A Simple and Effective Strategy for the Development of Robust Promoter-Centric Gene Expression Tools.

Promoter-centric genetic tools play a crucial role in controlling gene expression for various applications, such as strain engineering and synthetic biology studies. Hence, a critical need persists for the development of robust gene expression tools. Streptomyces are well-known prolific producers of natural products and exceptional surrogate hosts for the production of high-value chemical compounds and enzymes. In this study, we reported a straightforward and effective strategy for the creation of potent gene expression tools. This was primarily achieved by introducing an additional -35-like motif upstream of the original -35 region of the promoter, coupled with the integration of a palindromic cis-element into the 5'-UTR region. This approach has generated a collection of robust constitutive and inducible gene expression tools tailored for Streptomyces. Of particular note, the fully activated oxytetracycline-inducible gene expression system containing an engineered kasOp* promoter (OK) exhibited nearly an order of magnitude greater activity compared to the well-established high-strength promoter kasOp* under the tested conditions, establishing itself as a powerful gene expression system for Streptomyces. This strategy is expected to be applicable in modifying various other promoters to acquire robust gene expression tools, as evidenced by the enhancement observed in the other two promoters, PL and P21 in this study. Moreover, the effectiveness of these tools has been demonstrated through the augmented production of transglutaminase and daptomycin. The gene expression tools established in this study, alongside those anticipated in forthcoming research, are positioned to markedly advance pathway engineering and synthetic biology investigations in Streptomyces and other microbial strains.

ThermoFinder: A sequence-based thermophilic proteins prediction framework.

Thermophilic proteins are important for academic research and industrial processes, and various computational methods have been developed to identify and screen them. However, their performance has been limited due to the lack of high-quality labeled data and efficient models for representing protein. Here, we proposed a novel sequence-based thermophilic proteins prediction framework, called ThermoFinder. The results demonstrated that ThermoFinder outperforms previous state-of-the-art tools on two benchmark datasets, and feature ablation experiments confirmed the effectiveness of our approach. Additionally, ThermoFinder exhibited exceptional performance and consistency across two newly constructed datasets, one of these was specifically constructed for the regression-based prediction of temperature optimum values directly derived from protein sequences. The feature importance analysis, using shapley additive explanations, further validated the advantages of ThermoFinder. We believe that ThermoFinder will be a valuable and comprehensive framework for predicting thermophilic proteins, and we have made our model open source and available on Github at https://github.com/Luo-SynBioLab/ThermoFinder.

Elucidation of genes enhancing natural product biosynthesis through co-evolution analysis.

Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we proposed that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the 'coenzyme' category have been examined, including a gene cluster encoding for the cofactor pyrroloquinoline quinone. When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides an innovative engineering strategy for improving polyketide production and finding previously unidentified BGCs.

Engineering functional materials through bacteria-assisted living grafting.

Functionalizing materials with biomacromolecules such as enzymes has broad applications in biotechnology and biomedicine. Here, we introduce a grafting method mediated by living cells to functionalize materials. We use polymeric scaffolds to trap engineered bacteria and micron-sized particles with chemical groups serving as active sites for grafting. The bacteria synthesize the desired protein for grafting and autonomously lyse to release it. The released functional moieties are locally grafted onto the active sites, generating the materials engineered by living grafting (MELGs). MELGs are resilient to perturbations because of both the bonding and the regeneration of functional domains synthesized by living cells. The programmability of the bacteria enables us to fabricate MELGs that can respond to external input, decompose a pollutant, reconstitute synthetic pathways for natural product synthesis, and purify mismatched DNA. Our work establishes a bacteria-assisted grafting strategy to functionalize materials with a broad range of biological activities in an integrated, flexible, and modular manner. A record of this paper's transparent peer review process is included in the supplemental information.

Pathway Evolution Through a Bottlenecking-Debottlenecking Strategy and Machine Learning-Aided Flux Balancing.

The evolution of pathway enzymes enhances the biosynthesis of high-value chemicals, crucial for pharmaceutical, and agrochemical applications. However, unpredictable evolutionary landscapes of pathway genes often hinder successful evolution. Here, the presence of complex epistasis is identifued within the representative naringenin biosynthetic pathway enzymes, hampering straightforward directed evolution. Subsequently, a biofoundry-assisted strategy is developed for pathway bottlenecking and debottlenecking, enabling the parallel evolution of all pathway enzymes along a predictable evolutionary trajectory in six weeks. This study then utilizes a machine learning model, ProEnsemble, to further balance the pathway by optimizing the transcription of individual genes. The broad applicability of this strategy is demonstrated by constructing an Escherichia coli chassis with evolved and balanced pathway genes, resulting in 3.65 g L-1 naringenin. The optimized naringenin chassis also demonstrates enhanced production of other flavonoids. This approach can be readily adapted for any given number of enzymes in the specific metabolic pathway, paving the way for automated chassis construction in contemporary biofoundries.

A Semisynthesis Platform for the Efficient Production and Exploration of Didemnin-Based Drugs.

Plitidepsin (or dehydrodidemnin B), an approved anticancer drug, belongs to the didemnin family of cyclic depsipeptides, which are found in limited quantities in marine tunicate extracts. Herein, we introduce a new approach that integrates microbial and chemical synthesis to generate plitidepsin and its analogues. We screened a Tistrella strain library to identify a potent didemnin B producer, and then introduced a second copy of the didemnin biosynthetic gene cluster into its genome, resulting in a didemnin B titer of approximately 75 mg/L. Next, we developed two straightforward chemical strategies to convert didemnin B into plitidepsin, one of which involved a one-step synthetic route giving over 90 % overall yield. Furthermore, we synthesized 13 new didemnin derivatives and three didemnin probes, enabling research into structure-activity relationships and interactions between didemnin and proteins. Our study highlights the synergistic potential of biosynthesis and chemical synthesis in overcoming the challenge of producing complex natural products sustainably and at scale.

The effects of non-invasive brain stimulation on disorder of consciousness in patients with brain injury: A systematic review and meta-analysis of randomized controlled trial.

INTRODUCTION: Disorders of consciousness (DOC) result from neural system injury and manifest as changes in arousal or awareness. This systematic review and meta-analysis aimed to investigate the therapeutic effects of non-invasive brain stimulation (NIBS) techniques, such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), on consciousness dysfunction in patients with brain disorders. METHODS: Literature was systematically searched in Medline, Embase, Cochrane database, Web of Science, EBSCO from inception to May 2023. Only randomized controlled trial with NIBS as an intervention and participants with DOC were included. RESULTS: A total of 7 studies with 313 participants were included for meta-analysis. Compared with sham- or placebo-stimulation, NIBS can improve the Coma Recovery Scale-Revised scores significantly (mean difference [MD] = 1.96, 95 % confidence interval [CI] = [1.49; 2.43], P <.0001). CONCLUSION: NIBS has a significant positive effect in enhancing the symptoms of DOC. Nevertheless, it is imperative for further investigations comprising high-quality research designs and larger sample sizes in order to comprehensively elucidate the effects of NIBS techniques on diverse targets of stimulation within the population of individuals suffering from DOC.