased metabolomics (global metabolite analysis) approach was used to understand the effects of different levels of serum and glutamine on metabolite profiles associated with cell growth behaviour and insulin-like growth factor 1 (IGF1) protein production.
Methods: CHO-KI cells producing IGF1 were obtained from American Type Culture Collection (ATCC) and grown in T- ask (37°C, 5 % CO2) until 70-80 % conuent in optimized medium, RPMI 1640 with different levels of of serum (%) and glutamine (mM). The different compositions of serum and glutamine were based on three levels full factorial design generated by MODDE, SIMCA P+Version 12 (Umetrics). Samples were then taken at 8-hourly intervals for routine cell counting, biochemical responses, IGF1 protein concentration and global metabolite analysis (GCMS). Conditioned media from each time point were spun down before injection into GCMS. Data from GCMS were then transferred to SIMCAP+ Version 12 (Umetrics) for chemometric evaluation using Partial Least Square Discriminant Analysis (PLS-DA).
Results: Experiment Run 3 which produced the highest cell density was discriminated from other runs based on the extracellular metabolites. This indicates that Run 3 although supplied with very little glutamine, was able to undergo active metabolism including glycolysis and as such has efficiently used up the nutrient sources to produce the highest cell number. However, no clear relationship can be delineated for the IGF1 production.
Conclusion: Global metabolite analysis approach has been proven to be able to give more insights into the metabolism of cells as compared to routine biochemical studies where data was less informative.

Urinary Metabolite Profiles May be Predictive of Cognitive Performance under Conditions of Acute Sleep Deprivation by Nicholas J. DelRaso, Donald L. Harville, Mari L. Chamberlain, Paul E. Anderson, Isaie Sibomana, Michael L. Raymer, Nicholas V. Reo (63-77).
Continuous and sustained actions in military and civilian operational environments typically lead to reduced sleep normally required to perform optimally. Because cognitive fatigue leading to defects in performance is an occupational hazard, there is a recognized need for real-time detection technologies that minimize cognitive fatigue-induced mishaps. Here, 23 individuals were subjected to 36 h of continuous wakefulness, and cognitive psychomotor vigilance and automated neuropsychological assessment metric tests were conducted over the last 24 h of wakefulness. Urine was collected prior to and during the cognitive testing period for metabolite analysis using proton NMR spectroscopy. Multivariate statistical analysis showed that temporal changes in urinary metabolite profiles mirrored cognitive performance during continuous wakefulness. Additionally, subjects identified by cognitive assessments as having a high tolerance (n=6) or low tolerance (n=6) to sleep deprivation could be classified separately with statistical confidence (p<0.001) using urinary metabolite profiles. We identified 20 specific metabolites that could be used to classify cognitive fatigue tolerance at early (0 - 12 h) and late (28 h) times during the 36-h sleep deprivation period. Many of these metabolites (11 of 20) appeared to be associated with energy metabolism or nutritional status. Analysis of subject food logs suggested that increases in dietary protein intake prior to sleep deprivation leads to improved cognitive performance. Taken together, our results indicate that urinary metabolomics may be useful for identifying metabolite markers that can be incorporated into sensor platforms to screen for cognitive performance readiness, prior to scheduling tasks requiring a high level of cognitive function.

Blood plasma and serum are among the most common biofluids studied by metabolomics. The plethora of collection tubes available and variety of coatings on offer make it difficult to decide which collection tube offers the greatest reproducibility and least signal interference for metabolomics studies. We have acquired the NMR proton spectra of water washes of a range of monovettes available to the UK National Health Service (NHS) clinicians in order to demonstrate the variability between tubes, including different batches of the same type. This technical note provides clear evidence that sample collection tubes should be tested before selecting the most appropriate one for NMR metabolomic studies. From the collection tubes tested in this study we found Serum Z blood collection tube most suited to NMR metabolomics both in residual signals arising and also variance between batches.