Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

Abstract

In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membranebound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with D-mannitol, D-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with D-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQdependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.The acetic acid bacterium Gluconobacter oxydans is characterized by its ability to incompletely oxidize various sugars and alcohols by using membrane-associated dehydrogenases that contain pyrroloquinoline quinone (PQQ) as a cofactor (33). Examples are the quinoprotein glucose dehydrogenase (3) and the quinoprotein glycerol dehydrogenase, which also oxidizes D-gluconate, D-mannitol, D-sorbitol, and other polyols (34). The oxidation reactions take place in the periplasmic space and are coupled to the respiratory chain (19,33). Several oxidation reactions carried out by G. oxydans quinoproteins are of industrial importance, e.g., the conversion of D-gluconate to 5-D-ketogluconate, a precursor for the production of L-tartaric acid, and the formation of L-sorbose, an intermediate in the synthesis of vitamin C (reviewed in reference 9). PQQ has invoked considerable attention due to its positive physiological effects in mammals (47). A possible role of PQQ as a vitamin in mammals has been suggested but is controversially debated (13,28,40).Genes involved in PQQ synthesis have been characterized for several bacteria, including Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens AM1, and Pseudomonas sp. (reviewed in reference 19). In Klebsiella pneumoniae, the PQQ biosynthetic genes are clustered in the pqqABCDEF operon (35). In Pseudomonas aeruginosa, the pqqABCDE operon is separated from the pqqF operon (18,48). Methylobacterium extorquens AM1 contains a ...

Glucose Oxidation and PQQ-Dependent Dehydrogenases in <i>Gluconobacter oxydans</i>

“…One of these so far uncharacterized membrane-bound quinoproteins in G. oxydans ATCC 621H (DSM 2343) was recently identified as a quinoprotein myo-inositol dehydrogenase [Hölscher et al, 2006]. The enzyme is required for growth of G. oxydans on myo -inositol as sole energy source.…”

“…Recently, a PQQ-deficient G. oxydans ATCC 621H (DSM 2343) mutant was generated by Tn5 transposon mutagenesis, which showed a defect in a gene with high homology to the E. coli tldD gene [Hölscher and Görisch, 2006] ( fig. 2b ).…”

Mutations That Disrupt Either thepqqor thegdhGene ofRahnella aquatilisAbolish the Production of an Antibacterial Substance and Result in Reduced Biological Control of Grapevine Crown Gall

“…Genes associated with PQQ production have been identified for various bacteria, including Acinetobacter calcoaceticus (21), E. intermedium 60-2G (32), Gluconobacter oxydans (26), Klebsiella pneumoniae (40), Methylobacterium extorquens AM1 (51), P. fluorescens B16 (14), and P. fluorescens CHA0 (46). The pqqABCDEF genes are conserved in these bacteria, but the accurate biochemical functions of the encoded proteins remain to be determined.…”

An Efficient Separation of Pyrroloquinoline Quinone Using Chemical Complexation Extraction

“…strain MP688 [4][5][6][7][8]. Their synthesis gene cluster has also been revealed in these bacteria [4][5][6][7][8]. Fig.…”