Knockout and overexpression of pyrroloquinoline quinone biosynthetic genes in Gluconobacter oxydans 621H
Knockout and overexpression of pyrroloquinoline quinone biosynthetic genes in Gluconobacter oxydans 621H
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DOI：
10.1128/jb.01009-06
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发表时间：
2006-11-01
影响因子：
3.2
通讯作者：
Goerisch, Helmut
中科院分区：
文献类型：
--
作者：
Hoelscher, Tina;Goerisch, Helmut
在葡萄糖酸氧化物中，吡咯喹啉醌（PQQ）作为各种膜结合的脱氢酶的辅因子，氧化周质中的糖和醇。用于PQQ生物合成的蛋白质由pqqABCDE基因簇编码。我们的逆转录PCR和启动子分析数据表明，pqqA启动子是G. oxydans 621 H. PQQ在G.通过用质粒携带的pqqA基因或完整的pqqABCDE簇转化来获得氧化丹酶。A G.通过定点破坏pqqA基因获得了一株不能产生PQQ的oxydans突变体。与野生型菌株相反，pqqA突变体不能以D-甘露醇、D-葡萄糖或甘油作为唯一能源生长，表明在G. oxydans 621 H，PQQ是生长所必需的这些基板。然而，pqqA突变体的生长被发现与D-葡萄糖酸盐作为能源。pqqA突变体的生长行为与相应的PQQ依赖性膜结合脱氢酶活性的存在或不存在相关，表明这些酶在G.氧化物代谢通过Tn 5转座子诱变产生不同的PQQ缺陷型突变体。该突变体在与编码肽酶的大肠杆菌tldD基因具有高度同源性的基因中显示出缺陷。我们的结果表明，G. oxydans 621 H参与PQQ的生物合成，可能具有与在其他PQQ合成细菌中发现的pqqF基因类似的功能。
In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membrane-bound 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 PQQ-dependent 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.