Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany.
Appl Environ Microbiol. 2011 Feb;77(4):1325-34. doi: 10.1128/AEM.01977-10. Epub 2010 Dec 17.
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.
革兰氏阴性兼性化能自养细菌罗尔斯通氏菌（Ralstonia eutropha）菌株 H16 以其碳水化合物利用范围狭窄而闻名，这限制了其在生物技术生产聚羟基烷酸酯和可能利用可再生资源生产其他产品中的应用。为了拓宽其底物利用范围，即仅限于果糖、N-乙酰氨基葡萄糖和葡萄糖酸盐的碳水化合物和相关化合物，我们对菌株 H16 进行了工程改造，使其能够将甘露糖和葡萄糖用作生长的唯一碳源。来自运动发酵单胞菌（Zymomonas mobilis）的促进扩散蛋白（glf）基因和来自大肠杆菌（Escherichia coli）的葡糖激酶（glk）、甘露糖果糖激酶（mak）和磷酸甘露糖异构酶（pmi）基因，分别在新卡那霉素或 lac 启动子的控制下，在含有 broad-host-range 载体的 R. eutropha 菌株 H16 中组成型表达。携带 pBBR1MCS-3::glf::mak::pmi 或 pBBR1MCS-3::glf::pmi 的重组菌株能够在甘露糖上生长，而 pBBR1MCS-3::glf::mak 和 pBBR1MCS-3::glf 则不能赋予 R. eutropha 利用甘露糖作为碳源的能力。携带 pBBR1MCS-3::glf::pmi 的重组菌株在甘露糖上的生长速度比携带 pBBR1MCS-3::glf::mak::pmi 的重组菌株慢。这些数据表明，磷酸甘露糖异构酶对于将甘露糖-6-磷酸转化为果糖-6-磷酸以随后通过 Entner-Doudoroff 途径进行分解代谢是必需的。此外，所有质粒还赋予了 R. eutropha 在葡萄糖存在下生长的能力。带有质粒 pBBR1MCS-2::P(nk)::glk::glf 的重组 R. eutropha 菌株表现出最佳生长。此外，在转录和蛋白质水平以及通过测量甘露糖果糖激酶（0.622±0.063 U mg(-1)）、磷酸甘露糖异构酶（0.251±0.017 U mg(-1)）和葡糖激酶（0.518±0.040 U mg(-1)）的活性，证明了相应酶的表达。在 1%（wt/vol）葡萄糖或甘露糖作为唯一碳源的情况下，重组罗尔斯通氏菌（Ralstonia eutropha）菌株的细胞合成聚（3-羟基丁酸酯）至约 65%至 67%（wt/wt）的细胞干重。
