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Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16.

ABSTRACT: The biotechnical platform strain Ralstonia eutropha H16 was genetically engineered to express a cox subcluster of the carboxydotrophic Oligotropha carboxidovoransOM5, including (i) the structural genes coxM, -S and -L, coding for an aerobic carbon monoxide dehydrogenase (CODH) and (ii) the genes coxD, -E, -F and -G, essential for the maturation of CODH. The coxOc genes expressed under control of the CO2 -inducible promoter PL enabled R. eutropha to oxidize CO to CO2 for the use as carbon source, as demonstrated by 13 CO experiments, but the recombinant strains remained dependent on H2 as external energy supply. Therefore, a synthetic metabolism, which could be described as 'carboxyhydrogenotrophic', was established in R. eutropha. With this extension of the bacterium's substrate range, growth in CO-, H2 - and CO2 -containing artificial synthesis gas atmosphere was enhanced, and poly(3-hydroxybutyrate) synthesis was increased by more than 20%.

SUBMITTER: Heinrich D

PROVIDER: S-EPMC6011924 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16.

Heinrich Daniel D Raberg Matthias M Steinbüchel Alexander A

Microbial biotechnology 20171013 4

The biotechnical platform strain Ralstonia eutropha H16 was genetically engineered to express a cox subcluster of the carboxydotrophic Oligotropha carboxidovoransOM5, including (i) the structural genes coxM, -S and -L, coding for an aerobic carbon monoxide dehydrogenase (CODH) and (ii) the genes coxD, -E, -F and -G, essential for the maturation of CODH. The coxOc genes expressed under control of the CO2 -inducible promoter PL enabled R. eutropha to oxidize CO to ...[more]

PMID: 29027357

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Project description:The gram-negative facultative chemolithoautotrophic bacterium Ralstonia 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) from Zymomonas mobilis and for a glucokinase (glk), mannofructokinase (mak), and phosphomannose isomerase (pmi) from Escherichia coli were alone or in combination constitutively expressed in R. eutropha strain H16 under the control of the neokanamycin or lac 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 to R. 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 to R. eutropha the ability to grow in the presence of glucose. The best growth was observed with a recombinant R. eutropha strain harboring plasmid pBBR1MCS-2::P(nk)::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 of R. 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.

Dataset Information

Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16.

Publications

Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16.

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