Characterization and Transcriptional Regulation of the 2-Ketogluconate Utilization Operon in <i>Pseudomonas plecoglossicida</i>
<i>Pseudomonas plecoglossicida</i> JUIM01 is an industrial 2-keto-<span style="font-variant: small-caps;">d</span>-gluconate (2KGA)-producing strain. However, its regulation mechanism of 2KGA metabolism remains to be clarified. Among other reported <i>Pseudomo...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-12-01
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| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/12/12/2530 |
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| Summary: | <i>Pseudomonas plecoglossicida</i> JUIM01 is an industrial 2-keto-<span style="font-variant: small-caps;">d</span>-gluconate (2KGA)-producing strain. However, its regulation mechanism of 2KGA metabolism remains to be clarified. Among other reported <i>Pseudomonas</i> species, the 2-ketogluconate utilization operon (<i>kgu</i> operon) plays key roles in 2KGA catabolism. In this study, the structural genes of the <i>kgu</i> operon and its promoter in <i>P. plecoglossicida</i> JUIM01 were identified using reverse transcription PCR and <i>lacZ</i> reporter gene fusion. The results showed the <i>kgu</i> operon in <i>P. plecoglossicida</i> was composed of four structural genes: <i>kguE</i>, <i>kguK</i>, <i>kguT</i>, and <i>kguD</i>. The <i>ptxS</i> gene located upstream of <i>kguE</i> was excluded from the <i>kgu</i> operon. Then, the knockout and corresponding complementation strains of <i>kguE</i>, <i>kguK</i>, <i>kguT</i>, and <i>kguD</i> were constructed, respectively, to prove the <i>kgu</i> operon was involved in 2KGA catabolism of <i>P. plecoglossicida</i>. The knockout stains, especially JUIM01Δ<i>kguE</i>, showed potential as industrial production strains for 2KGA. Moreover, the transcriptional regulation mechanism of PtxS on the <i>kgu</i> operon was elucidated using multiple methods. In <i>P. plecoglossicida</i>, the LacI-family transcription regulator PtxS could recognize a 14 bp palindrome (5′-TGAAACCGGTTTCA-3′) within the promoter region of the <i>kgu</i> operon and specifically bind to a 26 bp region where the palindrome was located. As the binding sites overlapped with the transcription start site of the <i>kgu</i> operon, the binding of PtxS possibly hindered the binding of RNA polymerase, thus repressing the transcription of the <i>kgu</i> operon and further regulating 2KGA catabolism. 2KGA bound to PtxS as an effector to dissociate it from the <i>kgu</i> operon promoter region, so as to relieve the transcription repression. The results will provide strategies for improving the product accumulation in 2KGA industrial production and theoretical bases for the construction of a <i>Pseudomonas</i> chassis. |
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| ISSN: | 2076-2607 |