Metabolic Engineering of Escherichia coli for De Novo Production of 1,2-Butanediol.

1,2-Butanediol (1,2-BDO) is an important platform chemical widely utilized in the synthesis of polyester polyols, plasticizers, cosmetics, and pharmaceuticals. However, no natural metabolic pathway for its biosynthesis has been identified, and biological production of 1,2-BDO from renewable bioresources has not been reported so far. In this study, we designed and experimentally verified a feasible non-natural synthesis pathway for the de novo production of 1,2-BDO from renewable carbohydrates for the first time. This pathway extends the l-threonine synthesis pathway by introducing two artificial metabolic modules to sequentially convert l-threonine into 2-hydroxybutyric acid and 1,2-BDO. Following key enzyme screening and enhancement of l-threonine synthesis module in the chassis microorganism, the best engineered Escherichia coli strain was able to produce 0.15 g/L 1,2-BDO using glucose as the sole carbon source. This work lays the foundation for the bioproduction of 1,2-BDO from renewable resources.

Preserved vegetable consumption and its association with mortality among 440,415 people in the China Kadoorie Biobank.

BACKGROUND: Fresh vegetable consumption has been associated with lower incidence of cardiovascular disease (CVD). However, whether preserved vegetable consumption is linked with CVD and mortality remains unclear. This study aimed to assess the associations of preserved vegetable consumption with all-cause and cause-specific mortality. METHODS: A total of 440,415 participants free of major chronic diseases, aged 30-79 years, were enrolled from 10 diverse regions in China between 2004 and 2008 and were followed up for an average of 10 years. Preserved vegetable consumption was assessed using a validated food frequency questionnaire. Cause-specific hazard models with the consideration of competing risk from various deaths were performed to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) of mortality. RESULTS: During 4,415,784 person-years of follow-up, we documented 28,625 deaths. After adjustment for major risk factors, preserved vegetable consumption was marginally associated with higher CVD mortality (P = 0.041 for trend and P = 0.025 for non-linearity) but not associated with cancer mortality and total mortality. For specific causes of death, consuming preserved vegetables was associated with higher hemorrhagic stroke mortality. The multivariable-adjusted HRs (95% CIs) of hemorrhagic stroke mortality compared with non-consumers were 1.32 (1.17-1.50) for 1-3 days/week and 1.15 (1.00-1.31) for regular consumers (≥4 days/week) (P = 0.006 for trend and P < 0.001 for non-linearity). In addition, regular preserved vegetable consumption was associated with increased risk of digestive tract cancer mortality [HR (95% CI): 1.13 (1.00-1.28); P = 0.053 for trend] and esophageal cancer mortality [HR (95% CI): 1.45 (1.17-1.81); P = 0.002 for trend]. CONCLUSIONS: Frequent consumption of preserved vegetables was associated with higher risk of mortality from hemorrhagic stroke and esophageal cancer in China. Our findings suggest limiting preserved vegetable consumption might be protective for premature death from hemorrhagic stroke and digestive tract cancer.

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway.

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.

Associations of Hemoglobin Adducts of Acrylamide and Glycidamide with Prevalent Metabolic Syndrome in a Nationwide Population-Based Study.

Environmental and dietary exposures to acrylamide (AA) have been linked with various metabolic-related outcomes, but the results are mixed. However, the association between long-term exposure to AA and the prevalence of metabolic syndrome (MetS) remains unknown. In this study, we aimed to assess the relationship between hemoglobin adducts of AA, biomarkers of internal exposure to AA, and MetS prevalence among a U.S. nationwide population. MetS patients were defined by meeting three or more of the following five characteristics: elevated blood pressure, high fasting glucose, abdominal obesity, hypertriglyceridemia, and lower high-density lipoprotein cholesterol (HDL-C). Multivariate-adjusted logistic regression models and restricted cubic spline models were used to analyze the associations between AA hemoglobin biomarkers and MetS prevalence. A total of 1552 MetS cases were documented. After adjustment for the potential confounders, the odds ratios (95% confidence intervals) of MetS prevalence in the highest quartile of AA hemoglobin biomarkers were 0.60 (0.40-0.89), 1.26 (0.84-1.89), 0.93 (0.71-1.21), and 1.61 (1.18-2.20) for HbAA, HbGA, the sum of HbAA and HbGA (HbAA + HbGA), and the ratio of HbGA to HbAA (HbGA/HbAA), compared with the lowest quartile, respectively. HbAA was significantly and inversely associated with blood pressure, fasting glucose, abdominal obesity, hypertriglyceridemia, and low HDL-C, while the HbGA/HbAA ratio was also positively associated with abdominal obesity, hypertriglyceridemia, and low HDL-C. The restricted cubic spline models revealed a positive relationship between the HbGA/HbAA ratio and the prevalence of MetS, while the HbAA level was inversely associated with MetS prevalence. Our current findings provided epidemiological evidence that HbAA and the HbGA/HbAA ratio were significantly associated with MetS prevalence among general U.S. adults. Further studies should be conducted to examine the association between internal exposure to AA and MetS prevalence.

Eicosapentaenoic and docosahexaenoic acids attenuate hyperglycemia through the microbiome-gut-organs axis in db/db mice.

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been suggested to prevent the development of metabolic disorders. However, their individual role in treating hyperglycemia and the mechanism of action regarding gut microbiome and metabolome in the context of diabetes remain unclear. RESULTS: Supplementation of DHA and EPA attenuated hyperglycemia and insulin resistance without changing body weight in db/db mice while the ameliorative effect appeared to be more pronounced for EPA. DHA/EPA supplementation reduced the abundance of the lipopolysaccharide-containing Enterobacteriaceae whereas elevated the family Coriobacteriaceae negatively correlated with glutamate level, genera Barnesiella and Clostridium XlVa associated with bile acids production, beneficial Bifidobacterium and Lactobacillus, and SCFA-producing species. The gut microbiome alterations co-occurred with the shifts in the metabolome, including glutamate, bile acids, propionic/butyric acid, and lipopolysaccharide, which subsequently relieved ß cell apoptosis, suppressed hepatic gluconeogenesis, and promoted GLP-1 secretion, white adipose beiging, and insulin signaling. All these changes appeared to be more evident for EPA. Furthermore, transplantation with DHA/EPA-mediated gut microbiota mimicked the ameliorative effect of DHA/EPA on glucose homeostasis in db/db mice, together with similar changes in gut metabolites. In vitro, DHA/EPA treatment directly inhibited the growth of Escherichia coli (Family Enterobacteriaceae) while promoted Coriobacterium glomerans (Family Coriobacteriaceae), demonstrating a causal effect of DHA/EPA on featured gut microbiota. CONCLUSIONS: DHA and EPA dramatically attenuated hyperglycemia and insulin resistance in db/db mice, which was mediated by alterations in gut microbiome and metabolites linking gut to adipose, liver and pancreas. These findings shed light into the gut-organs axis as a promising target for restoring glucose homeostasis and also suggest a better therapeutic effect of EPA for treating diabetes. Video abstract.

Association of exposures to perchlorate, nitrate, and thiocyanate with allergic symptoms: A population-based nationwide cohort study.

Allergic diseases have been one of the leading causes of chronic disorders in the United States. Animal studies have suggested that exposures to perchlorate, nitrate, and thiocyanate could induce allergic inflammation. However, the associations have not been examined among general populations. Here, we investigated data of 7030 participants aged ≥6 years from the National Health and Nutritional Examination Survey (NHANES) 2005-2006. Urinary levels of perchlorate, nitrate, and thiocyanate were measured by ion chromatography combined with electrospray tandem mass spectrometry. Information on allergic symptoms (hay fever, allergy, rash, sneeze, wheeze, eczema, and current asthma) was collected by questionnaire. Allergic sensitization was defined by a concentration ≥150 kU/L for total immunoglobulin E (IgE) levels. The associations were estimated using multivariate-adjusted logistic regression models. A positive association was observed for urinary nitrate and eczema (p < 0.001 for the trend). Compared with quartile 1 (lowest quartile), the odds ratios of eczema with 95% confidence intervals [ORs (95% CIs)] from quartiles 2 to 4 were 1.72 (95% CI, 1.41, 2.09), 1.94 (1.53, 2.47) and 2.10 (1.49, 2.97) for urinary nitrate. In addition, urinary thiocyanate was positively related to sneeze (ORQ4 vs. Q1: 1.25, 95% CI: 1.01, 1.55; p = 0.015 for the trend). However, urinary perchlorate was not correlated with any allergic-related outcome. Additionally, the associations were different among subgroups in a four-level polytomous model. Thus, our results suggested that exposures to nitrate and thiocyanate may be associated with allergic symptoms. Further investigations are warranted to concentrate on the practical strategies to monitor exposure levels and the latent mechanisms of the relationship between exposure and allergy.

Egg and cholesterol consumption and mortality from cardiovascular and different causes in the United States: A population-based cohort study.

BACKGROUND: Whether consumption of egg and cholesterol is detrimental to cardiovascular health and longevity is highly debated. Data from large-scale cohort studies are scarce. This study aimed to examine the associations of egg and cholesterol intakes with mortality from all causes, cardiovascular disease (CVD), and other causes in a US population. METHODS AND FINDINGS: Overall, 521,120 participants (aged 50-71 years, mean age = 62.2 years, 41.2% women, and 91.8% non-Hispanic white) were recruited from 6 states and 2 additional cities in the US between 1995 and 1996 and prospectively followed up until the end of 2011. Intakes of whole eggs, egg whites/substitutes, and cholesterol were assessed by a validated food frequency questionnaire. Cause-specific hazard models considering competing risks were used, with the lowest quintile of energy-adjusted intake (per 2,000 kcal per day) as the reference. There were 129,328 deaths including 38,747 deaths from CVD during a median follow-up of 16 years. Whole egg and cholesterol intakes were both positively associated with all-cause, CVD, and cancer mortality. In multivariable-adjusted models, the hazard ratios (95% confidence intervals) associated with each intake of an additional half of a whole egg per day were 1.07 (1.06-1.08) for all-cause mortality, 1.07 (1.06-1.09) for CVD mortality, and 1.07 (1.06-1.09) for cancer mortality. Each intake of an additional 300 mg of dietary cholesterol per day was associated with 19%, 16%, and 24% higher all-cause, CVD, and cancer mortality, respectively. Mediation models estimated that cholesterol intake contributed to 63.2% (95% CI 49.6%-75.0%), 62.3% (95% CI 39.5%-80.7%), and 49.6% (95% CI 31.9%-67.4%) of all-cause, CVD, and cancer mortality associated with whole egg consumption, respectively. Egg white/substitute consumers had lower all-cause mortality and mortality from stroke, cancer, respiratory disease, and Alzheimer disease compared with non-consumers. Hypothetically, replacing half a whole egg with equivalent amounts of egg whites/substitutes, poultry, fish, dairy products, or nuts/legumes was related to lower all-cause, CVD, cancer, and respiratory disease mortality. Study limitations include its observational nature, reliance on participant self-report, and residual confounding despite extensive adjustment for acknowledged dietary and lifestyle risk factors. CONCLUSIONS: In this study, intakes of eggs and cholesterol were associated with higher all-cause, CVD, and cancer mortality. The increased mortality associated with egg consumption was largely influenced by cholesterol intake. Our findings suggest limiting cholesterol intake and replacing whole eggs with egg whites/substitutes or other alternative protein sources for facilitating cardiovascular health and long-term survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT00340015.

Exposure to acrylamide induces skeletal developmental toxicity in zebrafish and rat embryos.

Acrylamide is a well-known carcinogen and neurotoxic substance that has been discovered in frying or baking carbohydrate-rich foods and is widely found in soils and groundwater. The purpose of this study was to investigate the adverse effects of exposure to acrylamide on skeletal development. After treatment with acrylamide in zebrafish embryos, the survival and hatching rates decreased, and the body length shortened, with cartilage malformation and a decrease in skeletal area. Exposure to acrylamide in maternal rats during the lactation period disturbed bone mineral density, serum levels of parathyroid hormone, and the expression of skeletal development-related genes in neonates. Exposure to acrylamide in pregnant rats during the pregnancy period decreased the trabecular density and inhibited cartilage formation by delaying the differentiation of osteoblasts and promoting the maturation of osteoclasts in rat embryos. Furthermore, acrylamide intervention downregulated the expression of chondrocyte and osteoblast differentiation-related genes (sox9a, bmp2, col2a1, and runx2), and upregulated the expression of osteoclast marker genes (rankl and mcsf) in zebrafish and rat embryos at different gestational stages. Our results indicated that exposure to acrylamide dysregulated signature gene and protein expression profiles of skeletal development by suppressing the differentiation and maturation of osteoblasts and cartilage matrix and promoting the formation of osteoclasts, and ultimately induced skeletal abnormality in morphology, which brings increasing attention to the intergenerational toxicity of acrylamide via mother-to-child transmission.

Environmental exposure to perchlorate, nitrate, and thiocyanate in relation to obesity: A population-based study.

BACKGROUND: Perchlorate, nitrate, and thiocyanate are well-known thyroid disrupters and may contribute to changes in body weight. However, the associations between environmental exposure to these chemicals and obesity-related outcomes remain unclear. OBJECTIVES: We aim to examine the urinary levels of perchlorate, nitrate, and thiocyanate and their associations with obesity and abdominal obesity in the U.S. METHODS: Here, we investigated the data of 16,265 adults aged 20-85 years from the National Health and Nutritional Examination Survey (NHANES) in 2001-2014. Urinary levels of perchlorate, nitrate, and thiocyanate were measured by ion chromatography combined with electrospray tandem mass spectrometry. Obesity and abdominal obesity were defined by the body mass index and waist circumference, respectively. Logistic regression models were used to estimate the associations. RESULTS: Overall, 5794 (35.6%) cases of obesity and 9090 cases (55.9%) of abdominal obesity were observed among the participants. In multivariable-adjusted logistic regression models, urinary nitrate was inversely associated with obesity (p = 0.0022 for trend), while urinary thiocyanate was positively related to obesity (p < 0.001 for trend). Compared with the lowest quartile, the odds ratios with 95% confidence intervals (CIs) across increasing quartiles were 0.95 (95% CI, 0.83-1.08), 0.88 (0.75-1.03), and 0.74 (0.60-0.90) for urinary nitrate and 1.31 (1.16-1.48), 1.53 (1.36-1.73), and 1.73 (1.47-2.03) for urinary thiocyanate. Urinary perchlorate was not correlated with obesity. Similar associations were also found between exposure to these chemicals and abdominal obesity. CONCLUSIONS: A higher exposure to urinary nitrate was associated with a lower risk of obesity, while a positive association was observed for urinary thiocyanate. These findings emphasize the need to longitudinally evaluate environmental exposure to perchlorate, nitrate, and thiocyanate with respect to their effect on obesity in humans.

Exposure to acrylamide disrupts cardiomyocyte interactions during ventricular morphogenesis in zebrafish embryos.

Acrylamide (AA), a ubiquitous chemical that is present in surrounding environment and baked or fried carbohydrate-rich food, has recently been linked to cardiac developmental toxicity. However, the toxicological role of AA exposure in the cardiac development remains largely unknown. Here we showed the cardiotoxicity of AA and its role in cardiomyocyte interactions in zebrafish embryos during ventricular morphogenesis. Using the embryo model of transgenic zebrafish Tg(Tp1:d2GFP;myl7:mCherry), we found AA interfered the dynamics of Notch signaling in the endocardium during early cardiogenesis. Prolonged exposure to AA thickened the chamber wall and prevented the trabeculae from extending into the lumen of ventricular chamber. As a result, AA reduced the ventricular shortening fraction and spatial dimension via excessively activating the Notch signal in myocardium during cardiac maturation. Moreover, exposure to AA inhibited the re-distribution of N­cadherin and failed to coordinate cardiomyocyte interactions between the myocardium layers due to the lack of delaminated cardiomyocytes. Therefore, AA-treated embryos exhibited subcellular pathological states including disarrayed myofibrils and abnormal morphology of mitochondria despite normal proliferation of cardiomyocytes. In addition, we found overexpression of some cardiac-specific transcription factors, such as hand2 and nkx2.5, in hearts of AA-treated embryos compared with those in control group. Our study provided the evidence that the period of ventricular chamber morphogenesis might be a vulnerable window in zebrafish, and revealed new insights into how AA might exert cardiac developmental toxicity.

Cryptococcus laurentii controls gray mold of cherry tomato fruit via modulation of ethylene-associated immune responses.

This research aimed to investigate the roles of phytohormone ethylene in cherry tomato fruit immune response against gray mold caused by Botrytis cinerea. Pretreatment with antagonistic yeast Cryptococcus laurentii resulted in a significantly decreased disease incidence of B. cinerea infection, and accompanied by a burst of ethylene production in the whole fruit. Blocking the ethylene perception by adding 1-MCP (5 µL L-1 or greater) remarkably weaken the protection ability of fruit itself and suppressed the C. laurentii-stimulated host immune response. 5 µL L-1 1-MCP prefumigation decreased the expression of ethylene biosynthesis and perception related genes SlACO1, SlACS2, SlERF1, SlPti5 and SlMPK3, and ethylene production in C. laurentii treated fruit. Consequently, the expressions of SlCHI9, SlGlub, SlPAL3, SlPR1 and SlPR5 up-regulated by the yeast were all impaired to different degrees by the 1-MCP prefumigation. These findings demonstrate that ethylene contributes to fruit immunity and C. laurentii-mediated immune responses of cherry tomato.