Determination of Electrical and Mechanical Properties of Liquids Using a Resonator with a Longitudinal Electric Field.

The possibility of determining the elastic modules, viscosity coefficients, dielectric constant and electrical conductivity of a viscous conducting liquid using a piezoelectric resonator with a longitudinal electric field is shown. For the research, we chose a piezoelectric resonator made on an AT-cut quartz plate with round electrodes, operating with a shear acoustic mode at a frequency of about 4.4 MHz. The resonator was fixed to the bottom of a 30 mL liquid container. The samples of a mixture of glycerol and water with different viscosity and conductivity were used as test liquids. First, the frequency dependences of the real and imaginary parts of the electrical impedance of a free resonator were measured and, using the Mason electromechanical circuit, the elastic module, viscosity coefficient, piezoelectric constant and dielectric constant of the resonator material (quartz) were determined. Then, the container was filled with the test sample of a liquid mixture so that the resonator was completely covered with liquid, and the measurement of the frequency dependences of the real and imaginary parts of the electrical impedance of the loaded resonator was repeated. The dependences of the frequency of parallel and series resonances, as well as the maximum values of the electrical impedance and admittance on the conductivity of liquids for various viscosity values, were plotted. It was shown that these dependences can be used to unambiguously determine the viscosity and conductivity of the test liquid. Next, by fitting the theoretical frequency dependences of the real and imaginary parts of the electrical impedance of the resonator loaded with the liquid under study to the experimental dependences, the elastic module of the liquid and its dielectric constant were determined.

The Study of the Acoustic Characteristics of Chitosan Acetate Film Using a Radial Electric Field Excited Resonator.

Currently, the lateral electric field excited resonators are used for the creation of various sensors. We have recently proposed a new type of acoustic resonator called radial electric field excited disk acoustic resonator. The advantage of this type of resonator is its high sensitivity to mechanical and electrical boundary conditions on its free surface. This makes it possible to determine both the acoustic and electrical properties of a thin layer of material deposited on the free end of the resonator. In this work, we used a radial electric field excited disk acoustic resonator of Russian-made barium plumbum zirconate titanate (BPZT) piezoceramics. With the help of this resonator, the material constants for the piezoceramic sample were refined, and their temperature dependencies were determined. Then, this resonator was used to determine the elastic modulus, viscosity, and conductivity of the chitosan acetate film in air and ammonia vapors of various concentrations. It was shown that the chitosan acetate film under the influence of ammonia vapor significantly changes its mechanical properties and increases its electrical conductivity thousands of times, and then completely restores its properties.

The Effect of Glycerol-Based Suspensions on the Characteristics of Resonators Excited by a Longitudinal Electric Field.

This study examines the effect of suspensions based on pure glycerol and diamond powder with different concentrations on the characteristics of resonators with a longitudinal electric field. We used two disk resonators made of the quartz and langasite plates with round electrodes on both sides of the plate and resonant frequencies of 4.4 and 4.1 MHz, operating in shear and longitudinal acoustic modes, respectively. Each resonator was mounted on the bottom of a 30 mL liquid container. During the experiments, the container was filled with the suspension under study in such a way that the resonator was completely immersed in the suspension, and the frequency dependences of the real and imaginary parts of the electrical impedance of the resonator were measured. As a result, the shear modulus of the elasticity and shear coefficient of the viscosity of the studied suspensions were determined. The material constants of the suspensions were found by fitting the theoretical frequency dependences of the real and imaginary parts of the electrical impedance of the resonator to the experimentally measured ones, which was calculated using Mason's equivalent circuit. As a result, the dependencies of the density, shear modulus of elasticity, shear viscosity coefficient, and velocity of the shear acoustic wave on the volume concentration of the diamond particles were constructed.

Electroacoustic Biosensor Systems for Evaluating Antibiotic Action on Microbial Cells.

Antibiotics are widely used to treat infectious diseases. This leads to the presence of antibiotics and their metabolic products in the ecosystem, especially in aquatic environments. In many countries, the growth of pathogen resistance to antibiotics is considered a threat to national security. Therefore, methods for determining the sensitivity/resistance of bacteria to antimicrobial drugs are important. This review discusses the mechanisms of the formation of antibacterial resistance and the various methods and sensor systems available for analyzing antibiotic effects on bacteria. Particular attention is paid to acoustic biosensors with active immobilized layers and to sensors that analyze antibiotics directly in liquids. It is shown that sensors of the second type allow analysis to be done within a short period, which is important for timely treatment.

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%-51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.

Modulation of the cell wall protein Ecm33p in yeast Saccharomyces cerevisiae improves the production of small metabolites.

The cell wall is a dynamic organelle that determines the shape and provides the cell with mechanical strength. This study investigated whether modulation of cell wall composition can influence the production or secretion of small metabolites by yeast cell factories. We deleted and upregulated several cell wall-related genes KRE2, CWP1, CWP2, ECM33, PUN1, and LAS21 in yeast Saccharomyces cerevisiae engineered for p-coumaric acid or ß-carotene production. Deletions of las21∆ and ecm33∆ impaired the yeast growth on medium with cell wall stressors, calcofluor white, and caffeine. Both overexpression and deletion of ECM33 significantly improved the specific yield of p-coumaric acid and ß-carotene. We observed no change in secretion in any cell wall-altered mutants, suggesting the cell wall is not a limiting factor for small molecule secretion at the current production levels. We evaluated the cell wall morphology of the ECM33 mutant strains using transmission electron microscopy. The ecm33∆ mutants had an increased chitin deposition and a less structured cell wall, while the opposite was observed in ECM33-overexpressing strains. Our results point at the cell wall-related gene ECM33 as a potential target for improving production in engineered yeast cell factories.

Biosynthesis of insect sex pheromone precursors via engineered ß-oxidation in yeast.

Mating disruption with insect sex pheromones is an attractive and environmentally friendly technique for pest management. Several Lepidoptera sex pheromones have been produced in yeast, where biosynthesis could be accomplished by the expression of fatty acyl-CoA desaturases and fatty acyl-CoA reductases. In this study, we aimed to develop yeast Yarrowia lipolytica cell factories for producing Lepidoptera pheromones which biosynthesis additionally requires ß-oxidation, such as (Z)-7-dodecenol (Z7-12:OH), (Z)-9-dodecenol (Z9-12:OH), and (Z)-7-tetradecenol (Z7-14:OH). We expressed fatty acyl-CoA desaturases from Drosophila melanogaster (Dmd9) or Lobesia botrana (Lbo_PPTQ) and fatty acyl-CoA reductase from Helicoverpa armigera (HarFAR) in combinations with 11 peroxisomal oxidases of different origins. Yeast cultivations were performed with supplementation of methyl myristate (14:Me). The oxidase Lbo_31670 from L. botrana provided the highest titers of (Z)-7-dodecenoate, (Z)-9-dodecenoate, and (Z)-7-tetradecenoate. However, no chain-shortened fatty alcohols were produced. The mutation of fatty acid synthase (Fas2pI1220F) to increase myristate production did not lead to targeted fatty alcohol production. The problem was solved by directing the reductase into peroxisomes, where the strain with Dmd9 produced 0.10 ± 0.02 mg/l of Z7-12:OH and 0.48 ± 0.03 mg/l of Z7-14:OH, while the strain with Lbo_PPTQ produced 0.21 ± 0.03 mg/l of Z9-12:OH and 0.40 ± 0.07 mg/l of Z7-14:OH. In summary, the engineering of ß-oxidation in Y. lipolytica allowed expanding the portfolio of microbially produced insect sex pheromones.

Production of abscisic acid in the oleaginous yeast Yarrowia lipolytica.

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1,BcABA2,BcABA3,BcABA4 and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of the mevalonate pathway and ABA biosynthesis encoding genes, and we expressed plant ABA transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g dry cell weight (DCW) ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA; however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms Y. lipolytica as a potential alternative host for the ABA production.

An integrated yeast-based process for cis,cis-muconic acid production.

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae.

Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion-sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.

Microbial Acoustical Analyzer for Antibiotic Indication.

In this study, a compact acoustic analyzer for express analysis of antibiotics based on a piezoelectric resonator with a lateral electric field and combined with a computer was developed. The possibility of determining chloramphenicol in aqueous solutions in the concentration range of 0.5-15 µg/mL was shown. Bacterial cells that are sensitive to this antibiotic were used as a sensory element. The change in the electrical impedance modulus of the resonator upon addition of the antibiotic to the cell suspension served as an analytical signal. The analysis time did not exceed 4 min. The correlation of the experimental results of an acoustic sensor with the results obtained using the light phase-contrast microscopy and standard microbiological analysis was established. The compact biological analyzer demonstrated stability, reproducibility, and repeatability of results.

Neutrophil Activation by Mineral Microparticles Coated with Methylglyoxal-Glycated Albumin.

Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, H2O2 generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca2+) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.

Transportome-wide engineering of Saccharomyces cerevisiae.

Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae. We applied the method to study the transport of xenobiotic compounds, cis,cis-muconic acid (CCM), protocatechuic acid (PCA), and betaxanthins. We found 22 transporters that influenced the production of CCM or PCA. The transporter of the 12-spanner drug:H(+) antiporter (DHA1) family Tpo2p was further confirmed to import CCM and PCA in Xenopus expression assays. We also identified three transporter proteins (Qdr1p, Qdr2p, and Apl1p) involved in betaxanthins transport. In summary, the described method enables high-throughput transporter identification for small molecules in cell factories.

Tailored biosynthesis of gibberellin plant hormones in yeast.

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway. We then built a GA4-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA4. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA7 (trace amounts) and GA3 (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA3-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes. CLASSIFICATION: Biological Sciences, Applied Biological Sciences.

Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica.

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates.

ß-Carotene is a yellow-orange-red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for ß-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of ß-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding ß-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L ß-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest ß-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites.

Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

The Radial Electric Field Excited Circular Disk Piezoceramic Acoustic Resonator and Its Properties.

A new type of piezoceramic acoustic resonator in the form of a circular disk with a radial exciting electric field is presented. The advantage of this type of resonator is the localization of the electrodes at one end of the disk, which leaves the second end free for the contact of the piezoelectric material with the surrounding medium. This makes it possible to use such a resonator as a sensor base for analyzing the properties of this medium. The problem of exciting such a resonator by an electric field of a given frequency is solved using a two-dimensional finite element method. The method for solving the inverse problem for determining the characteristics of a piezomaterial from the broadband frequency dependence of the electrical impedance of a single resonator is proposed. The acoustic and electric field inside the resonator is calculated, and it is shown that this location of electrodes makes it possible to excite radial, flexural, and thickness extensional modes of disk oscillations. The dependences of the frequencies of parallel and series resonances, the quality factor, and the electromechanical coupling coefficient on the size of the electrodes and the gap between them are calculated.

Acoustical Slot Mode Sensor for the Rapid Coronaviruses Detection.

A method for the rapid detection of coronaviruses is presented on the example of the transmissible gastroenteritis virus (TGEV) directly in aqueous solutions with different conductivity. An acoustic sensor based on a slot wave in an acoustic delay line was used for the research. The addition of anti-TGEV antibodies (Abs) diluted in an aqueous solution led to a change in the depth and frequency of resonant peaks on the frequency dependence of the insertion loss of the sensor. The difference in the output parameters of the sensor before and after the biological interaction of the TGE virus in solutions with the specific antibodies allows drawing a conclusion about the presence/absence of the studied viruses in the analyzed solution. The possibility for virus detection in aqueous solutions with the conductivity of 1.9-900 µs/cm, as well as in the presence of the foreign viral particles, has been demonstrated. The analysis time did not exceed 10 min.

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production.

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.