Buntru Matthias, Hahnengress Nils, Croon Alexander, Schillberg Stefan
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
Institute of Phytopathology, Justus Liebig University, Giessen, Germany.
Front Plant Sci. 2022 Jan 28;12:794999. doi: 10.3389/fpls.2021.794999. eCollection 2021.
Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot transcription-translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 μg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.
无细胞表达系统能够在数小时或数天内生产蛋白质和代谢产物。去除细胞环境同时保留蛋白质生物合成装置提供了一个开放系统，该系统通过表达不同数量和组合的酶来安装和优化代谢途径。这有利于合成在基于细胞的系统中难以生产的次生代谢产物，因为它们对宿主细胞有毒或会立即转化为下游产物。最近，我们开发了一种源自烟草BY-2细胞悬浮培养物的无细胞裂解物用于生产重组蛋白。该系统具有显著的生产能力，在一锅法转录-翻译反应中产量高达3 mg/mL，并且包含高活性的能量和辅因子再生途径。在此，我们首次证明BY-2无细胞裂解物也能高效生产几类次生代谢产物。作为案例研究，我们合成了番茄红素、靛玉红、甜菜红素和甜菜黄素，它们在食品、化妆品、纺织和制药行业中都有用途。通过共表达多达三种代谢酶实现了生产。对于所有这四种产物，我们都获得了中等到高产率。然而，甜菜红素的产量（555 μg/mL）非常突出，比酵母细胞中报道的水平高出30多倍。我们的结果表明，BY-2无细胞裂解物不仅适用于代谢途径的验证和优化，还适用于中小规模次生代谢产物的高效生产。
