BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, The University of Nottingham, Nottingham, NG7 2RD, United Kingdom; Present address: Manchester Centre for Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, United Kingdom.
BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, The University of Nottingham, Nottingham, NG7 2RD, United Kingdom.
Metab Eng. 2022 Jul;72:24-34. doi: 10.1016/j.ymben.2022.02.003. Epub 2022 Feb 8.
Cupriavidus necator H16 is one of the most researched carbon dioxide (CO)-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 CO utilising precursors derived from the CBB cycle.
铜绿假单胞菌 H16 是研究最多的二氧化碳 (CO) 固定细菌之一。它可以将聚合物聚羟基丁酸酯形式的碳储存起来，并在自养氢氧化条件下通过需氧氢氧化产生能量，使铜绿假单胞菌成为从废气中生物生产有价值化合物的理想底盘。然而，尽管它具有巨大的潜力，但铜绿假单胞菌用于化学物质自养生物合成的实验证据有限。在这里，我们通过基因工程使铜绿假单胞菌能够从卡尔文-本森-巴斯汉姆 (CBB) 循环中间体中直接高水平从头合成工业相关的糖醇甘露醇。为了在铜绿假单胞菌中确定最佳甘露醇生产条件，应用甘露醇响应生物传感器筛选单功能和双功能甘露醇 1-磷酸脱氢酶 (MtlD) 和甘露醇 1-磷酸磷酸酶 (M1P)。我们发现，褐藻褐藻 Ectocarpus siliculosus 的 MtlD/M1P 在异养生长条件下总体表现最佳，并被选择进行染色体整合。因此，重组铜绿假单胞菌的自养发酵产生了高达 3.9 g/L 的甘露醇，比使用重组蓝藻的甘露醇生物合成有了显著提高。重要的是，我们证明在静止生长阶段开始时，近 100%的碳可以通过甘油醛 3-磷酸和果糖 6-磷酸中间体从 CBB 循环定向进入甘露醇。这项研究首次强调了铜绿假单胞菌利用源自 CBB 循环的前体从 CO 生成糖醇的潜力。
