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The B12-producing strains Pseudomonas nitroreducens DSM 1650 and Pseudomonas sp. CCUG 2519 (both formerly Pseudomonas denitrificans), with the most distributed pathway among bacteria for exogenous choline/betaine utilization, are promising recombinant hosts for the endogenous production of B12 precursor betaine by direct methylation of bioavailable glycine or non-proteinogenic ß-alanine. Two plasmid-based de novo betaine pathways, distinguished by their enzymes, have provided an expression of the genes encoding for N-methyltransferases of the halotolerant cyanobacterium Aphanothece halophytica or plant Limonium latifolium to synthesize the internal glycine betaine or ß-alanine betaine, respectively. These betaines equally allowed the recombinant pseudomonads to grow effectively and to synthesize a high level of cobalamin, as well as to increase their protective properties against abiotic stresses to a degree comparable with the supplementation of an exogenous betaine. Both de novo betaine pathways significantly enforced the protection of bacterial cells against lowering temperature to 15 °C and increasing salinity to 400 mM of NaCl. However, the expression of the single plant-derived gene for the ß-alanine-specific N-methyltransferase additionally increased the effectiveness of exogenous glycine betaine almost twofold on cobalamin biosynthesis, probably due to the Pseudomonas' ability to use two independent pathways, their own choline/betaine pathway and the plant ß-alanine betaine biosynthetic pathway.

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Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. For this reason, finding better producer strains and optimizing their bioprocesses have been the main focus of industrial producers over the last few decades. In this review, we initially provide a historical overview of vitamin B12 research and the main biosynthetic characteristics of the two microorganism families typically used for its industrial production: several strains of Propionibacterium freudenreichii and strains related to Pseudomonas denitrificans. Later, a complete summary of the current state of vitamin B12 industrial production as well as the main advances and challenges for improving it is detailed, with a special focus on bioprocess optimization, which aims not only to increase production but also sustainability. In addition, a comprehensive list of the most important and relevant patents for the present industrial strains is provided. Finally, the potential applications of vitamin B12 in different markets are discussed.

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BACKGROUND: Our previous comparative metabolomics research revealed that betaine (N,N,Ntrimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. OBJECTIVES: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. MATERIALS AND METHODS: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine in addition to P. denitrificans fermentation medium. RESULTS: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, precorrin-2 C(20)- methyltransferase, precorrin-8X methylmutase, and precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the latestage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. CONCLUSION: The present study demonstrated clearly that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

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RESUMEN La batata (Ipomoea batatas L.) se cultiva en todo el mundo como fuente de carbohidratos, y su producción comercial requiere un alto aporte de fertilizantes químicos, lo cual eleva los costos de producción. Los inoculantes microbianos, se emplean como una fuente alternativa de nutrición vegetal. El objetivo de esta investigación fue evaluar el efecto de Pseudomonas denitrificans IBVS2 y Azotobacter vinelandii IBVS13 con diferentes niveles fertilización química nitrogenada en el cultivo de batata en la microrregión del Valle del Sinú en el Caribe Colombiano. Para los montajes de los experimentos se utilizó un diseño completamente aleatorizado, ocho tratamientos y tres repeticiones usando como material vegetal plántulas obtenidas in vitro endurecidas en invernadero. Los resultados demostraron que la cepa Azotobacter vinelandii IBVS13 con un 75% de fertilización nitrogenada (FN) mejoró la capacidad de acumulación de materia seca en los tubérculos de batata, generando incrementos de 6,65 t/ha respecto al testigo químico y 3,18 t/ha en relación con el testigo absoluto, garantizando un incremento del rendimiento. Así mismo, el contenido de proteína bruta aumentó 13,93% al realizar la inoculación de las plantas con esta cepa. En el mismo sentido, la cepa Pseudomonas denitrificans IBVS2+ fertilización nitrogenada 50% presentó aumentos en la variable de fibra cruda 31,75% respecto al testigo absoluto, contribuyendo de manera eficaz como bioestimulante microbiano en la agricultura.

ABSTRACT Sweet potatoes (Ipomoea batatas L.) are grown worldwide as a source of carbohydrates, and their commercial production requires a high contribution of chemical fertilizers, which increases production costs. Microbial inoculants are used as an alternative source of plant nutrition. The objective of this research was to evaluate the effect of Pseudomonas denitrificans IBVS2 and Azotobacter vinelandii IBVS13 with different levels of nitrogen chemical fertilization in the sweet potato crop in the microregion of the Sinú Valley in the Colombian Caribbean. A completely randomized design was used for the experiment development, eight treatments was evaluated and three repetitions were carried out. In vitro hardened seedlings was used as a plant material. The results showed that the Azotobacter vinelandii IBVS1 3 strain with 75% nitrogen fertilization (FM) improved the accumulation capacity of dry matter in sweet potato roots, generating increases of 6.65 t / ha compared to the chemical control and 3.18 t / ha in relation to the absolute control, guaranteeing an increase in yield. The crude protein content was increased in 13.93% when inoculating the plants with this strain. In the same way, with the inoculation of strain Pseudomonas denitrificans IBVS2 + 50% nitrogen fertilization the crude fiber variable was increased in 31.75% compared to the absolute control, contributing effectively as a microbial biostimulant in agriculture.

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The use of acetate as carbon feedstock can enhance sustainability and economics of the current bio-productions. This study explored the potential of acetate for the production of 3-hydroxypropionic acid by engineered Pseudomonas denitrificans. Heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus and endogenous accABCD (encoding acetyl-CoA carboxylase) were overexpressed in P. denitrificans. Carbon flux to 3-HP synthesis at the malonyl-CoA node was promoted by suppressing fatty acid synthesis through addition of cerulenin or deletion of fabF gene. In addition, stimulation of glyoxylate shunt and/or TCA cycle were attempted. Recombinant P. denitrificans overexpressing mcr and accABCD produced 19.3 mM 3-HP with cerulenin addition, and 14.2 mM with fabF deletion, respectively. Furthermore, the non-growing cells devoid of fabF could continuously produce 3-HP up to 40.4 mM without losing its production activity for 22 h. This study demonstrates that acetate is a good substrate for 3-HP production by recombinant P. denitrificans.

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Excessive nitrate in aquaculture systems has attracted wide attention. To isolate novel aerobic denitrifying strain and characterize its nitrogen removal processes, a facultative anaerobic denitrification bacterium, identified as Pseudomonas denitrificans G1, was isolated from marine sediments. Strain G1 could grow and remove 90-98% of nitrate and 97-99% of nitrite under an aerobic or anaerobic condition in 24 h, with the total nitrogen removal rate of 33-38% (87-100 mg/L). The highest denitrification rate could reach 15.1 mg/(L·h). The suitable condition for the denitrification of G1 is C/N ratio 5-22, dissolved oxygen 0-4.68 mg/L, salinity 0-30 g NaCl/L, pH 7-9.5. Under the aerobic condition, G1 grew fast; however, the mass spectrographic analysis showed that the gas product was N2O. Under the anaerobic conditions, G1 grow relatively slowly, but could also achieve effective denitrification and the final product was N2. In denitrification of aquaculture wastewater, strain G1 can remove 60.57% of nitrate and 36.36% of total nitrogen; meanwhile, there was a slight accumulation of ammonia nitrogen. P. denitrificans strain G1 has potential in denitrification processes for the treatment of aquaculture wastewater. However, the regulation of reaction conditions and gas products needs to be further studied.

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Bio-production of 1,3-propanediol (1,3-PDO) from glycerol was studied using Pseudomonas denitrificans as host, which aerobically synthesizes coenzyme B12, an essential cofactor of glycerol dehydratase (GDHt). P. denitrificans was transformed with the 1,3-PDO synthesis pathway composed of GDHt and 1,3-PDO oxidoreductase (PDOR), and its putative 3-hydroxypropionaldehyde (3-HPA) dehydrogenase(s), leading to the production of 3-hydroxypropioninc acid form the intermediary 3-HPA, was identified and deleted. In addition, to improve the availability of NADH for PDOR, oxidation of NADH in the electron transport chain was disturbed by deletion of the nuo operon and/or ndh gene. Finally, acetate formation pathway was eliminated. One resulting strain could produce 68.95 mM 1,3-PDO with the yield of 0.92 mol 1,3-PDO/mol glycerol on flask scale and 440 mM with the yield of 0.89 mol 1,3-PDO/mol glycerol in a fed-batch bioreactor experiment. This study demonstrates that P. denitrificans is a promising recombinant host for the production of 1,3-PDO from glycerol.

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Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

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The relationship between the morphological character of Pseudomonas denitrificans and vitamin B12 synthesis based on real-time capacitance measurement and online specific oxygen consumption rate (Q O2) control was established for enhancing vitamin B12 production. Results demonstrated that the threshold Q O2 value lower than 2.0 mmol/gDCW/l would greatly stimulate the state transfer from the cell number growth phase to the cell elongation phase and promote rapid vitamin B12 biosynthesis, while the vitamin B12 biosynthesis rate could also be inhibited when the rate of cell's length-to-width ratio (ratio-LW) was higher than 10:1. Furthermore, the optimal morphology controlling strategy was achieved based on online Q O2 control, which increases the appropriate active cell numbers at the former phase, and then control the elongation of ratio-LW no more than 10:1 at the vitamin B12 biosynthesis phase. The maximal vitamin B12 production reached 239.7 mg/l at 168 h, which was improved by 14.7 % compared with the control (208 mg/l). This online controlling strategy would be effectively applied for improving industrial vitamin B12 fermentation.

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Coenzyme B12 (Vitamin B12 ) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B12 . These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B12 synthetic genes and successfully produced coenzyme B12 . However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B12 produced by the recombinant E. coli (0.21 ± 0.02 µg/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 ± 0.22 µg/g cdw). Optimization of the culture conditions to improve the production of coenzyme B12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 µg/g cdw) of coenzyme B12 was obtained. Interestingly, although the synthesis of coenzyme B12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B12 under anaerobic conditions.

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3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12 -dependent glycerol dehydratase and an NAD(P)(+) -dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol.

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Bacillus penetrans Mankau, 1975, previously described as Duboscqia penetrans Thorne 1940, is a candidate agent for biocontrol of nematodes. This review considers the life stages of this bacterium: vegetative growth phase, colony fragmentation, sporogenesis, soil phase, spore attachment, and penetration into larvae of root-knot nematodes. The morphology of the microthallus colonies and the unusual external features of the spore are discussed. Taxonomic affinities with the actinomycetes, particularly with the genus Pasteuria, are considered. Also discussed are other soil bacterial species that are potential biocontrol agents. Products of their bacterial fermentation in soil are toxic to nematodes, making them effective biocontrol agents.