Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. Here, we show that a strategy of terminal differentiation improves the evolutionary stability of burdensome functions in a general manner by realizing a reproductive and metabolic division of labor. To implement this strategy, we develop a genetic differentiation circuit in Escherichia coli using unidirectional integrase-recombination. With terminal differentiation, differentiated cells uniquely express burdensome functions driven by the orthogonal T7 RNA polymerase, but their capacity to proliferate is limited to prevent the propagation of advantageous loss-of-function mutations that inevitably occur. We demonstrate computationally and experimentally that terminal differentiation increases duration and yield of high-burden expression and that its evolutionary stability can be improved with strategic redundancy. Further, we show this strategy can even be applied to toxic functions. Overall, this study provides an effective, generalizable approach for protecting burdensome engineered functions from evolutionary degradation.

合成生物学、生物工程和计算的进步使我们能够快速可靠地对细胞进行编程，使其具有日益复杂和有用的功能。然而，由于我们设计细胞执行的功能通常会给细胞生长造成负担，因此它们可能会由于突变和自然选择的过程而迅速丧失。在这里，我们表明，终末分化策略通过实现生殖和代谢分工，以一般方式提高了繁重功能的进化稳定性。为了实施这一策略，我们使用单向整合酶重组在大肠杆菌中开发了遗传分化电路。通过终末分化，分化细胞独特地表达由正交 T7 RNA 聚合酶驱动的繁重功能，但它们的增殖能力受到限制，以防止不可避免地发生的有利功能丧失突变的传播。我们通过计算和实验证明，终末分化增加了高负荷表达的持续时间和产量，并且可以通过策略冗余来提高其进化稳定性。此外，我们表明这种策略甚至可以应用于毒性功能。总的来说，这项研究提供了一种有效的、可推广的方法来保护繁重的工程功能免遭进化退化。