LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Cross-feeding promotes heterogeneity within yeast cell populations.

Cellular heterogeneity in cell populations of isogenic origin is driven by intrinsic factors such as stochastic gene expression, as well as external factors like nutrient availability and interactions with neighbouring cells. Heterogeneity promotes population fitness and thus has important implications in antimicrobial and anticancer treatments, where stress tolerance plays a significant role. Here, we study plasmid retention dynamics within a population of plasmid-complemented ura3∆0 yeast cells, and show that the exchange of complementary metabolites between plasmid-carrying prototrophs and plasmid-free auxotrophs allows the latter to survive and proliferate in selective environments. This process also affects plasmid copy number in plasmid-carrying prototrophs, further promoting cellular functional heterogeneity. Finally, we show that targeted genetic engineering can be used to suppress cross-feeding and reduce the frequency of plasmid-free auxotrophs, or to exploit it for intentional population diversification and division of labour in co-culture systems.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Information Technology Tools for Mental Resilience Training in Sports: A Systematic Review / Herramientas de tecnología de la información para el entrenamiento de la resiliencia mental en los deportes: una revisión sistemática

The aim of this research study is to determine the information technology tools related to mental resilience training in sports. This research study determines the primary data for measuring the relation between dependent and independent. For this purpose, develop research questions related to information technology tools and mental resilience. Research determines through smart PLS software and generates informative results related to variables. The descriptive statistical analysis, the correlation coefficients, and the significant analysis also present the co-linearity statistical analysis between them. A systematic and deliberate procedure termed mental resilience training aims to increase a person's ability to deal with stress, obstacles, and adversity successfully. It entails developing the mental capacities and attitudes required to recover from failures, stay focused under duress, and persevere in the face of adversity. The overall research study found the significant and direct effect of information technology tools and mental resilience training in sports.(AU)

Rapid production of multimeric RNA aptamers stabilized by a designed pseudo-circular structure in E. coli.

RNA aptamers bind specifically and selectively to various macromolecules, cell surfaces, and viruses and find broad applications as biosensors, diagnostics, and in therapeutic treatments and drug delivery. Currently, RNA aptamer production is via in vitro methods. Herein, a new E. coli-based approach has been demonstrated for the rapid production of multimeric RNA aptamer transcripts that are protected from degradation by burying the 5' and 3' ends of the transcript in a designed double-stranded spacer. Multimeric and fluorescent RNA aptamers were produced stably in vivo and readily isolated from RNase III-deficient cells, and their full functionalities were shown by binding assays and fluorescence measurements. This approach shows promise as a rapid and scalable bioprocess for the production of RNA aptamers at low cost.

Crowder-directed interactions and conformational dynamics in multistimuli-responsive intrinsically disordered protein.

The consequences of crowding on the dynamic conformational ensembles of intrinsically disordered proteins (IDPs) remain unresolved because of their ultrafast motion. Here, we report crowder-induced interactions and conformational dynamics of a prototypical multistimuli-responsive IDP, Rec1-resilin. The effects of a range of crowders of varying sizes, forms, topologies, and concentrations were examined using spectroscopic, spectrofluorimetric, and contrast-matching small- and ultrasmall-angle neutron scattering investigation. To achieve sufficient neutron contrast against the crowders, deuterium-labeled Rec1-resilin was biosynthesized successfully. Moreover, the ab initio "shape reconstruction" approach was used to obtain three-dimensional models of the conformational assemblies. The IDP revealed crowder-specific systematic extension and compaction with the level of macromolecular crowding. Last, a robust extension-contraction model has been postulated to capture the fundamental phenomena governing the observed behavior of IDPs. The study provides insights and fresh perspectives for understanding the interactions and structural dynamics of IDPs in crowded states.

An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae.

Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.

Engineering eukaryote-like regulatory circuits to expand artificial control mechanisms for metabolic engineering in Saccharomyces cerevisiae.

Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L-1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.

Auxin-mediated induction of GAL promoters by conditional degradation of Mig1p improves sesquiterpene production in Saccharomyces cerevisiae with engineered acetyl-CoA synthesis.

The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l-1 in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose.

Auxin-mediated protein depletion for metabolic engineering in terpene-producing yeast.

In metabolic engineering, loss-of-function experiments are used to understand and optimise metabolism. A conditional gene inactivation tool is required when gene deletion is lethal or detrimental to growth. Here, we exploit auxin-inducible protein degradation as a metabolic engineering approach in yeast. We demonstrate its effectiveness using terpenoid production. First, we target an essential prenyl-pyrophosphate metabolism protein, farnesyl pyrophosphate synthase (Erg20p). Degradation successfully redirects metabolic flux toward monoterpene (C10) production. Second, depleting hexokinase-2, a key protein in glucose signalling transduction, lifts glucose repression and boosts production of sesquiterpene (C15) nerolidol to 3.5 g L-1 in flask cultivation. Third, depleting acetyl-CoA carboxylase (Acc1p), another essential protein, delivers growth arrest without diminishing production capacity in nerolidol-producing yeast, providing a strategy to decouple growth and production. These studies demonstrate auxin-mediated protein degradation as an advanced tool for metabolic engineering. It also has potential for broader metabolic perturbation studies to better understand metabolism.

Adaptation to Industrial Stressors Through Genomic and Transcriptional Plasticity in a Bioethanol Producing Fission Yeast Isolate.

Schizosaccharomyces pombe is a model unicellular eukaryote with ties to the basic research, oenology and industrial biotechnology sectors. While most investigations into S. pombe cell biology utilize Leupold's 972h- laboratory strain background, recent studies have described a wealth of genetic and phenotypic diversity within wild populations of S. pombe including stress resistance phenotypes which may be of interest to industry. Here we describe the genomic and transcriptomic characterization of Wilmar-P, an S. pombe isolate used for bioethanol production from sugarcane molasses at industrial scale. Novel sequences present in Wilmar-P but not in the laboratory S. pombe genome included multiple coding sequences with near-perfect nucleotide identity to Schizosaccharomyces octosporus sequences. Wilmar-P also contained a ∼100kb duplication in the right arm of chromosome III, a region harboring ght5+, the predominant hexose transporter encoding gene. Transcriptomic analysis of Wilmar-P grown in molasses revealed strong downregulation of core environmental stress response genes and upregulation of hexose transporters and drug efflux pumps compared to laboratory S. pombe Finally, examination of the regulatory network of Scr1, which is involved in the regulation of several genes differentially expressed on molasses, revealed expanded binding of this transcription factor in Wilmar-P compared to laboratory S. pombe in the molasses condition. Together our results point to both genomic plasticity and transcriptomic adaptation as mechanisms driving phenotypic adaptation of Wilmar-P to the molasses environment and therefore adds to our understanding of genetic diversity within industrial fission yeast strains and the capacity of this strain for commercial scale bioethanol production.

Structural elucidation of a hy-droxy-cineole product obtained from cytochrome P450 monooxygenase CYP101J2 catalysed transformation of 1,8-cineole.

1,8-Cineole is an abundant natural product that has the potential to be transformed into other building blocks that could be suitable alternatives to petroleum-based chemicals. Mono-hydroxy-lation of 1,8-cineole can potentially occur at eight different carbon sites around the bicyclic ring system. Using cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae B2, the hy-droxy-lation can be regioselectively directed at the C atom adjacent to the methyl-substituted quaternary bridgehead atom of 1,8-cineole. The unambiguous location of the hydroxyl functionality and the stereochemistry at this position was determined by X-ray crystal analysis. The mono-hydroxy-lated compound derived from this microorganism was determined to be (1S)-2a-hy-droxy-1,8-cineole (trivial name) or (1S,4R,6S)-1,3,3-trimethyl-2-oxabi-cyclo-[2.2.2]octan-6-ol (V) (systematic), C10H18O2. In the solid state this compound exhibits an inter-esting O-H⋯O hydrogen-bonding motif.

Crystal structure of a ß-aminopeptidase from an Australian Burkholderia sp.

ß-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal ß-amino acids from synthetic ß-peptides. ß-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of ß-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a ß-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0 Šand showed a tetrameric assembly typical of the ß-aminopeptidases. Each monomer consists of an α-subunit (residues 1-238) and a ß-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known ß-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV.

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.

Effect of Drying Methods on Protein and DNA Conformation Changes in Lactobacillus rhamnosus GG Cells by Fourier Transform Infrared Spectroscopy.

Microencapsulation protects cells against environmental stress encountered during the production of probiotics, which are used as live microbial food ingredients. Freeze-drying and spray-drying are used in the preparation of powdered microencapsulated probiotics. This study examines the ability of Fourier transform infrared (FTIR) spectroscopy to detect differences in cells exposed to freeze-drying and spray-drying of encapsulated Lactobacillus rhamnosus GG cells. The FTIR analysis clearly demonstrated there were more significant molecular changes in lipid, fatty acid content, protein, and DNA conformation of nonencapsulated compared to encapsulated bacterial cells. The technique was also able to differentiate between spray-dried and freeze-dried cells. The results also revealed the extent of protection from a protein-carbohydrate-based encapsulant matrix on the cells depending on the type drying process. The extent of this protection to the dehydration stress was shown to be less in spray-dried cells than in freeze-dried cells. This suggests that FTIR could be used as a rapid, noninvasive, and real-time measurement technique to detect detrimental drying effects on cells.

Engineering specific chemical modification sites into a collagen-like protein from Streptococcus pyogenes.

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017.

Formation of multimers of bacterial collagens through introduction of specific sites for oxidative crosslinking.

A range of non-animal collagens has been described, derived from bacterial species, which form stable triple-helical structures without the need for secondary modification to include hydroxyproline in the sequence. The non-animal collagens studied to date are typically smaller than animal interstitial collagens, around one quarter the length and do not pack into large fibrillar aggregates like those that are formed by the major animal interstitial collagens. A consequence of this for biomedical products is that fabricated items, such as collagen sponges, are not as mechanically and dimensionally stable as those of animal collagens. In the present study, we examined the production of larger, polymeric forms of non-animal collagens through introduction of tyrosine and cysteine residues that can form selective crosslinks through oxidation. These modifications allow the formation of larger aggregates of the non-animal collagens. When Tyr residues were incorporated, gels were obtained. And with Cys soluble aggregates were formed. These materials can be formed into sponges that are more stable than those formed without these modifications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2369-2376, 2016.

Energy evaluation of algal cell disruption by high pressure homogenisation.

The energy consumption of high pressure homogenisation (HPH) was analysed to determine the feasibility of rupturing algal cells for biodiesel production. Experimentally, the processing capacity (i.e. flow rate), power draw and cell disruption efficiency of HPH were independent of feed concentration (for Nannochloropsis sp. up to 25%w/w solids). Depending on the homogenisation pressure (60-150 MPa), the solids concentration (0.25-25%w/w), and triacylglyceride (TAG) content of the harvested algal biomass (10-30%), the energy consumed by HPH represented between 6% and 110-times the energy density of the resulting biodiesel. Provided the right species (weak cell wall and high TAG content) is selected and the biomass is processed at a sufficiently high solids concentration, HPH can consume a small fraction of the energy content of the biodiesel produced. This study demonstrates the feasibility of process-scale algal cell disruption by HPH based on its energy requirement.

Preparation and characterization of monomers to tetramers of a collagen-like domain from Streptococcus pyogenes.

The collagen like domain Scl2 from Streptococcus pyogenes has been proposed as a potential biomedical material. It is non-cytotoxic and non-immunogenic and can be prepared in good yield in fermentation. The Scl2 collagen domain is about a quarter of the length, 234 residues, of the main collagen type, mammalian type I collagen (1014 residues) that is currently used in biomedical devices. In the present study we have made constructs comprising 1 to 4 copies of the Scl2 collagen domain, plus these same constructs with a CysCys sequence at the C-terminal, analogous to that found in mammalian type III collagens. The yields of these constructs were examined from 2 L fermentation studies. The yields of both series declined with increasing size. Circular dichroism showed that the addition of further collagen domains did not lead to a change in the melting temperature compared to the monomer domain. Addition of the CysCys sequence led to a small additional stabilization of about 2-3°C for the monomer construct when the folding (V) domain was present.