Harnessing Interactional Sensory Genes for Rationally Reprogramming Chaotic Metabolism

Abstract

Rationally controlling cellular metabolism is of great importance but challenging owing to its highly complex and chaotic nature. Natural existing sensory proteins like histidine kinases (HKs) are understood as “sensitive nodes” of biological networks that can trigger disruptive metabolic reprogramming (MRP) upon perceiving environmental fluctuation. Here, the “sensitive node” genes were adopted to devise a global MRP platform consisting of a CRISPR interference-mediated dual-gene combinational knockdown toolbox and survivorship-based metabolic interaction decoding algorithm. The platform allows users to decode the interfering effects of n × n gene pairs while only requiring the synthesis of n pairs of primers. A total of 35 HK genes and 24 glycine metabolic genes were selected as the targets to determine the effectiveness of our platform in a Vibrio sp. FA2. The platform was applied to decode the interfering impact of HKs on antibiotic resistance in strain FA2. A pattern of combined knockdown of HK genes ( sasA_8 and 04288 ) was demonstrated to be capable of reducing antibiotic resistance of Vibrio by 108-fold. Patterns of combined knockdown of glycine pathway genes (e.g., gcvT and ltaE ) and several HK genes (e.g., cpxA and btsS ) were also revealed to increase glycine production. Our platform may enable an efficient and rational approach for global MRP based on the elucidation of high-order gene interactions. A web-based 1-stop service ( https://smrp.sjtu.edu.cn ) is also provided to simplify the implementation of this smart strategy in a broad range of cells.

Genome-scale CRISPRi screening: A powerful tool in engineering microbiology

“…The same group developed a CRISPR-dCas9-based multiple gene knockdown method in V. alginolyticus FA2 and created two small dual-gRNA libraries targeting 35 × 35 dual histidine kinase genes (1225 combinations) and 24 × 24 dual glycine-pathway genes (576 combinations), respectively. The dual-CRISPRi platform was used to decipher the interfering effect of histidine kinases on antibiotic resistance and that of glycine pathway genes on glycine production [84] .…”

Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code

“…We chose the SHKs as the design subject for both their biological significance and technical advantages. Histidine kinases are the most intensively studied and multitudinous membrane receptors in microorganisms, which control the greatest variety of cellular responses 23 and are promising to become new antimicrobial drug targets 19 , 24 , 25 . Importantly, their highly modular structure and signaling pattern allows easy and explicit examination of the functional effects introduced by design.…”

High-Performance Production of N-Acetyl-d-Neuraminic Acid with Whole Cells of Fast-Growing Vibrio natriegens via a Thermal Strategy

“…However, low efficiency remains ubiquitous in bioproduction systems. Developing high-performance production with biotechnology is also hindered by the chaotic nature of cellular metabolism, which consists of thousands of enzymes, metabolites, and their interactions, posing significant challenges to achieving high titer, productivity, and purity . Traditional solutions rely mainly on genetic manipulations including gene-knockout and gene-knockdown with genetic tools such as homologous recombination and CRISPR-Cas-mediated gene editing. , However, dozens of genes usually need to be simultaneously targeted, and uncharacterized reactions often exist, thus additionally increasing the execution difficulties .…”