Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon.

ABSTRACTRational engineering of gas-fermenting bacteria for high yields of bioproducts is vital for a sustainable bioeconomy. It will allow the microbial chassis to renewably valorize natural resources from carbon oxides, hydrogen, and/or lignocellulosic feedstocks more efficiently. To date, rational design of gas-fermenting bacteria such as changing the expression levels of individual enzymes to obtain the desired pathway flux is challenging, because pathway design must follow a verifiable metabolic blueprint indicating where interventions should be executed. Based on recent advances in constraint-based thermodynamic and kinetic models, we identify key enzymes in the gas-fermenting acetogen Clostridium ljungdahlii that correlate with the production of isopropanol. To this extent, we integrated a metabolic model in comparison with proteomics measurements and quantified the uncertainty for a variety of pathway targets needed to improve the bioproduction of isopropanol. Based on in silico thermodynamic optimization, minimal protein requirement analysis, and ensemble modeling-based robustness analysis, we identified the top two significant flux control sites, i.e., acetoacetyl-coenzyme A (CoA) transferase (AACT) and acetoacetate decarboxylase (AADC), overexpression of which could lead to increased isopropanol production. Our predictions directed iterative pathway construction, which enabled a 2.8-fold increase in isopropanol production compared to the initial version. The engineered strain was further tested under gas-fermenting mixotrophic conditions, where more than 4 g/L isopropanol was produced when CO, CO2, and fructose were provided as the substrates. In a bioreactor environment sparging with CO, CO2, and H2 only, the strain produced 2.4 g/L isopropanol. Our work highlighted that the gas-fermenting chasses can be fine-tuned for high-yield bioproduction by directed and elaborative pathway engineering.IMPORTANCE Highly efficient bioproduction from gaseous substrates (e.g., hydrogen and carbon oxides) will require systematic optimization of the host microbes. To date, the rational redesign of gas-fermenting bacteria is still in its infancy, due in part to the lack of quantitative and precise metabolic knowledge that can direct strain engineering. Here, we provide a case study by engineering isopropanol production in gas-fermenting Clostridium ljungdahlii. We demonstrate that a modeling approach based on the thermodynamic and kinetic analysis at the pathway level can provide actionable insights into strain engineering for optimal bioproduction. This approach may pave the way for iterative microbe redesign for the conversion of renewable gaseous feedstocks.

Gas fermentation using H2:CO2 and CO as feedstocks offers a sustainable alternative to fossil fuel-derived production of chemicals. Clostridium autoethanogenum is an industrially relevant acetogen for this process, but the effects of gas compositions — particularly H2:CO2 ratios — on product distribution are still unclear, hindering optimisation. Previous studies have not provided comprehensive analysis of metabolic responses during the coutilisation of multiple gaseous substrates in batch cultures. This study investigates how H2:CO2 ratios and low-level CO supplementation steer C. autoethanogenum metabolism, specifically influencing acetogenic and solventogenic pathways (acetate and ethanol production) and exogenous carboxylate reduction (conversion of butyrate to butanol). Batch cultures were conducted with H2:CO2 ratios of 1:1, 2.5:1, and 4:1, CO supplementation of 0%, 2%, and 5%, and butyrate addition of 0, 10, and 20 mM. Substrate consumption, product generation, and biomass were monitored over time. Flux balance analysis (FBA) was employed to estimate the changes in internal fluxes during different stages of the culture. Ethanol production positively correlated with initial H2:CO2 ratios, increasing from 0.7 mM ± 0.6 mM at 1:1 to 6.0 mM ±1.8 mM at 4:1. Acetate concentrations remained stable at 24.7 mM ± 4.7 mM. Higher initial CO pressures boosted biomass and CO uptake rates. Butyrate addition accelerated butanol but delayed ethanol production, indicating competition for reducing equivalents. Overall, this study demonstrates how H2:CO2:CO ratios can be strategically adjusted to steer C. autoethanogenum metabolism. The combination of factorial and Box–Behnken experimental design provided a flexible approach to generate data that can guide gas fermentation strategies towards targeted chemical production.

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Gas fermentation is a promising approach to reduce carbon emissions of production processes by using microorganisms to convert industrial gas streams into valuable carbon-based products. Here, isopropanol production by engineered Cupriavidus necator from CO2-rich gas streams is presented. Four different engineered strains were tested first on synthetic gas mixture, and one was selected, namely Re2133/pEG7d, for further evaluation on two different industrial CO2-rich gas streams. Up to 1.6 g L-1 of isopropanol was obtained in autotrophic gas flasks with this strain. Then, raw biogas from landfill containing 45% methane was successfully used as a CO2 source for isopropanol production with this strain Re2133/pEG7d producing up to 2.35 ± 0.25 g L-1, and methane enrichment was demonstrated. Finally, flue gases from a municipal incinerator were used as a source of CO2 and O2 for the autotrophic cultivation of the strain Re2133/pEG7d, obtaining a final isopropanol concentration of 2.5 ± 0.3 g L-1. This work highlighted the robustness of C. necator to potential industrial gas stream impurities and its versatility in using diverse gaseous feedstocks, promising future developments at larger scale.

Engineering Acetobacterium wieringae for acetone production from syngas

BackgroundImpactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts like Escherichia coli or yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential.ResultsIn this study, we report scarless genome editing of an industrially used model acetogen, Clostridium autoethanogenum, using the CRISPR/Cas9 system. Initial efforts to retrofit the CRISPR/Cas9 system for C. autoethanogenum resulted in poor efficiency likely due to uncontrolled expression of Cas9. To address this, we constructed and screened a small library of tetracycline-inducible promoters that can also be used to fine-tune gene expression. With a new inducible promoter, the efficiency of CRISPR/Cas9-mediated desired gene deletion in C. autoethanogenum was improved to over 50 %, making it a viable tool for engineering C. autoethanogenum.ConclusionsAddition of both an inducible promoter library and a scarless genome editing tool is an important expansion to the genetic tool box of industrial C. autoethanogenum strain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0638-3) contains supplementary material, which is available to authorized users.

Identification of a virulence-associated protein homolog gene and IS Ra1 in a plasmid of Riemerella anatipestifer

Aims: The MOB protein family is an evolutionarily highly conserved protein family that participates in the regulation of cell volume and proliferation. However, its function in tomato growth and development remains unclear. Clarifying the properties, structure and protein interaction network of SlMOB proteins is of great significance for exploring the regulatory mechanism of tomato growth and development. Study Design: To explore the characteristics and potential functions of the tomato SlMOB protein family, we conducted systematic bioinformatics analyses to predict and characterize the SlMOB proteins in Solanum lycopersicum. The results of this study laid a solid foundation for further in-depth exploration of the regulatory role of SlMOB proteins in tomato fruit development and the breeding of high-yield tomato varieties. Methodology: In this study, bioinformatics methods were used to comprehensively analyze the physicochemical properties, transmembrane structure, subcellular localization, signal peptide, secondary structure, conserved domain, open reading frame, 3D structure, protein interaction relationship and phylogenetic evolution of tomato SlMOB proteins. Results: The results showed that there were three members of the tomato SlMOB protein family, and all contained 215 amino acids, with no transmembrane regions or signal peptides, and were localized in the cytoplasm and nucleus. The secondary structure was mainly composed of α-helix, and all members contained the conserved Mob1_phocein domain. The 3D models of all SlMOB proteins were constructed with 5twg.1.A as the template, showing high evolutionary conservation. The proteins interacting with SlMOB family members mainly included serine/threonine protein kinase 38-like, serine/threonine protein kinase 39-like and other kinases involved in signal transduction and cell cycle regulation. Phylogenetic tree analysis revealed that tomato SlMOB proteins had a close evolutionary relationship with Camellia sinensis MOB proteins. Conclusions: The results of this study clarified the basic characteristics and evolutionary relationships of the tomato SlMOB protein family, enriched the information of the MOB protein family in plants, and provided a theoretical basis for further experimental verification of the function of SlMOB proteins and the molecular breeding of high-yield and high-quality tomatoes.

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism.

Molecular Characterization and Interstrain Variability of pHPS1, a Plasmid Isolated from the Sydney Strain (SS1) ofHelicobacter pylori

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector

The genus Aeromonas is increasingly implicated in human infections. However, accurate species-level identification remains challenging, particularly in clinical microbiology laboratories. This study aimed to develop a multiplex polymerase chain reaction (PCR) assay to identify four Aeromonas species-Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii, and Aeromonas dhakensis-most frequently associated with human infectious diseases. A total of 788 whole genome sequencing (WGS) data sets from 31 Aeromonas species were analyzed to identify open reading frames (ORFs) specifically present in A. hydrophila, A. caviae, A. veronii, and A. dhakensis. Primer sets were designed based on sequences of ORFs specific to each species to develop a multiplex PCR assay. To validate the efficacy of the assay, 256 clinical Aeromonas isolates were tested, and the results were compared with taxonomic affiliation inferred by WGS data, along with 19 type strains. The multiplex PCR successfully identified all strains of the four target species and produced no amplification in non-target species strains except the band for internal control. The multiplex PCR enables rapid and reliable identification of four Aeromonas spp. commonly involved in human infectious diseases.IMPORTANCEThe multiplex PCR assay facilitates accurate identification of clinically important Aeromonas spp. in clinical microbiology laboratories, providing crucial information to guide appropriate antimicrobial therapy and advance understanding of the epidemiology of Aeromonas spp.

Spn1 is a multifunctional histone chaperone that associates with RNA polymerase II during elongation and is essential for life in eukaryotes. While previous work has elucidated regions of the protein important for its many interactions, it is unknown how these domains contribute to the maintenance of chromatin structure. Here, we employ digestion by micrococcal nuclease followed by single-stranded library preparation and sequencing (MNase-SSP) to characterize chromatin structure in Saccharomyces cerevisiae expressing wild-type or mutants of Spn1 (spn1 K192N or spn1 141-305 ). We mapped protections of all sizes genome-wide. Surprisingly, we observed a widespread loss of short fragments over nucleosome-depleted regions (NDRs) at promoters in the spn1 K192N -containing strain, indicating critical functions of Spn1 in maintaining normal chromatin architecture outside open reading frames. Additionally, there are shifts in DNA protections in both Spn1 mutant expressing strains over open reading frames, which indicate changes in nucleosome and subnucleosome positioning. This was observed in markedly different Spn1 mutant strains, demonstrating that multiple functions of Spn1 are required to maintain proper chromatin structure in open reading frames. Changes in chromatin structure correlate positively with changes in gene expression as shown by RNA-seq analysis in the Spn1 mutant strains. Taken together, our results reveal a previously unknown role of Spn1 in the maintenance of NDR architecture and deepen our understanding of Spn1-dependent chromatin maintenance over transcribed regions.