Department of Molecular Sciences, Macquarie University, Sydney, Australia.
Department of Molecular Sciences, Macquarie University, Sydney, Australia; Biomolecular Discovery and Design Research Centre, Macquarie University, Sydney, Australia.
Biotechnol Adv. 2019 Jan-Feb;37(1):91-108. doi: 10.1016/j.biotechadv.2018.11.007. Epub 2018 Dec 3.
Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.
单一酶系统或工程微生物宿主已被使用了几十年，但将多个酶组装到无细胞合成途径中的概念是相对较新的发展。这种合成概念的广泛可能性使其成为生物制造精细和平台化学品、药物和生物燃料的快速发展且具有潜在高影响力的领域。然而，将单个酶反应转化为无细胞多酶途径并非易事。实际上，酶途径的动力学可能非常不足，并且多种酶的产生会给过程的经济性带来很大的负担。我们在这里检查了设计合成途径的策略，并提请注意底物、酶和辅因子再生系统的要求，以提高无细胞生物催化的有效性和可持续性。此外，我们还评论了固定化多酶途径成员的方法，以增强系统的生存能力。最后，我们专注于整合工具的最新发展，如计算机途径建模和高通量通量分析，旨在加强它们在无细胞生物催化途径未来的生物制造中的不可或缺的作用。
