Dinglasan Jaime Lorenzo N, Doktycz Mitchel J
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Graduate School of Genome Science and Technology, University of Tennessee-Knoxville, Knoxville, TN, USA.
Synth Biol (Oxf). 2023 Apr 17;8(1):ysad007. doi: 10.1093/synbio/ysad007. eCollection 2023.
Cell-free systems can expedite the design and implementation of biomanufacturing processes by bypassing troublesome requirements associated with the use of live cells. In particular, the lack of survival objectives and the open nature of cell-free reactions afford engineering approaches that allow purposeful direction of metabolic flux. The use of lysate-based systems to produce desired small molecules can result in competitive titers and productivities when compared to their cell-based counterparts. However, pathway crosstalk within endogenous lysate metabolism can compromise conversion yields by diverting carbon flow away from desired products. Here, the 'block-push-pull' concept of conventional cell-based metabolic engineering was adapted to develop a cell-free approach that efficiently directs carbon flow in lysates from glucose and toward endogenous ethanol synthesis. The approach is readily adaptable, is relatively rapid and allows for the manipulation of central metabolism in cell extracts. In implementing this approach, a block strategy is first optimized, enabling selective enzyme removal from the lysate to the point of eliminating by-product-forming activity while channeling flux through the target pathway. This is complemented with cell-free metabolic engineering methods that manipulate the lysate proteome and reaction environment to push through bottlenecks and pull flux toward ethanol. The approach incorporating these block, push and pull strategies maximized the glucose-to-ethanol conversion in an lysate that initially had low ethanologenic potential. A 10-fold improvement in the percent yield is demonstrated. To our knowledge, this is the first report of successfully rewiring lysate carbon flux without source strain optimization and completely transforming the consumed input substrate to a desired output product in a lysate-based, cell-free system.
无细胞系统可以通过绕过与使用活细胞相关的麻烦要求，加快生物制造过程的设计和实施。特别是，无细胞反应缺乏生存目标且具有开放性，这为工程方法提供了可能，使其能够有目的地引导代谢通量。与基于细胞的系统相比，使用基于裂解物的系统生产所需小分子可产生具有竞争力的滴度和生产率。然而，内源性裂解物代谢中的途径串扰可能会通过使碳流从所需产物转移而损害转化产率。在此，传统的基于细胞的代谢工程的“阻断-推动-拉动”概念被应用于开发一种无细胞方法，该方法能有效地引导裂解物中的碳流从葡萄糖流向内源性乙醇合成。该方法易于调整，相对快速，并且允许对细胞提取物中的中心代谢进行操作。在实施此方法时，首先优化阻断策略，从裂解物中选择性去除酶，直至消除形成副产物的活性，同时使通量通过目标途径。这辅以无细胞代谢工程方法，这些方法可操纵裂解物蛋白质组和反应环境以突破瓶颈并将通量拉向乙醇。结合这些阻断、推动和拉动策略的方法在最初具有低产乙醇潜力的裂解物中使葡萄糖到乙醇的转化率最大化。产率百分比提高了10倍。据我们所知，这是首次在基于裂解物的无细胞系统中成功重新布线裂解物碳通量而无需优化源菌株，并将消耗的输入底物完全转化为所需输出产物的报告。
