Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum.

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO2) into multicarbon biochemicals. Genotype-phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic performance; however, systemic investigation on the fitness contribution of individual gene has been limited. Here, we report genome-scale CRISPR interference screening using 41,939 guide RNAs designed from the E. limosum genome, one of the model acetogenic species, where all genes were targeted for transcriptional suppression. We investigated the fitness contributions of 96% of the total genes identified, revealing the gene fitness and essentiality for heterotrophic and autotrophic metabolisms. Our data show that the Wood-Ljungdahl pathway, membrane regeneration, membrane protein biosynthesis, and butyrate synthesis are essential for autotrophic acetogenesis in E. limosum. Furthermore, we discovered genes that are repression targets that unbiasedly increased autotrophic growth rates fourfold and acetoin production 1.5-fold compared to the wild-type strain under CO2-H2 conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.

RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics.

RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites (differential RNA-Seq), transcript 3' ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies.

Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth.

Acetogenic bacteria use cellular redox energy to convert CO2 to acetate using the Wood-Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum The hybrid system converts CO2 into acetate without the need for additional energy sources, such as H2, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications.

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.

Functional cooperation of the glycine synthase-reductase and Wood-Ljungdahl pathways for autotrophic growth of Clostridium drakei.

Among CO2-fixing metabolic pathways in nature, the linear Wood-Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genome-scale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.

Development of highly characterized genetic bioparts for efficient gene expression in CO2-fixing Eubacterium limosum.

Acetogenic bacteria demonstrate industrial potential for utilizing carbon dioxide (CO2) for biochemical production using the Wood-Ljungdahl pathway. However, the metabolic engineering of acetogenic bacteria has been hampered by the limited number of available genetic bioparts for gene expression. Here, we integrated RNA sequencing, ribosome profiling, differential RNA sequencing, and RNA 3'-end sequencing results of Eubacterium limosum to establish genetic bioparts, such as promoters, 5' untranslated regions, and transcript terminators, to regulate transcriptional and translational expression of genes composing of biosynthetic pathways. In addition, a transformation method for the strain was developed to efficiently deliver the obtained genetic bioparts into cells, resulting in a transformation efficiency of 2.5 × 105 CFU/µg DNA. Using this method, the genetic bioparts were efficiently introduced, and their strengths were measured, which were then applied to optimize the heterologous expression of acetolactate synthase and acetolactate decarboxylase for non-native biochemical acetoin production. The strategy developed in this study is the first report on integrating multi-omics data for biopart development of CO2 or syngas utilizing acetogenic bacteria, which lays a foundation for the efficient production of biochemicals from CO2 or syngas as a carbon feedstock under autotrophic growth conditions.

Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design.

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. thetaiotaomicron is not a natural producer of butyrate, we heterologously expressed a butyrate biosynthetic pathway in the strain, which led to the production of butyrate at the final concentration of 12 mg/L in a rich medium. Further optimization of butyrate production was achieved by a round of metabolic engineering guided by an expanded genome-scale metabolic model (GEM) of B. thetaiotaomicron. The in silico knock-out simulation of the expanded model showed that pta and ldhD were the potent knock-out targets to enhance butyrate production. The maximum titer and specific productivity of butyrate in the pta-ldhD double knockout mutant increased by nearly 3.4 and 4.8 folds, respectively. To our knowledge, this is the first engineering attempt that enabled butyrate production from a non-butyrate producing commensal B. thetaiotaomicron. The study also highlights that B. thetaiotaomicron can serve as an effective strain for live microbial therapeutics in human.

Genome-scale analysis of Acetobacterium bakii reveals the cold adaptation of psychrotolerant acetogens by post-transcriptional regulation.

Acetogens synthesize acetyl-CoA via CO2 or CO fixation, producing organic compounds. Despite their ecological and industrial importance, their transcriptional and post-transcriptional regulation has not been systematically studied. With completion of the genome sequence of Acetobacterium bakii (4.28-Mb), we measured changes in the transcriptome of this psychrotolerant acetogen in response to temperature variations under autotrophic and heterotrophic growth conditions. Unexpectedly, acetogenesis genes were highly up-regulated at low temperatures under heterotrophic, as well as autotrophic, growth conditions. To mechanistically understand the transcriptional regulation of acetogenesis genes via changes in RNA secondary structures of 5'-untranslated regions (5'-UTR), the primary transcriptome was experimentally determined, and 1379 transcription start sites (TSS) and 1100 5'-UTR were found. Interestingly, acetogenesis genes contained longer 5'-UTR with lower RNA-folding free energy than other genes, revealing that the 5'-UTRs control the RNA abundance of the acetogenesis genes under low temperature conditions. Our findings suggest that post-transcriptional regulation via RNA conformational changes of 5'-UTRs is necessary for cold-adaptive acetogenesis.

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.

Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway.

Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.

Genome-scale analysis of syngas fermenting acetogenic bacteria reveals the translational regulation for its autotrophic growth.

BACKGROUND: Acetogenic bacteria constitute promising biocatalysts for the conversion of CO2/H2 or synthesis gas (H2/CO/CO2) into biofuels and value-added biochemicals. These microorganisms are naturally capable of autotrophic growth via unique acetogenesis metabolism. Despite their biosynthetic potential for commercial applications, a systemic understanding of the transcriptional and translational regulation of the acetogenesis metabolism remains unclear. RESULTS: By integrating genome-scale transcriptomic and translatomic data, we explored the regulatory logic of the acetogenesis to convert CO2 into biomass and metabolites in Eubacterium limosum. The results indicate that majority of genes associated with autotrophic growth including the Wood-Ljungdahl pathway, the reduction of electron carriers, the energy conservation system, and gluconeogenesis were transcriptionally upregulated. The translation efficiency of genes in cellular respiration and electron bifurcation was also highly enhanced. In contrast, the transcriptionally abundant genes involved in the carbonyl branch of the Wood-Ljungdahl pathway, as well as the ion-translocating complex and ATP synthase complex in the energy conservation system, showed decreased translation efficiency. The translation efficiencies of genes were regulated by 5'UTR secondary structure under the autotrophic growth condition. CONCLUSIONS: The results illustrated that the acetogenic bacteria reallocate protein synthesis, focusing more on the translation of genes for the generation of reduced electron carriers via electron bifurcation, rather than on those for carbon metabolism under autotrophic growth.

Room-Temperature H2 Gas Sensing Characterization of Graphene-Doped Porous Silicon via a Facile Solution Dropping Method.

In this study, a graphene-doped porous silicon (G-doped/p-Si) substrate for low ppm H2 gas detection by an inexpensive synthesis route was proposed as a potential noble graphene-based gas sensor material, and to understand the sensing mechanism. The G-doped/p-Si gas sensor was synthesized by a simple capillary force-assisted solution dropping method on p-Si substrates, whose porosity was generated through an electrochemical etching process. G-doped/p-Si was fabricated with various graphene concentrations and exploited as a H2 sensor that was operated at room temperature. The sensing mechanism of the sensor with/without graphene decoration on p-Si was proposed to elucidate the synergetic gas sensing effect that is generated from the interface between the graphene and p-type silicon.

Reconstruction of Acetogenesis Pathway Using Short-Read Sequencing of Clostridium aceticum Genome.

Clostridium aceticum is an anaerobic homoacetogen, able to reduce CO2 to multi-carbon products using the reductive acetyl-CoA pathway. This unique ability to use CO2 or CO makes the microbe a potential platform for the biotech industry. However, the development of genetically engineered homoacetogen for the large-scale production of commodity chemicals is hampered by the limited amount of their genetic and metabolic information. Here we exploited next-generation sequencing to reveal C. aceticum genome. The short-read sequencing produced 44,871,196 high quality reads with an average length of 248 bases. Following sequence trimming step, 30,256,976 reads were assembled into 12,563 contigs with 168-fold coverage and 1,971 bases in length using de Bruijn graph algorithm. Since the k-mer hash length in the algorithm is an important factor for the quality of output contigs, a window of k-mers (k-51 to k-201) was tested to obtain high quality contigs. In addition to the assembly metrics, the functional annotation of the contigs was investigated to select the k-mer optimum. Metabolic pathway mapping using the functional annotation identified the majority of central metabolic pathways, such as the glycolysis and TCA cycle. Further, these analyses elucidated the enzymes consisting of Wood-Ljungdahl pathway, in which CO2 is fixed into acetyl-CoA. Thus, the metabolic reconstruction based on the draft genome assembly provides a foundation for the functional genomics required to engineer C. aceticum.

Strategies in engineering sustainable biochemical synthesis through microbial systems.

Growing environmental concerns and the urgency to address climate change have increased demand for the development of sustainable alternatives to fossil-derived fuels and chemicals. Microbial systems, possessing inherent biosynthetic capabilities, present a promising approach for achieving this goal. This review discusses the coupling of systems and synthetic biology to enable the elucidation and manipulation of microbial phenotypes for the production of chemicals that can substitute for petroleum-derived counterparts and contribute to advancing green biotechnology. The integration of artificial intelligence with metabolic engineering to facilitate precise and data-driven design of biosynthetic pathways is also discussed, along with the identification of current limitations and proposition of strategies for optimizing biosystems, thereby propelling the field of chemical biology towards sustainable chemical production.

Water-Based Generators with Cellulose Acetate: Uncovering the Mechanisms of Power Generation.

Power generation technologies based on water movement and evaporation use water, which covers more than 70% of the Earth's surface and can also generate power from moisture in the air. Studies are conducted to diversify materials to increase power generation performance and validate energy generation mechanisms. In this study, a water-based generator was fabricated by coating cellulose acetate with carbon black. To optimize the generator, Fourier-transform infrared spectroscopy, specific surface area, zeta potential, particle size, and electrical performance analyses were conducted. The developed generator is a cylindrical generator with a diameter of 7.5 mm and length of 20 mm, which can generate a voltage of 0.15 V and current of 82 µA. Additionally, we analyzed the power generation performance using three factors (physical properties, cation effect, and evaporation environment) and proposed an energy generation mechanism. Furthermore, we developed an eco-friendly and low-cost generator using natural fibers with a simple manufacturing process. The proposed generator can contribute to the identification of energy generation mechanisms and is expected to be used as an alternative energy source in the future.

Real-time analysis and prediction method of ion concentration using the effect of O-H stretching bands in aqueous solutions based on ATR-FTIR spectroscopy.

Analyzing the concentration of ions in aqueous solutions in real-time plays an important role in the fields of chemistry and biology. Traditional methods for measuring ion concentrations, such as concentration analysis by measuring electrical conductivity, inductively coupled plasma mass spectrometry, and ion chromatography, have been used in many research fields. However, these methods are limited in determining ion concentrations instantaneously. Fourier-transform infrared-attenuated total reflectance (ATR-FTIR) spectroscopy provides a new approach for determining ion concentrations in aqueous solutions. This allows for fast analysis without pretreatment and is scalable for real-time measurements. In this study, we present a method for measuring ion concentrations by examining ion-water interactions in the O-H stretching band of aqueous solutions using ATR-FTIR spectroscopy. Five aqueous solutions, namely LiCl + HCl, LiOH + HCl, LiOH, Li3PO4, and NaCl were used in the experiments and prepared at concentrations between 0.5-2 M. The ion concentrations in the prepared aqueous solutions were measured using ATR-FTIR spectroscopy. We observed that the difference in absorbance increased and decreased linearly with changes in concentration. The concentration of ions in the aqueous solution could be measured by validating the designed linear regression analysis function model. In this study, we proposed five linear regression analysis function models, all of which showed high coefficients of determination above 0.9, with the highest coefficient of determination reaching 0.9969. These results show that ATR-FTIR spectroscopy has the potential to be applied as a rapid and simple concentration analysis system.

Selenium reduction of ubiquinone via SQOR suppresses ferroptosis.

The canonical biological function of selenium is in the production of selenocysteine residues of selenoproteins, and this forms the basis for its role as an essential antioxidant and cytoprotective micronutrient. Here we demonstrate that, via its metabolic intermediate hydrogen selenide, selenium reduces ubiquinone in the mitochondria through catalysis by sulfide quinone oxidoreductase. Through this mechanism, selenium rapidly protects against lipid peroxidation and ferroptosis in a timescale that precedes selenoprotein production, doing so even when selenoprotein production has been eliminated. Our findings identify a regulatory mechanism against ferroptosis that implicates sulfide quinone oxidoreductase and expands our understanding of selenium in biology.

Evaporation-Driven Energy Generation Using an Electrospun Polyacrylonitrile Nanofiber Mat with Different Support Substrates.

Water evaporation-driven energy harvesting is an emerging mechanism for contributing to green energy production with low cost. Herein, we developed polyacrylonitrile (PAN) nanofiber-based evaporation-driven electricity generators (PEEGs) to confirm the feasibility of utilizing electrospun PAN nanofiber mats in an evaporation-driven energy harvesting system. However, PAN nanofiber mats require a support substrate to enhance its durability and stability when it is applied to an evaporation-driven energy generator, which could have additional effects on generation performance. Accordingly, various support substrates, including fiberglass, copper, stainless mesh, and fabric screen, were applied to PEEGs and examined to understand their potential impacts on electrical generation outputs. As a result, the PAN nanofiber mats were successfully converted to a hydrophilic material for an evaporation-driven generator by dip-coating them in nanocarbon black (NCB) solution. Furthermore, specific electrokinetic performance trends were investigated and the peak electricity outputs of Voc were recorded to be 150.8, 6.5, 2.4, and 215.9 mV, and Isc outputs were recorded to be 143.8, 60.5, 103.8, and 121.4 µA, from PEEGs with fiberglass, copper, stainless mesh, and fabric screen substrates, respectively. Therefore, the implications of this study would provide further perspectives on the developing evaporation-induced electricity devices based on nanofiber materials.

A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.

Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor.

Facile Fabrication of ZnO-ZnFe2O4 Hollow Nanostructure by a One-Needle Syringe Electrospinning Method for a High-Selective H2S Gas Sensor.

Herein, a facile fabrication process of ZnO-ZnFe2O4 hollow nanofibers through one-needle syringe electrospinning and the following calcination process is presented. The various compositions of the ZnO-ZnFe2O4 nanofibers are simply created by controlling the metal precursor ratios of Zn and Fe. Moreover, the different diffusion rates of the metal oxides and metal precursors generate a hollow nanostructure during calcination. The hollow structure of the ZnO-ZnFe2O4 enables an enlarged surface area and increased gas sensing sites. In addition, the interface of ZnO and ZnFe2O4 forms a p-n junction to improve gas response and to lower operation temperature. The optimized ZnO-ZnFe2O4 has shown good H2S gas sensing properties of 84.5 (S = Ra/Rg) at 10 ppm at 250 °C with excellent selectivity. This study shows the good potential of p-n junction ZnO-ZnFe2O4 on H2S detection and affords a promising sensor design for a high-performance gas sensor.