High value ferulic acid biosynthesis using modular design and spent coffee ground in engineered Escherichia coli chassis.

Sophisticated genetic engineering enables microbial hosts to derive high-value aromatics in a green manner. Ferulic acid (FA) is one of the noteworthy aromatics due to its potent pharmacokinetic properties. However, the current approaches to FA biosynthesis still decamp from time- and cost-effectiveness. Herein, FA pathway was artificially reconstructed in Escherichia coli using modular designs. Comprehensive screening of E. coli lineages was reckoned for efficient synthesis of p-coumaric acid (pCA) as a precursor and FA eventually. The modular design was further advanced by harboring tyrosine transporter, adapting the heterologous codon, utilizing pCA symporter, and enriching FADH2 cofactor pools via in vivo regeneration. Taken together with simultaneous optimization of culture condition, a remarkable FA yield of 972.6 mg/L with 89.4 % conversion was achieved in 48 h, circumventing the time-consuming issue. Moreover, this study successfully exported inexpensive precursor from spent coffee ground for the first time, paving the economical way of FA biosynthesis.

Sarcopenia in the Cirrhotic Patient: Current Knowledge and Future Directions.

Cirrhosis predisposes to abnormalities in energy, hormonal, and immunological homeostasis. Disturbances in these metabolic processes create susceptibility to sarcopenia or pathological muscle wasting. Sarcopenia is prevalent in cirrhosis and its presence portends significant adverse outcomes including the length of hospital stay, infectious complications, and mortality. This highlights the importance of identification of at-risk individuals with early nutritional, therapeutic and physical therapy intervention. This manuscript summarizes literature relevant to sarcopenia in cirrhosis, describes current knowledge, and elucidates possible future directions.

Reduced Nucleotides, Thiols and O2 in Cellular Redox Balance: A Biochemist's View.

In the present review, which is aimed at researchers, teachers and students in life sciences, we try to show how the physicochemical properties of the elements and molecules define the concept of redox balance. Living organism are open systems traversed by fluxes of energy and matter. During catabolic oxidative metabolism, matter-mostly hydrogenated organic molecules-is oxidized and ultimately released as CO2. Electrons are passed over to coupling molecules, such as NAD+ and FAD, whose reduced forms serve as electrons donors in anabolic reactions. Early photosynthetic activity led to the accumulation of O2 and the transformation of the reduction to an oxidizing atmosphere, favoring the development of oxidative metabolism in living organisms. We focus on the specific properties of O2 that provide the chemical energy for the combustion reactions occurring in living cells. We explain the concepts of redox potential and redox balance in complex systems such as living cells, we present the main redox couples involved in cellular redox balance and we discuss the chemical properties underlying their cellular roles and, in particular, their antioxidant properties in the defense against reactive oxygen species (ROS). Finally, we try to provide an integrative view emphasizing the interplay between metabolism, oxidative stress and metabolic compartmentation in mammalian cells.

Azo dyes degradation by microorganisms - An efficient and sustainable approach.

Synthetic aromatic compounds consisting of various functional groups are known as dyes. These colored compounds are often discharged in effluents, and they are very dangerous to aquatic life. Basically, the dye industry started by using natural plant and insect sources, and then suddenly turned into artificial manufacturing. Natural equilibrium of our environment gets changed by the reduction in photosynthetic activity due to the dyes. In China 900,000 tons of all kinds of dyes are usually produced, which are used in many industries like food, textile, food, paper and leather. Untreated wastewater contaminates aquatic bodies by causing eutrophication, change in water color, oxygen depletion which affect aquatic organisms to a great extent. Dye wastewater is now the key environmental pollution form. In recent eras an extensive study line has been developed to explore the dye decolorization and biodegradation under both aerobic as well as anaerobic conditions. In this review, the chemistry, toxicity and microbial biodegradation/decolorization are presented. Some recent studies along with the new techniques and methodologies of remediating the dye pollution are also discussed to provide the bases of their handling. Overall, efficient and high biodegradation potential make microbes an impending foundation for green chemistry to eradicate toxic dyes from industrial wastewater.

Molecular characteristics of the multi-functional FAO enzyme ACAD9 illustrate the importance of FADH2 /NADH ratios for mitochondrial ROS formation.

A decade ago I postulated that ROS formation in mitochondria was influenced by different FADH2 /NADH (F/N) ratios of catabolic substrates. Thus, fatty acid oxidation (FAO) would give higher ROS formation than glucose oxidation. Both the emergence of peroxisomes and neurons not using FAO, could be explained thus. ROS formation in NADH:ubiquinone oxidoreductase (Complex I) comes about by reverse electron transport (RET) due to high QH2 levels, and scarcity of its electron-acceptor (Q) during FAO. The then new, unexpected, finding of an FAO enzyme, ACAD9, being involved in complex I biogenesis, hinted at connections in line with the hypothesis. Recent findings about ACAD9's role in regulation of respiration fit with predictions the model makes: cementing connections between ROS production and F/N ratios. I describe how ACAD9 might be central to reversing the oxidative damage in complex I resulting from FAO. This seems to involve two distinct, but intimately connected, ACAD9 characteristics: (i) its upregulation of complex I biogenesis, and (ii) releasing FADH2 , with possible conversion into FMN, the crucial prosthetic group of complex I. Also see the video abstract here: https://youtu.be/N7AT_HBNumg.

Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties.

Polyethylene terephthalate (PET), the most common synthetic polyester today, is largely produced from fossil resources, contributing to global warming. Consequently, sustainable sources must be developed to meet the increasing demand for this useful polymer. Here, we demonstrate a cascaded value chain that provides green PET from lignin, the world's most underutilized renewable, via fermentative production of cis, cis-muconate (MA) from lignin-based aromatics as a central step. Catechol, industrially the most relevant but apparently also a highly toxic lignin-related aromatic, strongly inhibited MA-producing Pseudomonas putida MA-1. Assessed by 13C metabolic flux analysis, the microbe substantially redirected its carbon core fluxes, resulting in enhanced NADPH supply for stress defense but causing additional ATP costs. The reconstruction of MA production in a genome-reduced P. putida chassis yielded novel producers with superior pathway fluxes and enhanced robustness to catechol and a wide range of other aromatics. Using the advanced producer P. putida MA-10 catechol, MA could be produced in a fed-batch process from catechol (plus glucose as additional growth substrate) up to an attractive titer of 74 g L-1 and a space-time-yield of 1.4 g L-1 h-1. In terms of co-consumed sugar, the further streamlined strain MA-11 achieved the highest yield of 1.4 mol MA (mol glucose)-1, providing a striking economic advantage. Following fermentative production, bio-based MA was purified and used to chemically synthetize the PET monomer terephthalic acid and the comonomer diethylene glycol terephthalic acid through five steps, which finally enabled the first green PET from lignin.

Mitochondrial complex I as a therapeutic target for Alzheimer's disease.

Alzheimer's disease (AD), the most prominent form of dementia in the elderly, has no cure. Strategies focused on the reduction of amyloid beta or hyperphosphorylated Tau protein have largely failed in clinical trials. Novel therapeutic targets and strategies are urgently needed. Emerging data suggest that in response to environmental stress, mitochondria initiate an integrated stress response (ISR) shown to be beneficial for healthy aging and neuroprotection. Here, we review data that implicate mitochondrial electron transport complexes involved in oxidative phosphorylation as a hub for small molecule-targeted therapeutics that could induce beneficial mitochondrial ISR. Specifically, partial inhibition of mitochondrial complex I has been exploited as a novel strategy for multiple human conditions, including AD, with several small molecules being tested in clinical trials. We discuss current understanding of the molecular mechanisms involved in this counterintuitive approach. Since this strategy has also been shown to enhance health and life span, the development of safe and efficacious complex I inhibitors could promote healthy aging, delaying the onset of age-related neurodegenerative diseases.

Inefficient Batteries in Heart Failure: Metabolic Bottlenecks Disrupting the Mitochondrial Ecosystem.

Mitochondrial abnormalities have long been described in the setting of cardiomyopathies and heart failure (HF), yet the mechanisms of mitochondrial dysfunction in cardiac pathophysiology remain poorly understood. Many studies have described HF as an energy-deprived state characterized by a decline in adenosine triphosphate production, largely driven by impaired oxidative phosphorylation. However, impairments in oxidative phosphorylation extend beyond a simple decline in adenosine triphosphate production and, in fact, reflect pervasive metabolic aberrations that cannot be fully appreciated from the isolated, often siloed, interrogation of individual aspects of mitochondrial function. With the application of broader and deeper examinations into mitochondrial and metabolic systems, recent data suggest that HF with preserved ejection fraction is likely metabolically disparate from HF with reduced ejection fraction. In our review, we introduce the concept of the mitochondrial ecosystem, comprising intricate systems of metabolic pathways and dynamic changes in mitochondrial networks and subcellular locations. The mitochondrial ecosystem exists in a delicate balance, and perturbations in one component often have a ripple effect, influencing both upstream and downstream cellular pathways with effects enhanced by mitochondrial genetic variation. Expanding and deepening our vantage of the mitochondrial ecosystem in HF is critical to identifying consistent metabolic perturbations to develop therapeutics aimed at preventing and improving outcomes in HF.

Can All Major ROS Forming Sites of the Respiratory Chain Be Activated By High FADH2 /NADH Ratios?: Ancient evolutionary constraints determine mitochondrial ROS formation.

Aspects of peroxisome evolution, uncoupling, carnitine shuttles, supercomplex formation, and missing neuronal fatty acid oxidation (FAO) are linked to reactive oxygen species (ROS) formation in respiratory chains. Oxidation of substrates with high FADH2 /NADH (F/N) ratios (e.g., FAs) initiate ROS formation in Complex I due to insufficient availability of its electron acceptor (Q) and reverse electron transport from QH2 , e.g., during FAO or glycerol-3-phosphate shuttle use. Here it is proposed that the Q-cycle of Complex III contributes to enhanced ROS formation going from low F/N ratio substrates (glucose) to high F/N substrates. This contribution is twofold: 1) Complex III uses Q as substrate, thus also competing with Complex I; 2) Complex III itself will produce more ROS under these conditions. I link this scenario to the universally observed Complex III dimerization. The Q-cycle of Complex III thus again illustrates the tension between efficient ATP generation and endogenous ROS formation. This model can explain recent findings concerning succinate and ROS-induced uncoupling.

Biodegradation of 2,6-dibromo-4-nitrophenol by Cupriavidus sp. strain CNP-8: Kinetics, pathway, genetic and biochemical characterization.

Compound 2,6-dibromo-4-nitrophenol (2,6-DBNP) with high cytotoxicity and genotoxicity has been recently identified as an emerging brominated disinfection by-product during chloramination and chlorination of water, and its environmental fate is of great concern. To date, the biodegradation process of 2,6-DBNP is unknown. Herein, Cupriavidus sp. strain CNP-8 was reported to be able to utilize 2,6-DBNP as a sole source of carbon, nitrogen and energy. It degraded 2,6-DBNP in concentrations up to 0.7 mM, and the degradation of 2,6-DBNP conformed to Haldane inhibition model with µmax of 0.096 h-1, Ks of 0.05 mM and Ki of 0.31 mM. Comparative transcriptome and real-time quantitative PCR analyses suggested that the hnp gene cluster was likely responsible for 2,6-DBNP catabolism. Three Hnp proteins were purified and functionally verified. HnpA, a FADH2-dependent monooxygenase, was found to catalyze the sequential denitration and debromination of 2,6-DBNP to 6-bromohydroxyquinol (6-BHQ) in the presence of the flavin reductase HnpB. Gene knockout and complementation revealed that hnpA is essential for strain CNP-8 to utiluze 2,6-DBNP. HnpC, a 6-BHQ 1,2-dioxygenase was proposed to catalyze the ring-cleavage of 6-BHQ during 2,6-DBNP catabolism. These results fill a gap in the understanding of the microbial degradation process and mechanism of 2,6-DBNP.

Evolution of peroxisomes illustrates symbiogenesis.

Recently, the group of McBride reported a stunning observation regarding peroxisome biogenesis: newly born peroxisomes are hybrids of mitochondrial and ER-derived pre-peroxisomes. What was stunning? Studies performed with the yeast Saccharomyces cerevisiae had convincingly shown that peroxisomes are ER-derived, without indications for mitochondrial involvement. However, the recent finding using fibroblasts dovetails nicely with a mechanism inferred to be driving the eukaryotic invention of peroxisomes: reduction of mitochondrial reactive oxygen species (ROS) generation associated with fatty acid (FA) oxidation. This not only explains the mitochondrial involvement, but also its apparent absence in yeast. The latest results allow a reconstruction of the evolution of the yeast's highly derived metabolism and its limitations as a model organism in this instance. As I review here, peroxisomes are eukaryotic inventions reflecting mutual host endosymbiont adaptations: this is predicted by symbiogenetic theory, which states that the defining eukaryotic characteristics evolved as a result of mutual adaptations of two merging prokaryotes.

Alternating terminal electron-acceptors at the basis of symbiogenesis: How oxygen ignited eukaryotic evolution.

What kind of symbiosis between archaeon and bacterium gave rise to their eventual merger at the origin of the eukaryotes? I hypothesize that conditions favouring bacterial uptake were based on exchange of intermediate carbohydrate metabolites required by recurring changes in availability and use of the two different terminal electron chain acceptors, the bacterial one being oxygen. Oxygen won, and definitive loss of the archaeal membrane potential allowed permanent establishment of the bacterial partner as the proto-mitochondrion, further metabolic integration and highly efficient ATP production. This represents initial symbiogenesis, when crucial eukaryotic traits arose in response to the archaeon-bacterium merger. The attendant generation of internal reactive oxygen species (ROS) gave rise to a myriad of further eukaryotic adaptations, such as extreme mitochondrial genome reduction, nuclei, peroxisomes and meiotic sex. Eukaryotic origins could have started with shuffling intermediate metabolites as is still essential today.

The gene polymorphisms of MTHFR C677T and the susceptibility of chronic pulmonary heart disease / 天津医药

Objective To investigate the relationship between gene polymorphisms of homocysteine (Hcy), metabolic enzymes methylenetetrahydrofolate reductase MTHFR C677T and chronic pulmonary heart disease (CPHD). Methods The gene polymorphisms of MTHFR C677T were determined by the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)in CPHD patients (n=120) and healthy control (HC, n=120), and genotyping was carried on. The automatic biochemistry analyzer was used to detect the level of Hcy and other related biochemical indicators. Results There was significant difference in Hcy level between the CPHD group and HC group (P<0.05). The mutation frequencies of CC, CT and TT were 24.17%, 43.33%and 32.50%, 35.00%, 47.50%and 17.50%in the CPHD group and HC group. The mutation frequencies of allele C/T were 45.83%and 54.17%in HC group, and 58.75%and 41.25%in control group. There was significant difference in the overall frequency distribution between the three genotypes (χ2 =8.010, P<0.05). The frequency of T allele was significantly higher in CPHD group than that in control group (χ2=8.025,P<0.05). Conclusion The increased Hcy and its metabolic enzyme MTHFR C677T may be involved in the occurrence and development of CPHD.

Being right on Q: shaping eukaryotic evolution.

Reactive oxygen species (ROS) formation by mitochondria is an incompletely understood eukaryotic process. I proposed a kinetic model [BioEssays (2011) 33: , 88-94] in which the ratio between electrons entering the respiratory chain via FADH2 or NADH (the F/N ratio) is a crucial determinant of ROS formation. During glucose breakdown, the ratio is low, while during fatty acid breakdown, the ratio is high (the longer the fatty acid, the higher is the ratio), leading to higher ROS levels. Thus, breakdown of (very-long-chain) fatty acids should occur without generating extra FADH2 in mitochondria. This explains peroxisome evolution. A potential ROS increase could also explain the absence of fatty acid oxidation in long-lived cells (neurons) as well as other eukaryotic adaptations, such as dynamic supercomplex formation. Effective combinations of metabolic pathways from the host and the endosymbiont (mitochondrion) allowed larger varieties of substrates (with different F/N ratios) to be oxidized, but high F/N ratios increase ROS formation. This might have led to carnitine shuttles, uncoupling proteins, and multiple antioxidant mechanisms, especially linked to fatty acid oxidation [BioEssays (2014) 36: , 634-643]. Recent data regarding peroxisome evolution and their relationships with mitochondria, ROS formation by Complex I during ischaemia/reperfusion injury, and supercomplex formation adjustment to F/N ratios strongly support the model. I will further discuss the model in the light of experimental findings regarding mitochondrial ROS formation.

Correlation analysis about folate metabolism-related genes of pregnant women with fetal congenital defects / 中国医师杂志

Objective To investigate the relationship between folate metabolism-related gene polymorphism and fetal congenital defects,and discuss the effect of genetic factors on fetal congenital defects.Methods Retrospective analysis was used to investigate the genotype and gene frequency of 5,1O-methylenetetrahydrofolate reductase (MTHFR) C677T,A1298C gene loci and ethionine synthase reductase (MTRR) A66G gene locus in 132 cases of adverse pregnancy pregnant women (case group) and 150 cases normal pregnant women (control group) at the same period.The statistical differences were analyzed between the levels of their serum folate,vitamin B12 (Vit B12) and homocysteine (HCY).Results In the serum of case group,folate was positively correlated with Vit B12,and was negatively correlated with HCY,only HCY of skeletal system defects(6 cases) was higher (t =3.409,P ＜ 0.05).Comparing genotypes frequency of the MTHFR C677T,A1298C gene loci and MTRR A66G gene locus in case group with control group,the difference above was not statistically significant (P ＞ 0.05).In these three gene loci C/T,A/C and A/G allele frequency with the control group,the difference above was not statistically significant (all P ＞ 0.05).Different genotype combinations of MTHFR C667T and A1298C gene loci in control groups had no statistically different from the control group (P ＞ 0.05),and there was no synergy.Conclusions Maternal folate metabolism-related MTHFR and MTRR genes polymorphisms can affect the metabolic products levels accordingly.However,the correlation between the changes and the genetic mechanism of fetal congenital defects needs more large samples study in depth.

The relationship of the folate metabolism related gene polymorphisms of MTHFR and MTRR with unexplained recurrent spontaneous abortion / 天津医药

Objectives To investigate the relationship of methylenetetrahydrofolate reductase (MTHFR) and methioninesynthase reductase (MTRR) with unexplained recurrent spontaneous abortion (URSA). Methods Case control study was used to select 244 patients with URSA (miscarriage group) and 116 normal women (control group) who were admitted to Tianjin Medical University General Hospital and Tianjin Women’s and Children’s Health Center from January 2013 to March 2015. The oral mucosal epithelial cells were extracted using fluorescence quantitative PCR to detect MTHFR gene C677T, A1298C and MTRR gene loci of A66G single nucleotide polymorphisms (SNP). The relationship between folate metabolism related gene polymorphisms of MTHFR and MTRR and URSA was analysed. Results The frequency of C677T genotype MTHFR was significantly higher in URSA group than that in the control group, and the frequency of CT genotype was significantly lower than that of the control group (P<0.05). There was no significant difference in the frequencies of A1298C MTRR and A66G MTHFR between the two groups. The activity of MTHFR, red cell folate and plasma folate levels were significantly lower in URSA group than those of control group. Homocysteine levels were significantly higher in URSA group than those of control group (P<0.05). There were no significant differences in serum folic acid, red cell folate, homocysteine cysteine levels between patients <35 years old and ≥ 35 years old in URSA group. Conclusion C677TMTHFR gene polymorphism is associated with unexplained recurrent spontaneous abortion.

Birth of the eukaryotes by a set of reactive innovations: New insights force us to relinquish gradual models.

Of two contending models for eukaryotic evolution the "archezoan" has an amitochondriate eukaryote take up an endosymbiont, while "symbiogenesis" states that an Archaeon became a eukaryote as the result of this uptake. If so, organelle formation resulting from new engulfments is simplified by the primordial symbiogenesis, and less informative regarding the bacterium-to-mitochondrion conversion. Gradualist archezoan visions still permeate evolutionary thinking, but are much less likely than symbiogenesis. Genuine amitochondriate eukaryotes have never been found and rapid, explosive adaptive periods characteristic of symbiogenetic models explain this. Mitochondrial proteomes, encoded by genes of "eukaryotic origin" not easily linked to host or endosymbiont, can be understood in light of rapid adjustments to new evolutionary pressures. Symbiogenesis allows "expensive" eukaryotic inventions via efficient ATP generation by nascent mitochondria. However, efficient ATP production equals enhanced toxic internal ROS formation. The synergistic combination of these two driving forces gave rise to the rapid evolution of eukaryotes. Also watch the Video Abstract.

Association between maternal MTHFR C677T polymorphism and neural tube defects in offsprings:a Meta-analysis / 天津医药

Objective To explore the association between maternal methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism and neural tube defects (NTDs). Methods CBM, VIP, CNKI, Wanfang, PubMed and Web of Science databases from set up to March, 2014 were electronically searched to identify case-control studies on the relationship between maternal MTHFR C677T polymorphism and NTDs. The data were quantitatively analyzed by RevMan 5.0 software. Results A total of 25 studies were selected including 2 282 cases and 3 420 controls. Overall, the pooled OR (with 95%CI) under co-dominant model and allele contrast were 2.28(1.60-3.24), 1.25(1.02-1.53) and 1.42(1.21-1.67). Subgroup analysis showed significant association between maternal MTHFR C677T polymorphism and NTDs susceptibility in Asian populations. Conclusion The present meta-analysis suggests that MTHFR C677T polymorphism is significantly associated with maternal risk for NTDs, especially in Asian populations.

From genome to phenome-Simple inborn errors of metabolism as complex traits.

Sporadically, patients with a proven defect in either mFAO or OXPHOS are described presenting with a metabolic profile and clinical phenotype expressing concurrent defects in both pathways. Biochemical linkages between both processes are tight. Therefore, it is striking that concurrent dysfunction of both systems occurs so infrequent. In this review, the linkages between OXPHOS and mFAO and the hypothesized processes responsible for concurrent problems in both systems are reviewed, both from the point of view of primary biochemical connections and secondary cellular responses, i.e. signaling pathways constituting nutrient-sensing networks. We propose that affected signaling pathways may play an important role in the phenomenon of concurrent defects. Recent data indicate that interference in the affected signaling pathways may resolve the pathological phenotype even though the primary enzyme deficiency persists. This offers new (unexpected) prospects for treatment of these inborn errors of metabolism. This article is part of a Special Issue entitled: From Genome to Function.

How to deal with oxygen radicals stemming from mitochondrial fatty acid oxidation.

Oxygen radical formation in mitochondria is an incompletely understood attribute of eukaryotic cells. Recently, a kinetic model was proposed, in which the ratio between electrons entering the respiratory chain via FADH2 or NADH determines radical formation. During glucose breakdown, the ratio is low; during fatty acid breakdown, the ratio is high (the ratio increasing--asymptotically--with fatty acid length to 0.5, when compared with 0.2 for glucose). Thus, fatty acid oxidation would generate higher levels of radical formation. As a result, breakdown of fatty acids, performed without generation of extra FADH2 in mitochondria, could be beneficial for the cell, especially in the case of long and very long chained ones. This possibly has been a major factor in the evolution of peroxisomes. Increased radical formation, as proposed by the model, can also shed light on the lack of neuronal fatty acid oxidation and tells us about hurdles during early eukaryotic evolution. We specifically focus on extending and discussing the model in light of recent publications and findings.