Lupacchini Sara, Stauder Ron, Opel Franz, Klähn Stephan, Schmid Andreas, Bühler Bruno, Toepel Jörg
Department of Microbial Biotechnology, Helmholtz Centre for Environmental Research-UFZ, Permoserstrasse 15, 04318, Leipzig, Germany.
Department of Solar Materials Biotechnology, Helmholtz Centre for Environmental Research-UFZ, Permoserstrasse 15, 04318, Leipzig, Germany.
Biotechnol Biofuels Bioprod. 2025 Mar 28;18(1):41. doi: 10.1186/s13068-025-02634-5.
Cyanobacteria bear great biotechnological potential as photosynthetic cell factories. In particular, hydrogenases are promising with respect to light-driven H production as well as H-driven redox biocatalysis. Their utilization relies on effective strain design as well as a balanced synthesis and maturation of heterologous enzymes. In a previous study, the soluble O-tolerant hydrogenase complex from Cupriavidus necator (CnSH) could be introduced into the model cyanobacterium Synechocystis sp. PCC 6803. Due to its O-tolerance, it was indeed active under photoautotrophic growth conditions. However, the specific activity was rather low indicating that further engineering is required, for which we followed a two-step approach. First, we optimized the CnSH multigene expression in Synechocystis by applying different regulatory elements. Although corresponding protein levels and specific CnSH activity increased, the apparent rise in enzyme levels did not fully translate into activity increase. Second, the entire set of hyp genes, encoding CnSH maturases, was co-expressed in Synechocystis to investigate, if CnSH maturation was limiting. Indeed, the native CnSH maturation apparatus promoted functional CnSH synthesis, enabling a threefold higher H oxidation activity compared to the parental strain. Our results suggest that a fine balance between heterologous hydrogenase and maturase expression is required to ensure high specific activity over an extended time period.
蓝细菌作为光合细胞工厂具有巨大的生物技术潜力。特别是，氢化酶在光驱动产氢以及氢驱动的氧化还原生物催化方面很有前景。它们的利用依赖于有效的菌株设计以及异源酶的平衡合成和成熟。在之前的一项研究中，来自食酸铜绿假单胞菌（CnSH）的可溶性耐氧氢化酶复合物可以被引入模式蓝细菌聚球藻属PCC 6803中。由于其耐氧性，它在光合自养生长条件下确实具有活性。然而，比活性相当低，这表明需要进一步的工程改造，为此我们采用了两步法。首先，我们通过应用不同的调控元件优化了聚球藻中CnSH多基因的表达。尽管相应的蛋白质水平和特定的CnSH活性有所增加，但酶水平的明显上升并没有完全转化为活性的增加。其次，将编码CnSH成熟酶的整套hyp基因在聚球藻中共表达，以研究CnSH的成熟是否受到限制。事实上，天然的CnSH成熟装置促进了功能性CnSH的合成，与亲本菌株相比，使氢氧化活性提高了三倍。我们的结果表明，为了在较长时间内确保高比活性，需要在异源氢化酶和成熟酶表达之间实现精细平衡。
