Building a highly efficient Streptomyces super-chassis for secondary metabolite production by reprogramming naturally-evolved multifaceted shifts.

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae. We initially identified and characterized a set of multiplex targets (afsQ1, bldD, bldA, and miaA) that contribute to SM (jadomycin) production when subjected to ethanol shock. Subsequently, we developed an ethanol-induced orthogonal amplification system (EOAS), enabling dynamic and precise control over targets. Ultimately, we integrated these multiplex targets into functional units governed by the EOAS, generating a universal and plug-and-play Streptomyces super-chassis. In addition to achieving the unprecedented titer and yield of jadomycin B, we also evidenced the potential of this super-chassis for production of diverse heterologous SMs, including antibiotic oxytetracycline, anticancer drug doxorubicins, agricultural herbicide thaxtomin A, and plant growth regulator guvermectin, all with the yields of >10 mg/g glucose in a simple mineral medium. Given that the production of SMs all required complexed medium and the cognate yields were usually much lower, our achievement of using a universal super-chassis and engineering pipeline in a simple mineral medium is promising for convenient heterologous production of SMs.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool-through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy-we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

In vitro allosteric transcription factor-based biosensing.

A biosensor is an analytical device that converts a biological response into a measurable output signal. Bacterial allosteric transcription factors (aTFs) have been utilized as a novel class of recognition elements for in vitro biosensing, which circumvents the limitations of aTF-based whole-cell biosensors (WCBs) and helps to meet the increasing requirement of small-molecule biosensors for diverse applications. In this review, we summarize the recent advances related to the configuration of aTF-based biosensors in vitro. Particularly, we evaluate the advantages of aTFs for in vitro biosensing and highlight their great potential for the establishment of robust and easy-to-implement biosensing strategies. We argue that key technical innovations and generalizable workflows will enhance the pipeline for facile construction of diverse aTF-based small-molecule biosensors.

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering.

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/µg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.