Synthetic biology on acetogenic bacteria for highly efficient conversion of c1 gases to biochemicals

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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

A Novel Synthetic Method for N Doped TiO2 Nanoparticles Through Plasma-Assisted Electrolysis and Photocatalytic Activity in the Visible Region

Nitrogen doped TiO2 (N-TiO2) nanoparticles were synthesized via a novel plasma enhanced electrolysis method using bulk titanium (Ti) as a source material and nitric acid as the nitrogen dopant. This method possesses remarkable merits with regard to the direct-metal synthesis of nanoparticles with its one-step process, eco-friendliness, and its ability to be mass produced. The nanoparticles were synthesized from bulk Ti metal and dipped in 5–15 mmol of a nitric acid electrolyte under the application of AC 500 V, the minimum range of voltage to generate plasma. By controlling the electrolyte concentration, the nanoparticle size distribution could be tuned between 12.1 and 24.7 nm using repulsion forces via variations in pH. The prepared N-TiO2 nanoparticles were calcined at between 100 and 300°C to determine their photocatalytic efficiency within the visible-light region, which depended on their crystal structure and N doping content. Analysis showed that the temperature treatment yielded an anatase TiO2 crystalline structure when the N doping content was varied from 0.4 to 0.54 at.%. In particular, the 0.4 at.% N doped TiO2 catalyst exhibited the highest catalytic performance with quadruple efficiency compared to the P-25 standard TiO2 nanoparticles, which featured a 91% degradation of methyl orange organic dye within 300 min. This solid-liquid reaction based on plasma enhanced electrolysis could open new pathways with regard to high purity mass producible ceramic nanoparticles with advanced properties

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

Analysis of the core genome and pan-genome of autotrophic acetogenic bacteria

Acetogens are obligate anaerobic bacteria capable of reducing carbon dioxide (CO2) to multicarbon compounds coupled to the oxidation of inorganic substrates, such as hydrogen (H2) or carbon monoxide (CO), via the Wood-Ljungdahl pathway. Owing to the metabolic capability of CO2 fixation, much attention has been focused on understanding the unique pathways associated with acetogens, particularly their metabolic coupling of CO2 fixation to energy conservation. Most known acetogens are phylogenetically and metabolically diverse bacteria present in 23 different bacterial genera. With the increased volume of available genome information, acetogenic bacterial genomes can be analyzed by comparative genome analysis. Even with the genetic diversity that exists among acetogens, the Wood-Ljungdahl pathway, a central metabolic pathway, and cofactor biosynthetic pathways are highly conserved for autotrophic growth. Additionally, comparative genome analysis revealed that most genes in the acetogen-specific core genome were associated with the Wood-Ljungdahl pathway. The conserved enzymes and those predicted as missing can provide insight into biological differences between acetogens and allow for the discovery of promising candidates for industrial applications

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

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

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

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

Abstract 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