Instituto Superior de Agronomia (ISA), Centro de Botânica Aplicada à Agricultura (CBAA), Calçada da Tapada, 1349-017 Lisbon, Portugal.
Appl Microbiol Biotechnol. 2013 Jun;97(12):5635-43. doi: 10.1007/s00253-013-4920-z. Epub 2013 May 7.
Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae.
氢气（H2）是一种理想的燃料，具有高热值变化和缺乏温室效应的特点。这种生物燃料可以通过微藻中的质子还原反应产生，由氢化酶催化生成氢气。氢化酶所需的还原能力的主要竞争物是卡尔文循环，而 Rubisco 在其中起着关键作用。本研究旨在提高氢气产量，使用 Rubisco 水平、活性和稳定性降低的工程化衣藻作为研究基础。在缺硫或补硫的 Tris/乙酸盐/磷酸盐培养基中进行代谢工程突变细胞的生化监测，均在缺氧条件下进行。测量了光合作用活性、最大光化学效率、叶绿素和蛋白质水平。此外，还研究了 Rubisco、氢化酶、D1 和 Lhcb 的表达，并对 H2 进行了定量分析。在实验开始时，Rubisco 增加，随后强烈降解。Lhcb 蛋白在最初的 24 到 48 小时内表现出单体同工型，而 D1 在缺硫时表现出敏感性。Rubisco 突变体与对照相比，O2 释放显著减少。尽管缺硫培养基比其完全培养基更适合于 H2 生产，但在低-中度光照条件下，密封的 Rubisco 突变细胞补硫培养中也观察到了氢气释放。特别是，Rubisco 突变体 Y67A 在相同条件下比野生型高出 10-15 倍的氢气产量，并且还表现出不同的代谢参数。这些结果表明，Rubisco 是提高工程化微藻产氢率的有前途的目标。
