Pouzet Sylvain, Cruz-Ramón Jessica, Le Bec Matthias, Cordier Céline, Banderas Alvaro, Barral Simon, Castaño-Cerezo Sara, Lautier Thomas, Truan Gilles, Hersen Pascal
Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France.
Toulouse Biotechnology Institute, Université de Toulouse, Centre National de la Recherche Scientifique (CNRS), Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Institut National des Sciences Appliquées (INSA), Toulouse, France.
Front Bioeng Biotechnol. 2023 Feb 6;11:1085268. doi: 10.3389/fbioe.2023.1085268. eCollection 2023.
Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximizing yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase/phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale.
光遗传学作为一种精确控制微生物细胞工厂中基因回路的宝贵工具而兴起。光控有望优化生物生产方法并实现产量最大化，但其在菌株开发过程的不同步骤以及不同培养规模下的应用仍具有挑战性。在本研究中，我们旨在利用光遗传学控制β-胡萝卜素的生物合成，并研究其在不同培养规模下的性能表现。我们构建了四个实验室规模的照明装置，每个装置处理不同的培养体积，且各自具有特定的照明特性和培养条件。我们评估了不同装置间的光遗传学激活和β-胡萝卜素产量，并分别对它们进行了优化。然后，我们将光遗传学诱导与β-胡萝卜素生产相结合，构建了一种光诱导型β-胡萝卜素生产菌株。这是通过将双功能番茄红素环化酶/八氢番茄红素合酶CrtYB的转录置于由EL222-VP16光激活转录因子调控的pC120光遗传学启动子的控制之下实现的，而其他类胡萝卜素生成酶（CrtI、CrtE、tHMG）则组成型表达。我们发现，光照、培养体积和振荡对不同装置间的光遗传学激活和β-胡萝卜素生产有着不同的影响。这使我们能够确定在每个装置中实现光诱导β-胡萝卜素产量最大化的最佳培养条件。我们的研究例证了在不同实验室规模的装置中扩大光遗传学应用的关键所在，并揭示了光遗传学控制与代谢途径效率之间的相互作用和潜在冲突。作为一个通用原则，我们建议在将系统的两个组件组合成光遗传学生产菌株之前，先分别对它们进行优化，以避免大量的故障排除工作。我们预计，我们的研究结果有助于设计菌株和装置，最终可能促成更大规模的系统，从而推动光遗传学迈向工业规模。
