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| Content Provider | Springer Nature : BioMed Central |
|---|---|
| Author | Kawashima, Yui Orita, Izumi Nakamura, Satoshi Fukui, Toshiaki |
| Abstract | Background Phasin (PhaP), a kind of polyhydroxyalkanoate (PHA) granule-associated proteins, has a role in controlling the properties of PHA granules surface, and is thought to have influence on PHA biosynthesis in PHA-producing bacteria. This study focused on the phaP1 Re locus in Ralstonia eutropha as a site of chromosomal modification for production of flexible poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Results Considering the high expression level of phaP1 Re , phaJ Ac [encoding (R)-specific enoyl-CoA hydratase from Aeromonas caviae] was inserted into the downstream of phaP1 Re on chromosome 1 of R. eutropha strain NSDG harboring phaC NSDG (encoding PHA synthase with broad substrate specificity). The constructed strain efficiently accumulated P(3HB-co-3HHx) on soybean oil with higher 3HHx composition when compared to the previous strain having phaJ Ac within pha operon. Insertion of the second phaC NSDG along with phaJ Ac at the phaP1 Re locus led to incorporation of much larger 3HHx fraction into PHA chains, although the molecular weight was markedly reduced. The R. eutropha strains were further engineered by replacing phaP1 Re with phaP Ac (encoding phasin from A. caviae) on the chromosome. Interestingly, the phasin replacement increased 3HHx composition in the soybean oil-based PHA with keeping high cellular contents, nevertheless no modification was conducted in the metabolic pathways. Kinetic and Western blot analyses of PHA synthase using cellular insoluble fractions strongly suggested that the phasin replacement not only enhanced activity of PHA synthase from A. caviae but also increased affinity especially to longer (R)-3HHx-CoA. It was supposed that the increased affinity of PHA synthase to (R)-3HHx-CoA was responsible for the higher 3HHx composition in the copolyester. Conclusions The downstream of phaP1 Re was a useful site for integration of genes to be overexpressed during PHA accumulation in R. eutropha. The results also clarified that polymerization properties of PHA synthase was affected by the kind of phasin co-existed on the surface of PHA granules, leading to altered composition of the resulting P(3HB-co-3HHx). The phasin replacement is a novel engineering strategy for regulation of composition of PHA copolyesters. |
| Related Links | https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-015-0380-8.pdf |
| Ending Page | 12 |
| Page Count | 12 |
| Starting Page | 1 |
| File Format | HTM / HTML |
| ISSN | 14752859 |
| DOI | 10.1186/s12934-015-0380-8 |
| Journal | Microbial Cell Factories |
| Issue Number | 1 |
| Volume Number | 14 |
| Language | English |
| Publisher | BioMed Central |
| Publisher Date | 2015-11-23 |
| Access Restriction | Open |
| Subject Keyword | Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Ralstonia eutropha Polyhydroxyalkanoates PHA synthase Phasin |
| Content Type | Text |
| Resource Type | Article |
| Subject | Applied Microbiology and Biotechnology Bioengineering Biotechnology |
| Journal Impact Factor | 4.3/2023 |
| 5-Year Journal Impact Factor | 5.5/2023 |
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