Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology.

Bacteria exhibit a rich repertoire of RNA molecules that intricately regulate gene expression at multiple hierarchical levels, including small RNAs (sRNAs), riboswitches, and antisense RNAs. Notably, the majority of these regulatory RNAs lack or have limited protein-coding capacity but play pivotal roles in orchestrating gene expression by modulating transcription, post-transcription or translation processes. Leveraging and redesigning these regulatory RNA elements have emerged as pivotal strategies in the domains of metabolic engineering and synthetic biology. While previous investigations predominantly focused on delineating the roles of regulatory RNA in Gram-negative bacterial models such as Escherichia coli and Salmonella enterica, this review aims to summarize the mechanisms and functionalities of endogenous regulatory RNAs inherent to typical Gram-positive bacteria, notably Bacillus subtilis. Furthermore, we explore the engineering and practical applications of these regulatory RNA elements in the arena of synthetic biology, employing B. subtilis as a foundational chassis.

Coordinated optimization of the polymerization and transportation processes to enhance the yield of exopolysaccharide heparosan.

Heparosan as the precursor for heparin biosynthesis has attracted intensive attention while Escherichia coli Nissle 1917 (EcN) has been applied as a chassis for heparosan biosynthesis. Here, after uncovering the pivotal role of KfiB in heparosan biosynthesis, we further demonstrate KfiB is involved in facilitating KpsT to translocate the nascent heparosan polysaccharide chain. As a result, an artificial expression cassette KfiACB was constructed with optimized RBS elements, resulting in 0.77 g/L heparosan in shake flask culture. Moreover, in view of the intracellular accumulation of heparosan, we further investigated the effects of overexpression of the ABC transport system proteins on heparosan biosynthesis. By co-overexpressing KfiACB with KpsTME, the heparosan production in flask cultures was increased to 1.03 g/L with an extracellular concentration of 0.96 g/L. Eventually, the engineered strain EcN/pET-kfiACB3-galU-kfiD-glmM/pCDF-kpsTME produced 12.2 g/L heparosan in 5-L fed-batch cultures while the extracellular heparosan was about 11.2 g/L. The results demonstrate the high-efficiency of the strategy for co-optimizing the polymerization and transportation for heparosan biosynthesis. Moreover, this strategy should be also available for enhancing the production of other polysaccharides.

Construction of 5-Aminolevulinic Acid Microbial Cell Factories through Identification of Novel Synthases and Metabolic Pathway Screens and Transporters.

5-Aminolevulinic acid (5-ALA) plays a pivotal role in the biosynthesis of heme and chlorophyll and has garnered great attention for its agricultural applications. This study explores the multifaceted construction of 5-ALA microbial cell factories. Evolutionary analysis-guided screening identified a novel 5-ALA synthase from Sphingobium amiense as the best synthase. An sRNA library facilitated global gene screening that demonstrated that trpC and ilvA repression enhanced 5-ALA production by 74.3% and 102%, respectively. Subsequently, efflux of 5-ALA by the transporter Gdx increased 5-ALA biosynthesis by 25.7%. To mitigate oxidative toxicity, DNA-binding proteins from starved cells were employed, enhancing cell density and 5-ALA titer by 21.1 and 4.1%, respectively. Combining these strategies resulted in an Escherichia coli strain that produced 5-ALA to 1.51 g·L-1 in shake flask experiments and 6.19 g·L-1 through fed-batch fermentation. This study broadens the repertoire of available 5-ALA synthases and transporters and provides a new platform for optimizing 5-ALA bioproduction.

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities.

Gene regulation by trans-acting small RNAs (sRNAs) has considerable advantages over other gene regulation strategies. However, synthetic sRNAs mainly take natural sRNAs (MicC or SgrS) as backbones and comprise three functional elements folding into two or more stem-loop structures: an mRNA base pairing region, an Hfq-binding structure, and a rho-independent terminator. Due to limited numbers of natural sRNAs and complicated backbone structures, synthetic sRNAs suffer from low activity programmability and poor structural modularity. Moreover, natural sRNA backbone sequences may increase the possibility of unwanted recombination. Here, we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs (ssl-sRNAs), which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons. Particularly, ssl-sRNA requires no independent Hfq-binding structure, as the polyU tail fulfills the roles of binding Hfq. A thermodynamic-based scoring model and a web server sslRNAD (http://www.kangzlab.cn/) were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities. ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs. The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis. A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries. As examples, the E. coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs. ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis.

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B. subtilis are still not fully explored. Here, a bottom-up approach is proposed for designing artificial trans-acting sRNAs. By engineering the intrinsic sRNA SR6, a minimized core scaffold structure consisting of an 8 bp stem, a 4 nt loop, and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity (83%). Moreover, we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli (above 80%). A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences, which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence. By applying these auto-designed sRNAs, we rapidly modified the cell morphology and biofilm formation, and regulated metabolic flux toward acetoin biosynthesis. This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B. subtilis and E. coli but also enhanced our understanding of trans-acting sRNAs.

Construction of Osmotic Pressure Responsive Vacuole-like Bacterial Organelles with Capsular Polysaccharides as Building Blocks.

Many artificial organelles or subcellular compartments have been developed to tune gene expression, regulate metabolic pathways, or endow new cell functions. Most of these organelles or compartments were built using proteins or nucleic acids as building blocks. In this study, we demonstrated that capsular polysaccharide (CPS) retained inside bacteria cytosol assembled into mechanically stable CPS compartments. The CPS compartments were able to accommodate and release protein molecules but not lipids or nucleic acids. Intriguingly, we found that the CPS compartment size responds to osmotic stress and this compartment improves cell survival under high osmotic pressures, which was similar to the vacuole functionalities. By fine-tuning the synthesis and degradation of CPS with osmotic stress-responsive promoters, we achieved dynamic regulation of the size of CPS compartments and the host cells in response to external osmotic stress. Our results shed new light on developing prokaryotic artificial organelles with carbohydrate macromolecules.

Assessment of the reliability and quality of breast cancer related videos on TikTok and Bilibili: cross-sectional study in China.

Background: As the most common malignant tumor in the world, breast cancer also brings a huge disease burden to China. Ordinary people are increasingly inclined to use the Internet, especially video social platforms, as a source of health information. Educating the public to obtain correct information is important to reduce the incidence of breast cancer and improve the prognosis. However, the quality and reliability of breast cancer-related video content have not been fully studied. Objective: This study aims to evaluate the quality of the information of breast cancer-related videos on TikTok and Bilibili video sharing platforms and factors related to video quality. Methods: We collected the top 100 videos about breast cancer on TikTok and Bilibili, respectively. Categorize videos according to video source and video content. Video quality and reliability were assessed using Global Quality Score (GQS) and modified DISCERN (mDISCERN) tools. We also analyzed the correlation between video quality and video likes, comments, saves, and shares. Results: Although the quality and reliability of Bilibili's breast cancer videos were higher than TikTok (p = 0.002 and p = 0.001, respectively), the video quality of both video sharing platforms was not satisfactory, with a median GQS scores of 2.00 and 3.00 and mDISCERN scores of 1.00 and 2.00, respectively. In general, the quality and reliability of videos released by medical practitioners were higher than those of non-medical practitioners, and the quality and reliability of videos covering disease-related knowledge were higher than those of news reports (all p < 0.001). Among medical practitioners, the quality of videos uploaded by doctors in breast disease was significantly lower than that of doctors in other areas (p < 0.05). There was a significant positive correlation between video quality and duration (r = 0.240, p < 0.001), a weak negative correlation between video quality and likes (r = 0.191, p < 0.01), video quality and comments (r = 0.256, p < 0.001), video reliability and likes (r = 0.198, p < 0.001), video reliability and comments (r = 0.243, p < 0.01). Conclusion: Our study shows that the quality and reliability of breast cancer-related videos on TikTok and Bilibili are poor, and the overall quality is unsatisfactory. But videos uploaded by medical practitioners covering disease knowledge, prevention and treatment are of higher quality. Medical practitioners are encouraged to publish more high-quality videos, while video social platforms should formulate relevant policies to censor and supervise health education videos, so as to enable the public to obtain reliable health information.

Complete genome sequence of Exiguobacterium mexicanum A-EM, isolated from seafloor hydrothermal vents in Atlantic Ocean.

Exiguobacterium mexicanum A-EM was isolated from seafloor hydrothermal vents(Caifan field, 14.0S 14.4 W) and was shown to degrade toxins and contaminants. Here, we present the complete genome sequence of A-EM, consisting of 2,412,492 bp, with a GC content of 53.16%. A-EM sequence contains genes encoding enzymes that degrade toxins and contaminants. Complete genome sequence of the strain A-EM can further provide insights into microbial adaption to the seafloor hydrothermal system and the genomic basis for the biotechnological application of strain A-EM as an efficient agent to degrade environmental contaminants.

Engineering artificial cross-species promoters with different transcriptional strengths.

As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (Psh) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.