Open AccessExtension of the Substrate Utilization Range of Ralstonia eutropha Strain H16 by Metabolic Engineering To Include Mannose and Glucose
Author(s) -
Shanna Sichwart,
Stephan Hetzler,
Daniel Bröker,
Alexander Steinbüchel
Publication year - 2011
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.01977-10
Subject(s) - ralstonia , mannose , glucose 6 phosphate isomerase , zymomonas mobilis , biology , biochemistry , strain (injury) , isomerase , cupriavidus necator , plasmid , polyhydroxyalkanoates , recombinant dna , bacteria , microbiology and biotechnology , fermentation , enzyme , gene , ethanol fuel , genetics , anatomy
The Gram-negative facultative chemolithoautotrophic bacteriumRalstonia eutropha strain H16 is known for its narrow carbohydrate utilization range, which limits its use for biotechnological production of polyhydroxyalkanoates and possibly other products from renewable resources. To broaden its substrate utilization range, which is for carbohydrates and related compounds limited to fructose,N -acetylglucosamine, and gluconate, strain H16 was engineered to use mannose and glucose as sole carbon sources for growth. The genes for a facilitated diffusion protein (glf ) fromZymomonas mobilis and for a glucokinase (glk ), mannofructokinase (mak ), and phosphomannose isomerase (pmi ) fromEscherichia coli were alone or in combination constitutively expressed inR. eutropha strain H16 under the control of the neokanamycin orlac promoter, respectively, using an episomal broad-host-range vector. Recombinant strains harboring pBBR1MCS-3::glf ::mak ::pmi or pBBR1MCS-3::glf ::pmi grew on mannose, whereas pBBR1MCS-3::glf ::mak and pBBR1MCS-3::glf did not confer the ability to utilize mannose as a carbon source toR. eutropha . The recombinant strain harboring pBBR1MCS-3::glf ::pmi exhibited slower growth on mannose than the recombinant strain harboring pBBR1MCS-3::glf ::mak ::pmi . These data indicated that phosphomannose isomerase is required to convert mannose-6-phosphate into fructose-6-phosphate for subsequent catabolism via the Entner-Doudoroff pathway. In addition, all plasmids also conferred toR. eutropha the ability to grow in the presence of glucose. The best growth was observed with a recombinantR. eutropha strain harboring plasmid pBBR1MCS-2::Pnk ::glk ::glf . In addition, expression of the respective enzymes was demonstrated at the transcriptional and protein levels and by measuring the activities of mannofructokinase (0.622 ± 0.063 U mg−1 ), phosphomannose isomerase (0.251 ± 0.017 U mg−1 ), and glucokinase (0.518 ± 0.040 U mg−1 ). Cells of recombinant strains ofR. eutropha synthesized poly(3-hydroxybutyrate) to ca. 65 to 67% (wt/wt) of the cell dry mass in the presence of 1% (wt/vol) glucose or mannose as the sole carbon sources.