Dataset Information

Novel Pathway for Corrinoid Compounds Production in Lactobacillus.

ABSTRACT: Vitamin B12 or cobalamin is an essential metabolite for humans, which makes it an interesting compound for many research groups that focus in different producer-strains synthesis pathways. In this work, we report the influence of key intermediaries for cobalamin synthesis added to the culture medium in two Lactobacillus (L.) strains, L. reuteri CRL 1098 and L. coryniformis CRL 1001. Here, we report that addition of Co2+ and 5,6-dimethylbenzimidazole increased the corrinoid compounds production in both strains while addition of L-threonine increased only the corrinoid compounds production by CRL 1001 strain. Then, we purified and characterized by LC-MS the corrinoid compounds obtained. Physiological studies besides in silico analysis revealed that L. reuteri CRL 1098 and L. coryniformis CRL 1001 follow different pathways for the last steps of the corrinoid compounds synthesis.

SUBMITTER: Torres AC

PROVIDER: S-EPMC6167548 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Novel Pathway for Corrinoid Compounds Production in Lactobacillus.

Torres Andrea Carolina AC Vannini Verónica V Font Graciela G Saavedra Lucila L Taranto María Pía MP

Frontiers in microbiology 20180925

Vitamin B12 or cobalamin is an essential metabolite for humans, which makes it an interesting compound for many research groups that focus in different producer-strains synthesis pathways. In this work, we report the influence of key intermediaries for cobalamin synthesis added to the culture medium in two Lactobacillus (L.) strains, L. reuteri CRL 1098 and L. coryniformis CRL 1001. Here, we report that addition of Co2+ and 5,6-dimethylbenzimidaz ...[more]

PMID: 30319575

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Project description:The PduO-type adenosine 5'-triphosphate (ATP):corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co(1+)cobalamin (Cbl) and Co(1+)cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi(+)), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co(2+) ? Co(1+) reduction, the enzyme drastically weakens the axial ligand-Co(2+) bond so as to generate effectively four-coordinate (4c) Co(2+)corrinoid species. To explore how LrPduO generates these unusual 4c species, we have used magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopic techniques. The effects of active-site amino acid substitutions on the relative yield of formation of 4c Co(2+)corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an intersubunit salt bridge, and the hydrophobic region surrounding the bound corrin ring. A quantitative analysis of our MCD and EPR spectra indicates that the entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co(2+)corrinoids. Our data also show that a higher level of coordination among several LrPduO amino acid residues is required to exclude the dimethylbenzimidazole moiety of Co(II)Cbl from the active site than to remove the water molecule from Co(II)Cbi(+). Thus, the hydrophilic interactions around and above the corrin ring are more critical to form 4c Co(II)Cbl than 4c Co(II)Cbi(+). Finally, when ATP analogues were used as cosubstrate, only "unactivated" five-coordinate (5c) Co(II)Cbl was observed, disclosing an unexpectedly large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl. Collectively, our results indicate that the level of control exerted by LrPduO over the timing for the formation of the 4c Co(2+)corrinoid intermediates is even more exquisite than previously anticipated.

Project description:Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

Dataset Information

Novel Pathway for Corrinoid Compounds Production in Lactobacillus.

Publications

Novel Pathway for Corrinoid Compounds Production in <i>Lactobacillus</i>.

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