California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley, California 94158, USA.
Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, USA.
Nat Commun. 2017 Apr 26;8:15128. doi: 10.1038/ncomms15128.
Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.
嵌入宿主细胞的合成回路与细胞过程竞争有限的细胞内资源。在这里，我们展示了在宿主细胞的转录组被序列依赖性内切酶 MazF 完全降解后，如何通过将细胞资源集中到一个合成回路中来提高产量。通过对基因序列进行重新编码以消除识别位点，同时保留氨基酸序列，使靶基因免受 MazF 活性的影响。使用这种全基因组规模的控制策略，保护荧光报告基因的表达和高价值代谢物的通量得到了显著增强。蛋白质组学测量发现需要保护一种宿主因子来改善资源再分配活性。计算模型表明，MazF mRNA 衰减反馈回路能够在最优工作状态下对 MazF 进行比例控制。MazF 诱导细胞的转录组分析阐明了转录物丰度的动态变化，并发现了调控设计元素。总之，我们的研究结果表明，对细胞资源分配的操纵是合成回路设计的一个关键控制参数。
