Cupriavidus necator H16 is one of the most researched carbon dioxide (CO₂)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO₂ utilising precursors derived from the CBB cycle.

Cupriavidus necator H16 是研究最多的二氧化碳 (CO ₂ ) 固定细菌之一。它可以以聚合物聚羟基丁酸酯的形式储存碳，并在自养条件下通过需氧氢氧化产生能量，使C. necator 成为利用废气生物生产增值化合物的理想底盘。然而，尽管它具有巨大的潜力，但C. necator用于化学物质的自养生物合成的实验证据是有限的。在这里，我们对C. necator进行了基因工程，用于高水平的de novo直接从 Calvin-Benson-Bassham (CBB) 循环中间体生物合成工业相关的糖醇甘露醇。为了确定C. necator中的最佳甘露醇生产条件，甘露醇响应生物传感器用于筛选单功能和双功能甘露醇 1-磷酸脱氢酶 (MtlDs) 和甘露醇 1-磷酸磷酸酶 (M1Ps)。我们发现来自褐藻 Ectocarpus silculosus 的 MtlD/M1P在异养生长条件下总体表现最好，并被选择进行染色体整合。因此，重组C. necator的自养发酵产生高达 3.9 g/L 的甘露醇，与使用重组蓝细菌的甘露醇生物合成相比有了实质性的改进。重要的是，我们证明在稳定生长期开始时，几乎 100% 的碳可以通过 3-磷酸甘油醛和 6-磷酸果糖中间体从 CBB 循环进入甘露醇。该研究首次强调了C. necator利用源自​​ CBB 循环的前体从 CO ₂生成糖醇的潜力。