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Fungal systems, yeast as well as filamentous fungi, are effective platforms for producing recombinant eukaryotic proteins because of their efficient secretion, robust development features, and capacity for post-translational modification. However, to achieve optimum protein expression in fungal hosts, a precise regulation of gene expression levels is necessary. Promoters are critical cis-regulatory regions that drive gene expression. Therefore, understanding the structure and function of fungal promoters and the factors that influence their performance is an essential step in developing yeast and filamentous fungal platforms as hosts for the expression and secretion of eukaryotic proteins. However, literature on the characterization of filamentous fungal promoters is non-exhaustive. The present review attempts to provide a comprehensive account of available information and future applications of fungal promoters. The properties of promoters from different classes of fungi are discussed with respect to their general structure, the core and proximal components that constitute the fungal promoters, types of fungal promoters based on their functions etc. Furthermore, the utility of fungal promoters for applications in healthcare, biofuels, agriculture and biotechnology are also discussed. The comprehensive understanding of fungal promoters will help in developing tailored promoters, paving the way for the optimum production of economically important eukaryotic proteins in different host organisms., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The native membrane environment profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergents to disrupt and remove this vital membrane context, impeding our ability to decipher the local molecular context and its effect. Here we develop a membrane proteome-wide platform that enables rapid spatially resolved extraction of target MPs directly from cellular membranes into native nanodiscs that maintain the local membrane context, using a library of membrane-active polymers. We accompany this with an open-access database that quantifies the polymer-specific extraction efficiency for 2,065 unique mammalian MPs and provides the most optimized extraction condition for each. To validate, we demonstrate how this resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, we show how the database can be extended to capture overexpressed multiprotein complexes by taking two synaptic vesicle MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane 'nano-scoops' containing a target MP and interface with structural, functional and bioanalytical approaches., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA--PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels. [ABSTRACT FROM AUTHOR]

Atomic-resolution structural studies of membrane-associated proteins and peptides in a membrane environment are important to fully understand their biological function and the roles played by them in the pathology of many diseases. However, the complexity of the cell membrane has severely limited the application of commonly used biophysical and biochemical techniques. Recent advancements in NMR spectroscopy and cryoEM approaches and the development of novel membrane mimetics have overcome some of the major challenges in this area. For example, the development of a variety of lipid-nanodiscs has enabled stable reconstitution and structural and functional studies of membrane proteins. In particular, the ability of synthetic amphipathic polymers to isolate membrane proteins directly from the cell membrane, along with the associated membrane components such as lipids, without the use of a detergent, has opened new avenues to study the structure and function of membrane proteins using a variety of biophysical and biological approaches. This review article is focused on covering the various polymers and approaches developed and their applications for the functional reconstitution and structural investigation of membrane proteins. The unique advantages and limitations of the use of synthetic polymers are also discussed. [ABSTRACT FROM AUTHOR]

Desalination is considered one of the strongest alternatives to face global water scarcity. Since conventional reverse osmosis is an energy-intensive process, Aquaporin biomimetic membranes (ABM) emerge as an effective alternative to increase productivity and reduce energy consumption. Despite the highly theoretical water permeability and selectivity of aquaporins (Aqps), in practice, ABM did not achieve the expected performance. This review is focused on each step of the ABM fabrication process in order to find critical points where efforts should be made for future studies. Fabrication procedures include Aqp production, reconstitution into vesicles, immobilization in porous substrates and filtration performance. The principal identified challenge is the need for implementing monitoring and optimization techniques. Also, new strategies for protein production, stabilization, reconstitution, and support affinity could offer significant breakthroughs for developing ABMs as a viable emerging technology. [ABSTRACT FROM AUTHOR]

Currently, searching for new protease inhibitors is of interest to many scientists around the world because they play an important role in controlling harmful proteases. As terrestrial bioactive resources are becoming increasingly depleted, studies are aimed at finding other sources, for example, the ocean. Some recent reports indicate that sponge- associated microbes produce substances with high biological activity, such as anticancer and anti-inflammation, protease inhibition, etc. In this report, a new serpin gene screened from the metagenomics database of microorganism-associated Spheciospongia vesparium QT2 collected sea of Quang Tri province (Vietnam), was successful expressed in Pichia pastoris SMD1168. The obtained result showed that the maximum amount of recombinant protein secreted in the medium was received after 72 hours of induction with methanol. The PAGE electrophoresis supplemented with 0.1% casein and the Trypsin-sepharose 4B affinity chromatography column were performed, and the result was confirmed that the recombinant protein expressed in the P. pastoris SMD1168 (molecular weight about 50 kDa) was the target protein. The purified recombinant protein PI-QT exhibited inhibitor activity on trypsin, -chymotrypsin and thermolysin and the inhibition was 88.7%, 69% and 43%, respectively. In addition, PI-QT protein is stable and has optimal activity in the pH range 79, the temperature is below 60 C. [ABSTRACT FROM AUTHOR]

The bacterium Escherichia coli is among the most popular hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently generated the specialized MP‐producing E. coli strain SuptoxD, which upon co‐expression of the effector gene djlA, is capable of alleviating two major bottlenecks in bacterial recombinant MP production: it suppresses the toxicity that frequently accompanies the MP‐overexpression process and it markedly increases the cellular accumulation of membrane incorporated and properly folded recombinant MP. Combined, these two positive effects result in dramatically enhanced volumetric yields for various recombinant MPs of both prokaryotic and eukaryotic origin. Based on the observation that djlA is found in the genomes of various pathogenic bacteria, the aim of the present work was to investigate (a) whether other naturally occurring DjlA variants can exert the MP toxicity‐suppressing and production‐promoting effects similarly to the E. coli DjlA and (b) if we can identify a DjlA variant whose efficiency surpasses that of the E. coli DjlA of SuptoxD. We report that a quite surprisingly broad variety of homologous DjlA proteins exert beneficial effects on recombinant MP when overexpressed in E. coli. Furthermore, we demonstrate that the Salmonella enterica DjlA is an even more potent enhancer of MP productivity compared with the E. coli DjlA of SuptoxD. Based on this, we constructed a second‐generation SuptoxD strain, termed SuptoxD2.0, whose MP‐production capabilities surpass significantly those of the original SuptoxD, and we anticipate that SuptoxD2.0 will become a broadly utilized expression host for recombinant MP production in bacteria. [ABSTRACT FROM AUTHOR]

The aim of this paper is to make the point on the fortieth years study on the β‐glycosidase from Sulfolobus solfataricus. This enzyme represents one of the thermophilic biocatalysts, which is more extensively studied as witnessed by the numerous literature reports available since 1980. Comprehensive biochemical studies highlighted its broad substrate specificity for β‐d‐galacto‐, gluco‐, and fuco‐sides and also showed its remarkable exo‐glucosidase and transglycosidase activities. The enzyme demonstrated to be active and stable over a wide range of temperature and pHs, withstanding to several drastic conditions comprising solvents and detergents. Over the years, a great deal of studies were focused on its homotetrameric tridimensional structure, elucidating several structural features involved in the enzyme stability, such as ion pairs and post‐translational modifications. Several β‐glycosidase mutants were produced in the years in order to understand its peculiar behavior in extreme conditions and/or to improve its functional properties. The β‐glycosidase overproduction was also afforded reporting numerous studies dealing with its production in the mesophilic host Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and Lactococcus lactis. Relevant applications in food, beverages, bioenergy, pharmaceuticals, and nutraceutical fields of this enzyme, both in free and immobilized forms, highlighted its biotechnological relevance. [ABSTRACT FROM AUTHOR]

Vacuolar-type adenosine triphosphatases (V-ATPases) 1-3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases 4,5 . They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes 1,3 . In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle 6,7 . To shed light on this bona fide single-molecule biological process, we investigated electrogenic proton-pumping by single mammalian-brain V-ATPases in single synaptic vesicles. Here we show that V-ATPases do not pump continuously in time, as suggested by observing the rotation of bacterial homologues 8 and assuming strict ATP-proton coupling. Instead, they stochastically switch between three ultralong-lived modes: proton-pumping, inactive and proton-leaky. Notably, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. ATP regulates V-ATPase activity through the switching probability of the proton-pumping mode. By contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. A direct consequence of mode-switching is all-or-none stochastic fluctuations in the electrochemical gradient of synaptic vesicles that would be expected to introduce stochasticity in proton-driven secondary active loading of neurotransmitters and may thus have important implications for neurotransmission. This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode-switching., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3-15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca 2+ , Co 2+ , and Mg 2+ were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a V m value of 11.29 μmol/min and a K m value of 31.2 μmol/L. In short, due to excellent K m and V max , the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three‐fold increase in recombinant protein yield compared with those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml −1. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms. Aw and Polizzi developed a cell‐free protein synthesis (CFPS) system based on extracts from the industrially relevant yeast, Pichia pastoris. Genetically‐encoded biosensors were used to determine the optimal harvest time and screen engineered strains for sustained ribosome content. Overexpression of a single transcription factor led to a 3‐fold increase in recombinant protein yield. The work adds to the growing number of CFPS systems from diverse organisms and also provides a blueprint for rapidly engineering new strains with increased CFPS productivity. [ABSTRACT FROM AUTHOR]

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity., (© 2022. The Author(s).)

Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.