Insights into the biosynthesis of palladium nanoparticles for oxygen reduction reaction by genetically engineered bacteria of Shewanella oneidensis MR-1.

Owing to the increasing need for green synthesis and environmental protection, the utilization of biological organism-derived carbons as supports for noble-metal electrocatalysts has garnered public interest. Nevertheless, the mechanism by which microorganisms generate nanometals has not been fully understood yet. In the present study, we used genetically engineered bacteria of Shewanella oneidensis MR-1 (∆SO4317, ∆SO4320, ∆SO0618 and ∆SO3745) to explore the effect of surface substances including biofilm-associated protein (bpfA), protein secreted by type I secretion systems (TISS) and type II secretion systems (T2SS), and lipopolysaccharide in microbial synthesis of metal nanoparticles. Results showed Pd/∆SO4317 (the catalyst prepared with the mutant ∆SO4317) shows better performance than other biocatalysts and commercial Pd/C, where the mass activity (MA) and specific activity (SA) of Pd/∆SO4317 are 3.1 and 2.1 times higher than those of commercial Pd/C, reaching 257.49 A g-1 and 6.85 A m-2 respectively. It has been found that the exceptional performance is attributed to the smallest particle size and the presence of abundant functional groups. Additionally, the absence of biofilms has been identified as a crucial factor in the formation of high-quality bio-Pd. Because the absence of biofilm can minimize metal agglomeration, resulting in uniform particle size dispersion. These findings provide valuable mechanical insights into the generation of biogenic metal nanoparticles and show potential industrial and environmental applications, especially in accelerating oxygen reduction reactions.

Tumor-killing viruses score rare success in late-stage trial.

Bladder cancer results suggest that after decades of failure, the viruses could finally reach their potential.

The first in-depth exploration of the genome of the engineered bacterium, Gluconobacter thailandicus.

Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.

Multiplexed CRISPR-mediated engineering of protein secretory pathway genes in the thermotolerant methylotrophic yeast Ogataea thermomethanolica.

CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.

A single synonymous nucleotide change impacts the male-killing phenotype of prophage WO gene wmk.

Wolbachia are the most widespread bacterial endosymbionts in animals. Within arthropods, these maternally transmitted bacteria can selfishly hijack host reproductive processes to increase the relative fitness of their transmitting females. One such form of reproductive parasitism called male killing, or the selective killing of infected males, is recapitulated to degrees by transgenic expression of the prophage WO-mediated killing (wmk) gene. Here, we characterize the genotype-phenotype landscape of wmk-induced male killing in D. melanogaster using transgenic expression. While phylogenetically distant wmk homologs induce no sex-ratio bias, closely-related homologs exhibit complex phenotypes spanning no death, male death, or death of all hosts. We demonstrate that alternative start codons, synonymous codons, and notably a single synonymous nucleotide in wmk can ablate killing. These findings reveal previously unrecognized features of transgenic wmk-induced killing and establish new hypotheses for the impacts of post-transcriptional processes in male killing variation. We conclude that synonymous sequence changes are not necessarily silent in nested endosymbiotic interactions with life-or-death consequences.

Set4 regulates stress response genes and coordinates histone deacetylases within yeast subtelomeres.

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status identified a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, where the loss of Set4 decreases cell fitness and cell wall integrity. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for Set4 in regulating repressive, heterochromatin-like environments.

Limited Plasmodium sporozoite gliding motility in the absence of TRAP family adhesins.

BACKGROUND: Plasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter and cross several cellular and tissue barriers for which they employ a set of surface proteins. Three of these proteins are members of the thrombospondin related anonymous protein (TRAP) family. Here, potential additive, synergistic or antagonistic roles of these adhesion proteins were investigated. METHODS: Four transgenic Plasmodium berghei parasite lines that lacked two or all three of the TRAP family adhesins TRAP, TLP and TREP were generated using positive-negative selection. The parasite lines were investigated for their capacity to attach to and move on glass, their ability to egress from oocysts and their capacity to enter mosquito salivary glands. One strain was in addition interrogated for its capacity to infect mice. RESULTS: The major phenotype of the TRAP single gene deletion dominates additional gene deletion phenotypes. All parasite lines including the one lacking all three proteins were able to conduct some form of active, if unproductive movement. CONCLUSIONS: The individual TRAP-family adhesins appear to play functionally distinct roles during motility and infection. Other proteins must contribute to substrate adhesion and gliding motility.

Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters.

Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H2O2. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2'-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.

A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.

The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.

Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Antitumor Effect of IL-2 and TRAIL Proteins Expressed by Recombinant Salmonella in Murine Bladder Cancer Cells.

BACKGROUND/AIMS: Cancer is the second most deadly disease in the world. The bladder cancer is one of the most aggressive types and shows a continuous increase in the number of cases. The use of bacteria as live vectors to deliver molecules directly to the tumor is a promising tool and has been used as an adjuvant treatment against several types of cancer. The aim of this study was to investigate the antitumor effect of Interleukin 2 (IL-2), TNF-related apoptosis-inducing ligand (TRAIL) and protein MIX against murine bladder cancer cells, lineage MB49. METHODS: The attenuated Salmonella strain SL3261 was transformed by inserting the IL-2 and TRAIL genes. The effects of proteins on cell viability (MTT method), cell morphology (optical microscopy), cell recovery (clonogenic assay), cell membrane (lactate dehydrogenase release - LDH), on oxidative stress pathway (levels of nitric oxide, NO) and apoptosis (flow cytometry and high resolution epifluorescence images) were evaluated at intervals of 24 and 48 hours of action. RESULTS: The results showed that there was a decrease in cell viability via damage to the cell membrane, alteration of cell morphology, non-recovery of cells, increase in the production of NO and incubate for of cells in the state of apoptosis in the two periods analyzed. CONCLUSION: The data presented suggest that IL-2, TRAIL and their MIX proteins in MB49 cells have cytotoxic potential and that this is associated with oxidative stress and apoptosis pathways. These results may contribute to the development of new therapeutic strategies for bladder cancer.

Synthetic Cellobiose-Inducible Regulatory Systems Allow Tight and Dynamic Controls of Gene Expression in Streptomyces.

Precise control of microbial gene expression is crucial for synthetic biotechnological applications. This is particularly true for the bacterial genus Streptomyces, major producers of diverse natural products including many antibiotics. Although a plethora of genetic tools have been developed for Streptomyces, there is still an urgent need for effective gene induction systems. We herein created two novel cellobiose-inducible regulatory systems referred to as Cel-RS1 and Cel-RS2. The regulatory systems are based upon the well-characterized repressor/operator pair CebR/cebO from Streptomyces scabies and the well-defined constitutive kasO* promoter. Both Cel-RS1 and Cel-RS2 exhibit a high level of induced reporter activity and virtually no leaky expression in three model Streptomyces species, which are commonly used as surrogate hosts for expression of natural product biosynthetic gene clusters. Cel-RS2 has been proven successful for programmable control of gene expression and controllable production of specialized metabolites in multiple Streptomyces species. The strategy can be used to expand the toolkit of inducible regulatory systems that will be broadly applicable to various Streptomyces.

Engineering analysis of multienzyme cascade reactions for 3'-sialyllactose synthesis.

Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialo-oligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3'-sialyllactose (3SL) synthesis from N-acetyl- d-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5'-triphosphate, via the reactions of cytidine 5'-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl- d-mannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase. We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h). Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Resveratrol Production in Yeast Hosts: Current Status and Perspectives.

Resveratrol is a plant secondary metabolite known for its therapeutic applications as an antioxidant, anti-cancer, anti-inflammatory, anti-aging, cardio-protective, and neuroprotective agent. Topical formulas of resveratrol are also used for skin disease management and in cosmetic industries. Due to its importance, high resveratrol production is urgently required. Since the last decade, intensive efforts have been devoted to obtaining resveratrol from microorganisms by pathway and metabolic engineering. Yeasts were proven to be excellent host candidates for resveratrol production. In addition to the similar intracellular compartments between yeasts and plants, yeasts exhibit the ability to express genes coding for plant-derived enzymes and to perform post-translational modification. Therefore, this review summarizes the attempts to use yeasts as a platform for resveratrol synthesis as the next promising route in producing high titers of resveratrol from genetically engineered strains.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Development of a real-time PCR marker targeting a new unauthorized genetically modified microorganism producing protease identified by DNA walking.

A PCR-based DNA walking analysis was performed on a protease product suspected to contain a new unauthorized genetically modified microorganism (GMM). Though the characterization of unnatural associations of sequences between the pUB110 shuttle vector and a Bacillus amyloliquefaciens gene coding for a protease, the presence of the GMM was shown. Based on these sequences of interest, a real-time PCR marker was developed to target specifically the newly discovered GMM, namely GMM protease2. The performance of the real-time PCR marker was assessed in terms of specificity and sensitivity. The applicability of the real-time PCR GMM protease2 marker was also demonstrated on microbial fermentation products. To confirm its use by other GMO enforcement laboratories, the transferability of the in-house validated real-time PCR marker was demonstrated by assays performed by an external laboratory.

Resveratrol Production from Hydrothermally Pretreated Eucalyptus Wood Using Recombinant Industrial Saccharomyces cerevisiae Strains.

Resveratrol is a phenolic compound with strong antioxidant activity, being promising for several applications in health, food, and cosmetics. It is generally extracted from plants or chemically synthesized, in both complex and not sustainable processes, but microbial biosynthesis of resveratrol can counter these drawbacks. In this work, resveratrol production by microbial biosynthesis from lignocellulosic materials was assessed. Three robust industrial Saccharomyces cerevisiae strains known for their thermotolerance and/or resistance to inhibitory compounds were identified as suitable hosts for de novo resveratrol production from glucose and ethanol. Through the CRISPR/Cas9 system, all industrial strains, and a laboratory one, were successfully engineered with the resveratrol biosynthetic pathway via the phenylalanine intermediate. All strains were further screened at 30 °C and 39 °C to evaluate thermotolerance, which is a key feature for Simultaneous Saccharification and Fermentation processes. Ethanol Red RBP showed the best performance at 39 °C, with more than 2.6-fold of resveratrol production in comparison with the other strains. This strain was then used to assess resveratrol production from glucose and ethanol. A maximum resveratrol titer of 187.07 ± 19.88 mg/L was attained from a medium with 2% glucose and 5% ethanol (w/v). Lastly, Ethanol Red RBP produced 151.65 ± 3.84 mg/L resveratrol from 2.95% of cellulose from hydrothermally pretreated Eucalyptus globulus wood, at 39 °C, in a Simultaneous Saccharification and Fermentation process. To the best of our knowledge, this is the first report of lignocellulosic resveratrol production, establishing grounds for the implementation of an integrated lignocellulose-to-resveratrol process in an industrial context.

An optogenetic assay method for electrogenic transporters using Escherichia coli co-expressing light-driven proton pump.

In organisms, nutrients and wastes move across the cellular membrane, in which membrane-embedded transporters facilitate and inhibit the movement. Despite the physiological significances, the currently used assay methods for transporter activities require tedious preparation and analytical processes. In this study, we report the isotope-free and label-free measurement system for the transport activities of electrogenic transporters. In the system, two molecules, a light-driven inward proton pump rhodopsin, xenorhodopsin (XeR), and a representative of an electrogenic transporter, an oxalate transporter (OxlT), were co-expressed in Escherichia coli cells. The light illumination of the cells co-expressing XeR and OxlT showed an increase in the pH of the bulk solution and that the extent of the pH change is significantly enhanced by adding the oxalate, suggesting the light-induced inward proton transport by XeR coupled to the negative electrogenic transport by OxlT. Such a pH increase was dependent on the oxalate concentration, but not on the XeR expression level. Of note, pH increase was not observed for the nonfunctional mutants of OxlT, R272A, and K355Q, supporting the validity of the system. Thus, we successfully developed an optogenetic assay method for electrogenic transporters using E. coli co-expressing light-driven proton pump.

Inactivated infectious pancreatic necrosis virus (IPNV) vaccine and E.coli-expressed recombinant IPNV-VP2 subunit vaccine afford protection against IPNV challenge in rainbow trout.

Infectious pancreatic necrosis (IPN) is a highly contagious disease causing high mortality in juvenile trouts. Since there is no effective way to treatment against IPNV, early diagnosis and prevention play an important role in combating the disease. The different types of IPNV vaccines (inactive, live, recombinant, DNA, etc) have been produced from local isolates and have been used in developed countries. In Turkey, there is no commercial licensed vaccines against IPNV. Due to this reason, IPNV vaccine is needed in Turkey. The production of recombinant VP2 subunit vaccine (IPNV-VP2) and inactivated whole particle virus vaccine (IPNV-WPV) were attempted from selected isolate belong to sp serotype. For this purpose; the virus was produced in RTG-2 cell line and RT-PCR amplification was performed by using primers with restriction enzymes. The whole VP2 gene was cloned into a plasmid vector and VP2 was expressed by using E. coli expression system. A trial was conducted to determine the immunity ability of IPNV-VP2 and IPNV-WPV in rainbow trout. According to the SN50 assay, the IPNV-WPV stimulates immune response faster than the IPNV-VP2 vaccine. Besides, the relative percent of Survive (RPS) was detected as 79% in fish vaccinated with IPNV-WPV and 70% in fish vaccinated with IPNV-VP2. Thus, we can say that the recombinant vaccine of IPNV-VP2 is almost protected against IPNV infection as well as the inactive vaccine.