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Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

ABSTRACT: In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.

SUBMITTER: Raux E

PROVIDER: S-EPMC1223173 | biostudies-other | 2003 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundHeparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin. In addition to its utility as the starting material for this important anticoagulant and anti-inflammatory drug, heparosan is a versatile compound that possesses suitable chemical and physical properties for making a variety of high-quality tissue engineering biomaterials, gels and scaffolds, as well as serving as a drug delivery vehicle. The selected production host was the Gram-positive bacterium Bacillus megaterium, which represents an increasingly used choice for high-yield production of intra- and extracellular biomolecules for scientific and industrial applications.ResultsWe have engineered the metabolism of B. megaterium to produce heparosan, using a T7 RNA polymerase (T7 RNAP) expression system. This system, which allows tightly regulated and efficient induction of genes of interest, has been co-opted for control of Pasteurella multocida heparosan synthase (PmHS2). Specifically, we show that B. megaterium MS941 cells co-transformed with pT7-RNAP and pPT7_PmHS2 plasmids are capable of producing heparosan upon induction with xylose, providing an alternate, safe source of heparosan. Productivities of ~ 250 mg/L of heparosan in shake flasks and ~ 2.74 g/L in fed-batch cultivation were reached. The polydisperse Pasteurella heparosan synthase products from B. megaterium primarily consisted of a relatively high molecular weight (MW) heparosan (~ 200-300 kD) that may be appropriate for producing certain biomaterials; while the less abundant lower MW heparosan fractions (~ 10-40 kD) can be a suitable starting material for heparin synthesis.ConclusionWe have successfully engineered an asporogenic and non-pathogenic B. megaterium host strain to produce heparosan for various applications, through a combination of genetic manipulation and growth optimization strategies. The heparosan products from B. megaterium display a different range of MW products than traditional E. coli K5 products, diversifying its potential applications and facilitating increased product utility.

Project description:The environmental deterioration produced by heavy metals derived from anthropogenic activities has gradually increased. The worldwide dissemination of toxic metals in crop soils represents a threat for sustainability and biosafety in agriculture and requires strategies for the recovery of metal-polluted crop soils. The biorestoration of metal-polluted soils using technologies that combine plants and microorganisms has gained attention in recent decades due to the beneficial and synergistic effects produced by its biotic interactions. In this context, native and heavy metal-resistant plant growth-promoting bacteria (PGPB) play a crucial role in the development of strategies for sustainable biorestoration of metal-contaminated soils. In this study, we present a genomic analysis and characterization of the rhizospheric bacterium Bacillus megaterium HgT21 isolated from metal-polluted soil from Zacatecas, Mexico. The results reveal that this autochthonous bacterium contains an important set of genes related to a variety of operons associated with mercury, arsenic, copper, cobalt, cadmium, zinc and aluminum resistance. Additionally, halotolerance-, beta-lactam resistance-, phosphate solubilization-, and plant growth-promotion-related genes were identified. The analysis of resistance to metal ions revealed resistance to mercury (HgII+), arsenate [AsO4]³-, cobalt (Co2+), zinc (Zn2+), and copper (Cu2+). Moreover, the ability of the HgT21 strain to produce indole acetic acid (a phytohormone) and promote the growth of Arabidopsis thaliana seedlings in vitro was also demonstrated. The genotype and phenotype of Bacillus megaterium HgT21 reveal its potential to be used as a model of both plant growth-promoting and metal multiresistant bacteria. IMPORTANCE Metal-polluted environments are natural sources of a wide variety of PGPB adapted to cope with toxic metal concentrations. In this work, the bacterial strain Bacillus megaterium HgT21 was isolated from metal-contaminated soil and is proposed as a model for the study of metal multiresistance in spore-forming Gram-positive bacteria due to the presence of a variety of metal resistance-associated genes similar to those encountered in the metal multiresistant Gram-negative Cupriavidus metallidurans CH34. The ability of B. megaterium HgT21 to promote the growth of plants also makes it suitable for the study of plant-bacteria interactions in metal-polluted environments, which is key for the development of techniques for the biorestoration of metal-contaminated soils used for agriculture.

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Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

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