Metabolic Engineering of Ralstonia eutropha for the Production of Polyhydroxyalkanoates From Sucrose

Abstract

A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes -fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that -fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20g/L of sucrose, a high P(3HB) content of 73.2wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5mol% LA) to a polymer content of 19.5wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52x10(5) (M-n) and 2.19x10(4) (M-n), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources. Biotechnol. Bioeng. 2015;112: 638-643.