Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces

Nucleic Acids Research
Jinzhong TianWeihong Jiang

Abstract

Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growt...Continue Reading

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Citations

Jan 26, 2021·Current Opinion in Biotechnology·Melissa M MitchlerGavin J Williams
Feb 11, 2021·Applied Microbiology and Biotechnology·Yuqing LiuXiangjing Wang
Apr 7, 2021·Biotechnology Advances·Youxiang Liang, Huimin Yu
Sep 14, 2021·Microbiology·Agustina UndabarrenaJana K Schniete

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Methods Mentioned

BETA
biosensor
PCR
Protein Assay

Software Mentioned

CRISPy
BLASTp
EQCi

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