Biosynthesis of L-5-methyltetrahydrofolate by genetically engineered Escherichia coli.

L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 µg g-1 of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.

A Cytoplasmic NAD(P)H-Dependent Polysulfide Reductase with Thiosulfate Reductase Activity from the Hyperthermophilic Bacterium Thermotoga maritima.

Thermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H2 by fermenting carbohydrates. High concentration of H2 inhibits the growth of T. maritima, and S0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx, yeeE, and rnf genes when the strain grew in S0- or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments. IMPORTANCE The reduction of S0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S0, polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S0- or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

A Heterodimeric Reduced-Ferredoxin-Dependent Methylenetetrahydrofolate Reductase from Syngas-Fermenting Clostridium ljungdahlii.

The strict anaerobe Clostridium ljungdahlii can ferment CO or H2/CO2 via the Wood-Ljungdahl pathway to acetate, ethanol, and 2,3-butanediol. This ability has attracted considerable interest, since it can be used for syngas fermentation to produce biofuels and biochemicals. However, the key enzyme methylenetetrahydrofolate reductase (MTHFR) in the Wood-Ljungdahl pathway of the strain has not been characterized, and its physiological electron donor is unclear. In this study, we purified the enzyme 46-fold with a benzyl viologen reduction activity of 41.2 U/mg from C. ljungdahlii cells grown on CO. It is composed of two subunits, MetF (31.5 kDa) and MetV (23.5 kDa), and has an apparent molecular mass of 62.2 kDa. The brownish yellow protein contains 0.73 flavin mononucleotide (FMN) and 7.4 Fe, in agreement with the prediction that MetF binds one flavin and MetV binds two [4Fe4S] clusters. It cannot use NAD(P)H as its electron donor or catalyze an electron-bifurcating reaction in combination with ferredoxin as an electron acceptor. The reduced recombinant ferredoxin, flavodoxin, and thioredoxin of C. ljungdahlii can serve as electron donors with specific activities of 91.2, 22.1, and 7.4 U/mg, respectively. The apparent Km values for reduced ferredoxin and flavodoxin were around 1.46 µM and 0.73 µM, respectively. Subunit composition and phylogenetic analysis showed that the enzyme from C. ljungdahlii belongs to MetFV-type MTHFR, which is a heterodimer, and uses reduced ferredoxin as its electron donor. Based on these results, we discuss the energy metabolism of C. ljungdahlii when it grows on CO or H2 plus CO2. IMPORTANCE Syngas, a mixture of CO, CO2, and H2, is the main component of steel mill waste gas and also can be generated by the gasification of biomass and urban domestic waste. Its fermentation to biofuels and biocommodities has attracted attention due to the economic and environmental benefits of this process. Clostridium ljungdahlii is one of the superior acetogens used in the technology. However, the biochemical mechanism of its gas fermentation via the Wood-Ljungdahl pathway is not completely clear. In this study, the key enzyme, methylenetetrahydrofolate reductase (MTHFR), was characterized and found to be a non-electron-bifurcating heterodimer with reduced ferredoxin as its electron donor, representing another example of MetFV-type MTHFR. The findings will form the basis for a deeper understanding of the energy metabolism of syngas fermentation by C. ljungdahlii, which is valuable for developing metabolic engineering strains and efficient syngas fermentation technologies.

Behavioural Problems Amongst Pre-School Children in Chongqing, China: Current Situation and Influencing Factors.

INTRODUCTION: Behavioural problems in pre-school children are closely related to their mental health. Such problems include attention deficit, personality disorder, overdependency, poor adaptability and conduct problems. METHODS: From December 2018 to January 2019, we conducted a cross-sectional survey of parents of pre-schoolers. The survey covered sixteen kindergartens in six districts of Chongqing, China. A total of 2200 participants participated in the survey, and 1895 questionnaires were returned. After screening, 1496 valid questionnaires were compiled in the data analysis (n=1496). RESULTS: Analysis of the maximum likelihood estimation revealed that age, preterm birth, household income, amount of daily interaction with parents, and scolding frequency affected behavioural problems in the pre-schoolers. Behavioural problems tend to be attenuated as children grow. Preterm children had a higher probability of developing behavioural problems than did non-preterm children. Children from families with monthly household incomes between $1130-$1695 USD and $1696-$2260 USD were more prone to developing behavioural problems. Children whose parents spent less time interacting with them (39.26% of parents interacted with children less than 1 hour per day) and children who were scolded more often had greater behavioural problems (13.44% of parents often scolded their children). DISCUSSION: This study was conducted to evaluate the influence of parenting methods on pre-school children and the education provided by parents on their pre-school children's behavioural problems to provide insights for Chinese parents and mental health professionals to improve treatment of behavioural problems.

Parental Influence in Forming Preschool Children's Eating Behaviors-A Cross-Sectional Survey in Chongqing, China.

Children's eating habits are closely related to their health problems and the outlook for children's nutritional statuses appears poor. A cross-sectional survey was conducted among parents of preschool children from December 2018 to January 2019. Sixteen representative kindergartens in 6 districts of Chongqing, China, were included in the study. We took 2200 samples and collected information by questionnaire and after screening, 1781 questionnaires were valid and finally included in the data analysis (n = 1781). Ordinal logistic regression analysis found that age, fathers' education level, forced diet and perception of children's body shape were factors associated with children's eating behaviors (ordered logistic regression/three-level eating behavior; odds ratios p < 0.05). 80.24% of preschool children may have unhealthy eating behavior in this survey and 80.35% of parents had forced their children to eat. Eating behaviors of preschool children in Chongqing are closely related to family factors. This study provides important insight for parents and health care workers in China to improve preschool-aged children's nutritional behaviors. Intervention programs should focus on parents with low income, low education levels, coercive dietary behaviors and deviated body shape perceptions to reduce children's eating behavioral problems.

Distribution, Evolution, Catalytic Mechanism, and Physiological Functions of the Flavin-Based Electron-Bifurcating NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase.

NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) is an electron-bifurcating enzyme first discovered in the strict anaerobes Clostridium kluyveri and Moorella thermoacetica. In vivo, Nfn catalyzes the endergonic reduction of NADP+ with NADH coupled to the exergonic reduction of NADP+ with reduced ferredoxin. Most Nfn homologs consist of two subunits, although in certain species Nfn homologs are fused. In contrast to other electron-bifurcating enzymes, Nfn possess a simpler structure. Therefore, Nfn becomes a perfect model to determine the mechanism of flavin-based electron bifurcation, which is a novel energy coupling mode distributed among anaerobic bacteria and archaea. The crystal structures of Nfn from Thermotoga maritima and Pyrococcus furiosus are known, and studies have shown that the FAD molecule of the NfnB (b-FAD) is the site of electron bifurcation, and other cofactors, including a [2Fe2S] cluster, two [4Fe4S] clusters, and the FAD molecule on the NfnA subunit, contribute to electron transfer. Further, the short-lived anionic flavin semiquinone (ASQ) state of b-FAD is essential for electron bifurcation. Nfn homologs are widely distributed among microbes, including bacteria, archaea, and probably eukaryotes, most of which are anaerobes despite that certain species are facultative microbes and even aerobes. Moreover, potential evidence shows that lateral gene transfer may occur in the evolution of this enzyme. Nfn homologs present four different structural patterns, including the well-characterized NfnAB and three different kinds of fused Nfn homologs whose detailed properties have not been characterized. These findings indicate that gene fusion/fission and gene rearrangement may contribute to the evolution of this enzyme. Under physiological conditions, Nfn catalyzes the reduction of NADP+ with NADH and reduced ferredoxin, which is then used in certain NADPH-dependent reactions. Deletion of nfn in several microbes causes low growth and redox unbalance and may influence the distribution of fermentation products. It's also noteworthy that different Nfn homologs perform different functions according to its circumstance. Physiological functions of Nfn indicate that it can be a potential tool in the metabolic engineering of industrial microorganisms, which can regulate the redox potential in vivo.

Green route to synthesis of valuable chemical 6-hydroxynicotine from nicotine in tobacco wastes using genetically engineered Agrobacterium tumefaciens S33.

BACKGROUND: Tobacco is widely planted as an important nonfood economic crop throughout the world, and large amounts of tobacco wastes are generated during the tobacco manufacturing process. Tobacco and its wastes contain high nicotine content. This issue has become a major concern for health and environments due to its toxicity and complex physiological effects. The microbial transformation of nicotine into valuable functionalized pyridine compounds is a promising way to utilize tobacco and its wastes as a potential biomass resource. Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways, in which several intermediates, such as 6-hydroxynicotine, can be used as renewable precursors to synthesize drugs and insecticides. This provides an opportunity to produce valuable chemical 6-hydroxynicotine from nicotine via biocatalysis using strain S33. RESULTS: To accumulate the intermediate 6-hydroxynicotine, we firstly identified the key enzyme decomposing 6-hydroxynicotine, named 6-hydroxynicotine oxidase, and then disrupted its encoding gene in A. tumefaciens S33. With the whole cells of the mutant as a biocatalyst, we tested the possibility to produce 6-hydroxynicotine from the nicotine of tobacco and its wastes and optimized the reaction conditions. At 30 °C and pH 7.0, nicotine could be efficiently transformed into 6-hydroxynicotine by the whole cells cultivated with glucose/ammonium/6-hydroxy-3-succinoylpyridine medium. The molar conversion and the specific catalytic rate reached approximately 98% and 1.01 g 6-hydroxynicotine h-1 g-1 dry cells, respectively. The product could be purified easily by dichloromethane extraction with a recovery of 76.8%, and was further confirmed by UV spectroscopy, mass spectroscopy, and NMR analysis. CONCLUSIONS: We successfully developed a novel biocatalytic route to 6-hydroxynicotine from nicotine by blocking the nicotine catabolic pathway via gene disruption, which provides an alternative green strategy to utilize tobacco and its wastes as a biomass resource by converting nicotine into valuable hydroxylated-pyridine compounds.