Improvement of Poly(3-Hydroxybutyrate) [P(3HB)] Production in Corynebacterium glutamicum by Codon Optimization, Point Mutation and Gene Dosage of P(3HB) Biosynthetic Genes

In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon ( phaCAB Re) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been appli...

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Bibliographic Details
Published in:Journal of bioscience and bioengineering 2007-12, Vol.104 (6), p.457-463
Main Authors: Jo, Sung-Jin, Matsumoto, Ken'Ichiro, Leong, Chean Ring, Ooi, Toshihiko, Taguchi, Seiichi
Format: Article
Language:English
Subjects:
Biological and medical sciences
BIOPOLIMEROS
BIOPOLYMERE
BIOPOLYMERS
BIOSINTESIS
BIOSYNTHESE
BIOSYNTHESIS
Biotechnology
Codon - genetics
codon optimization
CORYNEBACTERIUM
Corynebacterium glutamicum
Corynebacterium glutamicum - physiology
Escherichia coli
Fundamental and applied biological sciences. Psychology
GENE
gene dosage
Gene Dosage - genetics
GENES
Genetic Enhancement - methods
Hydroxybutyrates - metabolism
Mutagenesis, Site-Directed
NUCLEOTIDE SEQUENCE
poly(3-hydroxybutyrate)
Polyesters - metabolism
Protein Engineering - methods
Ralstonia eutropha
rate-determining step
Recombinant Proteins - metabolism
SECUENCIA NUCLEOTIDICA
SEQUENCE NUCLEOTIDIQUE
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Summary:In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon ( phaCAB Re) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C. glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC Re) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC Re with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaC Re and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C. glutamicum enhanced the production of PhaC Re and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, β-ketothiolase (PhaA Re) and acetoacetyl-CoA reductase (PhaB Re). This finding prompted us to carry out a gene dosage of phaAB Re for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB Re with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(3HB) was increased by more than 4-fold compared with the non-P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.
ISSN:1389-1723
1347-4421
DOI:10.1263/jbb.104.457