title: LplR, a repressor belonging to the TetR family, regulates expression of the L-pantoyl lactone dehydrogenase gene in Rhodococcus erythropolis.
abstract: The L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) gene (lpldh) has been cloned from Rhodococcus erythropolis AKU2103, and addition of 1,2-propanediol (1,2-PD) was shown to be required for lpldh expression in this strain. In this study, based on an exploration of the nucleotide sequence around lpldh, a TetR-like regulator gene, which we designated lplR, was found upstream of lpldh, and three putative open reading frames existed between the two genes. Disruption of lplR led to 22.8 times higher lpldh expression, even without 1,2-PD induction, than that in wild-type R. erythropolis AKU2103 without 1,2-PD addition. Introduction of a multicopy vector carrying lplR (multi-lplR) into the wild-type and ΔlplR strains led to no detectable LPLDH activity even in the presence of 1,2-PD. The results of an electrophoretic mobility shift assay revealed that purified LplR bound to a 6-bp inverted-repeat sequence located in the promoter/operator region of the operon containing lpldh. These results indicated that LplR is a negative regulator in lpldh expression. Based on the clarification of the expression mechanism of lpldh, recombinant cells showing high LPLDH activity were constructed and used as a catalyst for the conversion of L-PL to ketopantoyl lactone. Finally, a promising production process of D-PL from DL-PL was constructed.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Dayong
authors.initials: D
authors.lastname: Si
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Urano
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kataoka
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D019946:Propylene Glycol / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D012097:Repressor Proteins; C008232:keto-pantoyllactone; D019946:Propylene Glycol; C005412:pantolactone; D000429:Alcohol Oxidoreductases; C073297:pantoyl lactone dehydrogenase; D015107:4-Butyrolactone