Overproduction Of Metabolites Research Articles

Deletions of conserved extracytoplasmic function sigma factors-encoding genes in Streptomyces have a major impact on secondary metabolism

BackgroundEthanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, which encodes a 19-amino acid peptide with an unknown function.ResultsTo establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism.ConclusionsThese findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.

Improving Muscat Hamburg Wine Quality with Innovative Fermentation Strategies Using Schizosaccharomyces pombe Derived from Fermented Grains of Sauce-Flavor Baijiu.

This study investigates innovative approaches to improve the quality and aroma characteristics of Muscat Hamburg wine production by substituting the conventional Saccharomyces cerevisiae yeast with an efficient fermentation strain of Schizosaccharomyces pombe. The typical use of S. cerevisiae in Muscat Hamburg wine often leads to uniformity and prolonged processing times, requiring subsequent malolactic fermentation to degrade excessive malic acid. The study advocates for the replacement of S. cerevisiae with a specific S. pombe strain, Sp-410, isolated from the fermented grains of sauce-flavor Baijiu, a Chinese spirit. Muscat Hamburg wine fermented with the S. pombe strain demonstrates decreased malic acid levels, offering a potential alternative to malolactic fermentation. However, exclusive S. pombe fermentation may result in an overproduction of acetic acid metabolites, leading to a monotonous taste. In response, the study proposes a mixed fermentation approach, combining the S. pombe strain with a Saccharomyces uvarum strain and a non-Saccharomyces yeast, Torulaspora delbrueckii. The optimized mixed fermentation strategies (M:SP+TD and M60SP+TD) involve specific proportions and intervals of inoculation, aiming to enhance the quality and aroma complexity of Muscat Hamburg wine. In conclusion, this research contributes to advancing the production of high-quality Muscat Hamburg wines, utilizing S. pombe as the primary yeast strain and implementing mixed fermentation methodologies.

New paradigms in the study of the cholinergic system and metabolic diseases: Acetyl-and-butyrylcholinesterase.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes that belong to the neuromuscular cholinergic system, their main function is to hydrolyze the neurotransmitter acetylcholine (ACh), through their hydrolysis these enzymes regulate the neuronal and neuromuscular cholinergic system. They have recently attracted considerable attention due to the discovery of new enzymatic and nonenzymatic functions. These discoveries have aroused the interest of numerous scientists, consolidating the relevance of this group of enzymes. Recent investigations have revealed a positive correlation between several risk factors for metabolic syndrome (MetS) and the expression of cholinesterases (ChE's), which underscore the impact of high ChE's activity on the pro-inflammatory state associated with MetS. In addition, the excessive hydrolysis of ACh and other choline esters (succinylcholine, propionylcholine, butyrylcholine, etc.) by both ChE's results in the overproduction of fatty acid precursor metabolites, which facilitate the synthesis of very low-density lipoproteins and triacylglycerols. Participation in these processes may represent the link between ChE's and metabolic disorders. However, further scientific research is required to fully elucidate the involvement of ChE's in metabolic diseases. This review aims to collect recent research studies that contribute to understanding the association between the cholinergic system and metabolic diseases.

Upregulated synthesis and production of bioactive compounds in Lotus arabicus L. by in vitro feeding with dried powder of date palm seeds

BackgroundPlants are considered the primary source of many principal bioactive compounds that have been utilized in a wide range of applications including the pharmaceutical and biotechnological industries. Therefore, there is an imperative need to modulate the production of natural bioactive components. The present study aimed to determine the importance of dried and pulverized date palm seeds (DPS) as a natural elicitor for the synthesis of secondary metabolites in Lotus arabicus L.ResultsThe presence of various antioxidant compounds, simple sugars, amino acids, fatty acids and reasonable mineral contents was distinct in the phytochemical characterization of DPS. The major components detected in DPS analysis were the 5-(hydroxymethyl) furfural and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone. The induced callus of L. arabicus (seven weeks old) was supplemented with DPS at different concentrations (0, 2, 4, 8 and 10 g/l) in culture media. Treatment with 8 g/l DPS induced the highest antioxidant capacity, ascorbic acid content and secondary metabolites (total phenolics and flavonoids) in the produced callus. Stress biomarkers (hydrogen peroxide and malondialdehyde) were found in the control ranges except at 10 g/l DPS. The expression patterns of key genes involoved in secondary metabolism modulation, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and deoxyxylulose phosphate reductoisomerase (DXR), were triggered after DPS treatments. Moreover, the quantitative profiling of phenolic and flavonoid compounds showed that supplementation with DPS, especially at 8 g/l, led to pronounced increases in most of the measured compounds.ConclusionThe marked upregulation of eliciting-responsive genes and overproduction of secondary metabolites provide molecular-based evidence for intensifying the principal pathways of phenylpropanoid, flavonoid and terpenoid biosynthesis. Overall, the present in vitro study highlights the stimulating capacity of DPS utilization to improve the bioactive components of L. arabicus at the physiological and molecular levels, enhancing its potential as a medicinal herb.

Targeting Gut Dysbiosis and Microbiome Metabolites for the Development of Therapeutic Modalities for Neurological Disorders.

The gut microbiota, composed of numerous species of microbes, works in synergy with the various organ systems in the body to bolster our overall health and well-being. The most well-known function of the gut microbiome is to facilitate the metabolism and absorption of crucial nutrients, such as complex carbohydrates, while also generating vitamins. In addition, the gut microbiome plays a crucial role in regulating the functioning of the central nervous system (CNS). Host genetics, including specific genes and single nucleotide polymorphisms (SNPs), have been implicated in the pathophysiology of neurological disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), and autism spectrum disorder (ASD). The gut microbiome dysbiosis also plays a role in the pathogenesis of these neurodegenerative disorders, thus perturbing the gut-brain axis. Overproduction of certain metabolites synthesized by the gut microbiome, such as short-chain fatty acids (SCFAs) and p-cresyl sulfate, are known to interfere with microglial function and trigger misfolding of alpha-synuclein protein, which can build up inside neurons and cause damage. By determining the association of the gut microbiome and its metabolites with various diseases, such as neurological disorders, future research will pave the way for the development of effective preventive and treatment modalities.

Safety evaluation of indigenous probiotic Limosilactobacillus fermentum NCDC 400 using whole genome sequences and in vitro approaches

The safety of probiotic bacterial strains has been the subject of considerable discussion due to emerging health risks associated with their use in various health conditions. Herein, we integrate and execute both in silico and in vitro approaches to evaluate the safety of an indigenous probiotic strain, Limosilactobacillus fermentum NCDC 400. Our analysis of the strain's whole genome sequencing using established pipelines and universally accepted databases find no potential antibiotic-resistance or virulence genes of public health concern. Functional annotation of the genome indicates that NCDC 400 does not produce unusual metabolites or harmful enzymes. In vitro testing confirms that the strain does not exhibit any harmful phenotypes, activities of harmful enzymes, or overproduction of harmful metabolites. The strain is sensitive to the antagonistic properties of human serum as well as to all antibiotics enlisted by EFSA guidelines. Targeted PCR assays confirm the absence of genes conferring unusual antibiotic resistance and virulence factors. Cell culture assays reveal that NCDC 400 is non-cytotoxic to Caco-2 cells and does not affect their cellular integrity. Overall, these results demonstrate the safe and non-toxic nature of NCDC 400 and call for further in vivo trials to evaluate and establish its safety in healthy and diseased preclinical models.

Co-Expression of Transcriptional Regulators and Housekeeping Genes in Streptomyces spp.: A Strategy to Optimize Metabolite Production.

The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance. Streptomyces spp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites in Streptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains of Streptomyces spp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistant Streptomyces strains (mutations known to enhance secondary metabolism in Streptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains' metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulate Streptomyces metabolism and explore their wide secondary metabolites production potential.

Ozone exposure response on physiological and biochemical parameters vis-a-vis secondary metabolites in a traditional medicinal plant Sida cordifolia L.

Tropospheric ozone is a phytotoxic air pollutant that alters the production of bioactive compounds and key metabolites of plants. A field study was established on a medicinal plant, Sida cordifolia L. (Bala) under ambient (AO; 43.3 ppb) and elevated level of ozone (ambient+20 ppb; EO) at different developmental stages. The objective of study was to analyze the changes in physiology and biochemical parameters with secondary metabolites of S. cordifolia under EO treatment using open-top chambers. Physiological parameters such as photosynthesis, chlorophyll fluorescence, water use efficiency, stomatal conductance, and intercellular CO2 concentration demonstrated a negative response at all growth stages in EO-treated plants. Trichome density increased by 83.56%, whereas photosynthetic pigments decreased under EO exposure. Total phenolics and thiols increased at 90 days after treatment (DAT) under EO, while non-enzymatic antioxidants such as flavonoids and proline induced at all growth stages. Reactive oxygen species showed a differential response at different growth stages under EO as compared with AO treatment. Enzymatic antioxidants such as ascorbate peroxidase, catalase, peroxidase, glutathione reductase, and phenylalanine ammonia-lyase increased at all growth stages under EO. However, superoxide dismutase was induced only at 30 DAT. A significant increase was observed in the contents of lignin, tannin, saponin, and alkaloids in leaves and roots of the test plant under EO exposure. Phytosterol increased under EO at all the growth stages in roots, and at 90 DAT in leaves. Despite diverse physiological, ROS, and antioxidant responses, the results demonstrated that elevated ozone boosted the production of secondary metabolites, medicinally important compounds in Sida cordifolia. The overproduction of secondary metabolites under futuristic levels of ozone could be a useful resource for the exploitation of bioactive, medicinally important compounds from S. cordifolia.

Enzyme-constrained metabolic model and in silico metabolic engineering of Clostridium ljungdahlii for the development of sustainable production processes

Constraint-based genome-scale models (GEMs) of microorganisms provide a powerful tool for predicting and analyzing microbial phenotypes as well as for understanding how these are affected by genetic and environmental perturbations. Recently, MATLAB and Python-based tools have been developed to incorporate enzymatic constraints into GEMs. These constraints enhance phenotype predictions by accounting for the enzyme cost of catalyzed model´s reactions, thereby reducing the space of possible metabolic flux distributions. In this study, enzymatic constraints were added to an existing GEM of Clostridium ljungdahlii, a model acetogenic bacterium, by including its enzyme turnover numbers (kcats) and molecular masses, using the Python-based AutoPACMEN approach. When compared to the metabolic model iHN637, the enzyme cost-constrained model (ec_iHN637) obtained in our study showed an improved predictive ability of growth rate and product profile. The model ec_iHN637 was then employed to perform in silico metabolic engineering of C. ljungdahlii, by using the OptKnock computational framework to identify knockouts to enhance the production of desired fermentation products. The in silico metabolic engineering was geared towards increasing the production of fermentation products by C. ljungdahlii, with a focus on the utilization of synthesis gas and CO2. This resulted in different engineering strategies for overproduction of valuable metabolites under different feeding conditions, without redundant knockouts for different products. Importantly, the results of the in silico engineering results indicated that the mixotrophic growth of C. ljungdahlii is a promising approach to coupling improved cell growth and acetate and ethanol productivity with net CO2 fixation.

Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana

Nowadays, overproduction of secondary metabolites in remedial herbs through giving biotic/abiotic stresses is an interesting area of research. In the current study, the influences of various concentrations of silver nanoparticles (Ag NPs) were evaluated on several morphological and physio-biochemical traits, such as the steviol glycosides level in Stevia. The findings showed that the herbs incubated with 400-ppm Ag NPs own the highest dry and fresh weight of shoot, while those incubated with 80- up to 200- ppm Ag NPs own the highest steviol glycosides content. As a result, we successfully improve the content of stevioside glycoside up to 1.75-fold by applying the 80- up to 200-ppm Ag NPs in Stevia medicinal plant. Moreover, our findings revealed that low concentrations the Ag NPs lead to an increase of glutathione content and total antioxidant capacity, and a decrease of MDA, whereas treatments at higher concentrations induced adverse effects for the plant. As a result, the treatment with Ag NPs low concentrations had a favorable efficacy on physio-biochemical and morphological characteristics of Stevia. These achievements are very promising, because they revealed a considerable capability for the Ag NPs application in enhancing the secondary metabolites in Stevia remedial herb. The present study is the first case assessing the desirable influences of Ag NPs on the Stevia, in regard with shifting of biosynthetic pathway of steviol glycosides in a concentration-dependent manner.

Systems metabolic engineering of Streptomyces venezuelae for the enhanced production of pikromycin.

Pikromycin is an important precursor of drugs, for example, erythromycin. Hence, systems metabolic engineering for the enhanced pikromycin production can contribute to the development of pikromycin-related drugs. In this study, metabolic genes in Streptomyces venezuelae were systematically engineered for enhanced pikromycin production. For this, a genome-scale metabolic model of S. venezuelae was reconstructed and simulated, which led to the selection of 11 metabolic gene targets. These metabolic genes, including four overexpression targets and seven knockdown targets, were individually engineered first. Next, two overexpression targets and two knockdown targets were selected based on the 11 strains' production performances to engineer two to four of these genes together for the potential synergistic effects on the pikromycin production. As a result, the NM1 strain with AQF52_RS24510 (methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase) overexpression and AQF52_RS30320 (sulfite reductase) knockdown showed the best production performance among all the 22 strains constructed in this study. Fed-batch fermentation of the NM1 strain produced 295.25 mg/L of pikromycin, by far the best production titer using the native producer S. venezuelae, to the best of our knowledge. The systems metabolic engineering strategy demonstrated herein can also be applied to the overproduction of other secondary metabolites using S. venezuelae.

Improvement of Rimocidin Biosynthesis by Increasing Supply of Precursor Malonyl-CoA via Over-expression of Acetyl-CoA Carboxylase in Streptomyces rimosus M527.

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed accsr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the accsr gene and by the concentration of malonyl-CoA, the accsr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed accsr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by accsr gene over-expression led to the improvement in rimocidin production.

“Kynurenine switch” and obesity

Aim. To assess the concentrations of bacterial and eukaryotic metabolites mainly involved in indole, kynurenine, and serotonin pathways of tryptophan metabolism in a cohort of patients with obesity. Materials and methods. Using high-performance liquid chromatography with mass spectrometric detection, the concentrations of several serum metabolites, such as kynurenine, kynurenic acid, anthranilic acid, xanthurenic acid, quinolinic acid, 5-hydroxyindole-3-acetate, tryptamine, serotonin, indole-3-lactate, indole-3-acetate, indole-3- butyrate, indole-3-carboxaldehyde, indole-3-acrylate, and indole-3-propionate, were analyzed in a cohort of obese patients compared with healthy volunteers.Results. It was found that serum levels of tryptophan metabolites of microbial and eukaryotic origin were significantly increased in obese patients. Therefore, the concentration of kynurenine in the blood serum in obese patients was 2,413 ± 855 nmol / l, while in healthy volunteers of the same age group, the level of kynurenine in the blood serum was 2,122 ± 863 nmol / l. In obese patients, two acids formed due to kynurenine metabolism; the concentrations of kynurenic and quinolinic acids were increased in the blood serum. The concentration of kynurenic acid in the blood serum in obese patients was 21.1 ± 9.26 nmol / l, and in healthy patients, it was 16.8 ± 8.37 nmol / l. At the same time, the level of quinolinic acid in the blood serum in obese patients was 73.1 ± 54.4 nmol / l and in healthy volunteers – 56.8 ± 34.1 nmol / l. Normally, the level of quinolinic acid is 3.4 times higher than the concentration of kynurenic acid, and in case of obesity, there is a comparable increase in these acids in the blood serum.From indole derivatives, mainly of microbial origin, the concentrations of indole-3-lactate, indole-3-butyrate, and indole-3-acetate were significantly increased in the blood serum of obese patients. In obese patients, the serum concentration of 5-hydroxyindole-3-acetate was elevated to 74.6 ± 75.8 nmol / l (in healthy volunteers – 59.4 ± 36.6 nmol / l); indole-3-lactate – to 523 ± 251 nmol / l (in healthy volunteers – 433 ± 208 nmol / l); indole-3-acetate – to 1,633 ± 1,166 nmol / l (in healthy volunteers – 1,186 ± 826 nmol / l); and indole-3-butyrate – to 4.61 ± 3.31 nmol / l (in healthy volunteers – 3.85 ± 2.51 nmol / l).Conclusion. In case of obesity, the utilization of tryptophan was intensified by both the microbiota population and the macroorganism. It was found that obese patients had higher concentrations of kynurenine, quinolinic and kynurenic acids, indole-3-acetate, indole-3-lactate, indole-3-butyrate, and 5-hydroxyindole-3-acetate. Apparently, against the background of increased production of proinflammatory cytokines by adipocytes in obese patients, the “kynurenine switch” was activated which contributed to subsequent overproduction of tryptophan metabolites involved in the immune function of the macroorganism.

POTENTIAL THERAPEUTIC OPTIONS TARGETING THE GUT DYSBIOSIS IN CHRONIC KIDNEY DISEASE.

The gut microbiota plays an important physiological role in controlling not only the function of the gastrointestinal tract, but also in maintaining systemic homeostasis. Quantitative and /or qualitative disturbances of the gut microbiota (dysbiosis) are an important element in the complex pathogenesis of many diseases, including chronic kidney disease (CKD). In the disease, the mutual interactions between disturbed gut microbiota and the progression of CKD (pathophysiological "kidney-gut axis") have been demonstrated. The kidney failure causes water and nitrogen waste retention which leads to disturbances of motility, secretion and absorption in the gastrointestinal tract. These abnormalities contribute to the development of gut dysbiosis, accompanied by overproduction of toxic bacterial metabolites, with their translocation to the peripheral blood and development of endotoxemia. As a consequence, chronic kidney "low-grade" inflammation and oxidative stress develop, with further deterioration of kidney function in the mechanism of the "vicious cycle" of the kidney-gut axis. Considering the key role of gut dysbiosis and the kidney-gut axis, the attempts to restore the gut eubiosis seem to have an important role in the treatment of CKD and may be even regarded as a form of causal therapeutic intervention. The paper briefly discusses the basics of the pathophysiological kidney-gut axis in CKD and potential methods of modulating the abnormal gut microbiota in this disease, including the use of probiotic or prebiotic preparations, agents that absorb bacterial-derived toxins in the intestinal lumen, fecal microbiota transplantation and drugs used so far for other indications (acarbose, meclofenamate, lubiprostone).

Optimizing Thiopurine Therapy with a Xanthine Oxidase Inhibitor in Patients with Systemic Autoimmune Diseases: A Single-Centre Experience.

Thiopurines are a mainstay of therapy for autoimmune diseases. However, up to 20% to 30% of patients experience overproduction of the methylated metabolites, known as 6-MMP, to the detriment of the active metabolite, 6-thioguanine nucleotide (6-TGN). These patients, commonly referred to as "shunters", are predisposed to thiopurine resistance and hepatotoxicity. In patients with inflammatory bowel diseases, the combination of thiopurine with a xanthine oxidase inhibitor (XOI) is used to reverse this skewed metabolism and to prevent treatment failure or hepatotoxicity. Data on the use of this strategy for patients with other diseases are limited. To investigate and describe the use of thiopurine-XOI combination therapy in shunters with systemic autoimmune diseases. Shunters treated in the study hospital between January 1, 2005, and December 31, 2015, were identified using the hospital's laboratory database, and clinical data were collected retrospectively. For each patient with optimization of thiopurine therapy, clinical and laboratory data were assessed over a 6-month period. Thirty-four patients were identified as shunters; for 14 of these patients, thiopurine therapy was optimized with an XOI. In these 14 patients, the median dose of azathioprine was reduced from 1.95 to 0.78 mg/kg with combination therapy. In addition, median 6-TGN level increased from 135 to 385 pmol/8 × 108 erythrocytes (p = 0.001); furthermore, 6-TGN levels rose to above 235 pmol/8 ×108 erythrocytes for 11 of the 14 patients. Conversely, the median 6-MMP level decreased from 6267 to 271 pmol/8 × 108 erythrocytes (p = 0.001). Except for a 12% increase in mean corpuscular volume, no clinically significant changes in blood count were recorded. Notable infections were reported in 3 patients, and 1 patient had to discontinue treatment because of cytopenia. After 6 months, median prednisone daily dose was reduced by 74%, from 16.7 mg to 4.4 mg (p = 0.005), and 4 patients had been weaned off corticosteroids. Of the 14 patients, 11 (79%) were in full remission, and 2 (14%) were in partial remission. Optimizing thiopurine therapy with an XOI may be a safe and effective strategy for patients with systemic autoimmune diseases.

Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

BackgroundTailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering.ResultsIn this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L).ConclusionsAll these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

The effect of abiotic and biotic stresses on the production of bioactive compounds in tea (Camellia sinensis (L.) O. Kuntze)

Tea (Camellia sinensis (L.) O. Kuntze) is the most famous, flavored, functional, and therapeutic non-alcoholic beverage, with two-thirds of the world's population drinking it. Tea leaves are demonstrated to contain many bioactive compounds, including polyphenols, amino acids, volatile compounds, and alkaloids, all of which have a good potential of biomedical uses. In plants under stress, including diverse elicitors or signal molecules, accumulation of such metabolites occurs frequently. In adapting plants to the ecosystem and alleviating stress conditions, phytochemicals play a significant role. Ecological factors may be categorized into biotic and abiotic factors which may be artificially induced to activate plant defense responses due to the generation of bioactive compounds. Different biotic and abiotic factors are directly linked to the productivity of tea. Abiotic and biotic factors such as temperature, cold, nutrition, shading, humidity, light intensity, water supply, mechanical, nutrition influence the growth of the plant and the production of the bioactive compounds in tea. Biotic and abiotic factors are versatile tools for the overproduction of valuable metabolites in tea. In this review, an update will be provided on current research focusing on the response of tea to an integration of various stresses. In specific, we will discuss how various stress responses are integrated and how they affect plant growth and physiological characteristics.

Metabolic reprogramming and mitochondrial dysfunction in HepG2 cells as potential drivers of AIP pathophysiology

Introduction and Aims: Acute intermittent porphyria (AIP) results from the porphobilinogen deaminase (PBGD) haploinsufficiency and affects hepatic heme biosynthesis. Life-threatening acute neurologic attacks occur in response to stressors (fasting) and were mainly attributed to overproduction of heme-derived neurotoxic metabolites. However, emerging data in both preclinical models and humans highlighted that alterations in liver metabolism, potentially involving insulin resistance and mitochondrial dysfunction, may drive AIP pathophysiology. Aim of this study will be to investigate the metabolic aberrancies in response to PBGD downregulation in vitro, with particular focus on mitochondria, in which many of metabolic processes occur.

Bisphenol A impaired cell adhesion by altering the expression of adhesion and cytoskeleton proteins on human podocytes

Bisphenol A (BPA), a chemical -xenoestrogen- used in food containers is present in the urine of almost the entire population. Recently, several extensive population studies have proven a significant association between urinary excretion of BPA and albuminuria. The alteration of glomerular podocytes or "podocytopathy" is a common event in chronic albuminuric conditions. Since many podocytes recovered from patients' urine are viable, we hypothesized that BPA could impair podocyte adhesion capabilities. Using an in vitro adhesion assay, we observed that BPA impaired podocyte adhesion, an effect that was abrogated by Tamoxifen (an estrogen receptor blocker). Genomic and proteomic analyses revealed that BPA affected the expression of several podocyte cytoskeleton and adhesion proteins. Western blot and immunocytochemistry confirmed the alteration in the protein expression of tubulin, vimentin, podocin, cofilin-1, vinculin, E-cadherin, nephrin, VCAM-1, tenascin-C, and β-catenin. Moreover, we also found that BPA, while decreased podocyte nitric oxide production, it lead to overproduction of ion superoxide. In conclusion, our data show that BPA induced a novel type of podocytopathy characterizes by an impairment of podocyte adhesion, by altering the expression of adhesion and cytoskeleton proteins. Moreover, BPA diminished production of podocyte nitric oxide and induced the overproduction of oxygen-free metabolites. These data provide a mechanism by which BPA could participate in the pathogenesis and progression of renal diseases.

CRISPR interference-guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli

BackgroundOne major mission of microbial breeding is high-level production of desired metabolites. Overproduction of intermediate metabolites in core pathways is challenging as it may impair cell growth and viability.ResultsHere we report that aconitic acid, an intermediate metabolite in tricarboxylic acid (TCA) cycle, can be overproduced by an engineered CRISPR interference (CRISPRi) system in Escherichia coli. This CRISPRi system was designed to simultaneously target pyruvate kinase (PK) and isocitrate dehydrogenase (IDH), two enzymes in glycolytic pathway and TCA cycle, respectively. Reverse transcription and quantitative PCR and enzyme activity assays showed that this engineered CRISPRi system significantly repressed the genes encoding IDH and PK, resulting in simultaneous reduction in the activities of IDH and PK. In shake-flask and fed-batch cultivation, this CRISPRi strain produced 60-fold (362.80 ± 22.05 mg/L) and 15-fold (623.80 ± 20.05 mg/L) of aconitic acid relative to the control strain, respectively. In addition, this two-target CRISPRi strain maintained low levels of acetate and lactate, two problematic byproducts.ConclusionsThis work demonstrates that CRISPRi system can improve aconitic acid production by coordinating glycolysis and TCA cycle. This study provides insights for high-level production of the intermediate metabolites in central pathways.