Department of Bioengineering, California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720.
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158.
Proc Natl Acad Sci U S A. 2021 Mar 23;118(12). doi: 10.1073/pnas.2017719118.
The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.
组合 DNA 文库的随机化和筛选是一种强大的技术，可以用于理解序列-功能关系并优化生物合成途径。虽然很难先验地预测哪些序列组合编码功能单元，但通常可以省略那些会增加文库大小和筛选工作量的不需要的组合。然而，当需要对序列空间进行复杂的扫描时，定义文库的生成就变得困难了。为了克服这一挑战，我们设计了一种混合阀和液滴式微流控系统，该系统可以在皮升级别的液滴中确定性地组装 DNA 片段，从而减少试剂消耗和偏差。使用该系统，我们构建了一个基于细菌 CpxA 的工程化组氨酸激酶 (HK) 的组合文库。我们的文库编码了设计的跨膜 (TM) 结构域，这些结构域调节 CpxA 的细胞质结构域的活性以及位于催化结构域附近的结构上较远的“S 螺旋”的变体。我们发现 S 螺旋设定了一个基本活性，进一步由 TM 结构域调节。令人惊讶的是，我们还发现，给定的 TM 基序可以对不同 S-螺旋变体的催化活性产生相反的影响。我们得出的结论是，中间的 HAMP 结构域被动地传递信号，并根据相邻结构域的细微变化来塑造信号响应。这种灵活性使 HK 能够根据不断变化的进化压力而产生丰富的功能输出。
