Revealing the promoting effect of betaine on vitamin B12 biosynthetic pathway of Pseudomonas denitrificans by using a proteomics analysis

Background: Our previous comparative metabolomics research revealed that betaine (N,N,N-trimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had a 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. Objective: 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 the 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 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 late-stage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. Conclusions: The present study clearly demonstrated 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.

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

Groundwater Nitrate Removal Performance of Selected Pseudomonas Strains Carrying nosZ Gene in Aerobic Granular Sequential Batch Reactors

Four granular sequencing batch reactors (GSBRs) were inoculated with four denitrifying Pseudomonas strains carrying nosZ to study the process of granule formation, the operational conditions of the bioreactors, and the carbon concentration needed for nitrate removal. The selected Pseudomonas strains were P. stutzeri I1, P. fluorescens 376, P. denitrificans Z1, and P. fluorescens PSC26, previously reported as denitrifying microorganisms carrying the nosZ gene. Pseudomonas denitrificans Z1 produced fluffy, low-density granules, with a decantation speed below 10 m h−1. However, P. fluorescens PSC26, P. stutzeri I1, and P. fluorescens 376 formed stable granules, with mean size from 7 to 15 mm, related to the strain and carbon concentration. P. stutzeri I1 and P. fluorescens 376 removed nitrate efficiently with a ratio in the range of 96%, depending on the source and concentration of organic matter. Therefore, the findings suggest that the inoculation of GSBR systems with denitrifying strains of Pseudomonas spp. containing the nosZ gene enables the formation of stable granules, the efficient removal of nitrate, and the transformation of nitrate into nitrogen gas, a result of considerable environmental interest to avoid the generation of nitrous oxide.

Betaine biosynthesis in a heterologous expression system based on the B12 producer Pseudomonas denitrificans

Glycine betaine is an important donor of methyl groups in various metabolic processes of the cell and acts as an osmoprotector when exposed to various abiotic stresses in pro- and eukaryotic organisms. Moreover, exogenous application of betaine activates the production of vitamin B12 in industrial strains-producers. In this work, we have developed a new technology for microbiological betaine synthesis that can be used in biotechnology to activate B12 biosynthesis during large-scale fermentation of Pseudomonas denitrificans.

Inoculantes microbianos incorporados al cultivo de Ipomoea batatas L. en el Valle del Sinú

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.