Heterologous Expression Research Articles

High-efficiency expression of alginate lyase in Pichia pastoris facilitated by Vitreoscilla hemoglobin

Vitreoscilla hemoglobin (VHb) can enhance the ability of recombinant strains to express heterologous proteins under low-oxygen conditions. However, its mechanism of action in the Pichia pastoris expression system remains unclear. In this study, three VHb construction strategies were designed to elucidate the mechanisms by which VHb promotes heterologous protein expression in P. pastoris. Notably, the co-expression pattern involving the sequential expression of the 102C300C gene followed by the Vgb gene significantly improved enzyme activity in the recombinant strain X33-102C300C-Vgb. The enzyme activity was 203.4 ± 0.57 U/mL at 180 h of fermentation in the 5-L system, which was 20.7 % higher than that of the starting strain X33-102C300C. Fluorescent labeling experiments revealed for the first time that a dual-transcription unit approach achieved superior VHb expression, indicating its potential for further development. Furthermore, transcriptomic and metabolomic analyses demonstrated that VHb enhanced the growth of recombinant yeast colonies by improving respiration-related metabolism under low-oxygen conditions. This, in turn, alleviated the repression of the expression alcohol oxidase (AOX) at high methanol concentrations, resulting in increased alginate lyase activity. This study provides a theoretical foundation for improving the target protein expression in recombinant P. pastoris during high-density fermentation.

Combined class I and class III antiarrhythmic effects of sotagliflozin - insights from heterologous expressions systems and atrial cardiomyocytes

Abstract Background The demand for more effective treatment options for atrial fibrillation (AF), the most common sustained arrhythmia, is driven by its direct impact on morbidity and mortality. Current therapies, such as antiarrhythmic drugs and catheter ablation, are limited by suboptimal efficacy and adverse effects or periinterventional complications. Therefore, safe and effective treatment of AF remains a major unmet clinical need. Sodium-glucose cotransporter (SGLT) inhibitors have shown promise in reducing the incidence of AF in large cardiovascular endpoint trials. However, the exact mechanisms of action remain unclear. While only two SGLT2 inhibitors are approved and clinically used in Europe, sotagliflozin, which inhibits both SGLT1 and SGLT2, might also have potential for the treatment of AF. Purpose This study aims to elucidate the effects of sotagliflozin on cardiac action potential formation. This understanding could optimise its clinical use, identify new targets for the treatment of atrial fibrillation and ultimately improve the quality of life of patients suffering from AF. Methods The effects of sotagliflozin on cardiac ionic currents were assessed using two-electrode voltage clamp measurements of several sodium and potassium channels heterologously expressed in Xenopus laevis oocytes. Subsequently, ascending concentrations of sotagliflozin were administered to isolated human atrial cardiomyocytes during patch-clamp measurements to investigate its effect on atrial action potential morphology. Results At a concentration of 100 µM, sotagliflozin showed significant inhibitory effects on three out of ten ion channels tested: NaV1.5 sodium channel current was reduced by 22,3 % %, hERG and KV4.3 potassium currents were reduced by 30,4 % and 22,5 % respectively. Patch-clamp experiments, performed on isolated human atrial cardiomyocytes showed a dose-dependent reduction in upstroke velocity and action potential amplitude by 20,8 % and 13,8 % at a concentration of 20 µM, reflecting class-I-antiarrhythmic effects. Furthermore, a prolongation of AP duration measured at 50 % (APD50) and 90 % (APD90) of repolarization by 57,7 % and 27,9 % at a sotagliflozin concentration of 20 µM was noted, implying additional class-III-antiarrhythmic action. Conclusions Sotagliflozin exerts direct inhibitory effects on heterologously expressed NaV1.5, hERG and KV4.3 channels, resulting in a reduction in upstroke velocity and action potential amplitude, as well as a prolongation of APD50 and APD90 levels in isolated human atrial cardiomyocytes. These results extend the pharmacological profile of sotagliflozin and suggest a potential role for the clinical use of combined SGLT1/SGLT2 inhibitors in the treatment of AF.

Heterologous expressing melittin in a probiotic yeast to evaluate its function for promoting NSC-34 regeneration

Melittin is a bioactive peptide and the predominant component in bee venom (BV), studied for its many medical properties, such as antibacterial, anti-inflammatory, anti-arthritis, nerve damage reduction, and muscle cell regeneration. Melittin is primarily obtained through natural extraction and chemical synthesis; however, both methods have limitations and cannot be used for mass production. This study established a heterologous melittin expression system in the probiotic yeast Kluyveromyces marxianus. This yeast was selected for its advantages in stress tolerance and high secreted protein yields, simplifying purification. A > 95% high-purity melittin (MET) and its precursor promelittin (ProMET) were successfully produced and purified at 1.68 μg/mL and 3.33 μg/mL concentrations and verified through HPLC and mass spectrum. The functional test of the NSC-34 cell regeneration revealed that MET achieved the best activity compared to ProMET and the natural-extracted BV groups. Growth-related gene expressions were evaluated, including microtubule-associated protein 2 (MAP2), microtubule-associated protein Tau (MAPT), growth-associated protein 43 (GAP-43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholine esterase (AChE). The results indicated that treating MET increased MAP2, GAP-43, and VAChT expressions, in which cholinergic signaling is related to neurological functions. A heterologously expressed melittin in a probiotic yeast and its potential for promoting NSC-34 regeneration described here facilitate commercial and therapeutic use.Key points• MET and its precursor ProMET were successfully hetero-expressed in K. marxianus• > 95% high-purity MET and ProMET were purified at 1.68 μg/mL and 3.33 μg/mL• MET has no cytotoxicity toward NSC-34 and significantly promotes NSC-34 growth

CsGAT1 modulates GABA metabolism and positively regulates cold resistance in tea plants

Tea plants (Camellia sinensis) are perennial woody economic crops that are often exposed to a range of abiotic stresses, especially low temperatures, during development. γ-Aminobutyric acid (GABA) is a nonprotein amino acid widely distributed in plants that is involved in the low-temperature response of plants. Here, we found that CsGAT1 was upregulated in tea leaves subjected to low-temperature stress according to transcriptomic data. Heterologous expression of CsGAT1 in a yeast mutant revealed that it specifically transports GABA. Subcellular localization assays revealed that CsGAT1 was located on the plasma membrane. The organizational localization experiments revealed that the expression level of CsGAT1 was relatively high in the old leaves and roots and relatively low in the flowers. Testing of different cold-tolerant tea germplasm resources revealed that cultivars with relatively low cold resistance presented relatively low CsGAT1 expression and GABA levels. In addition, Arabidopsis plants overexpressing CsGAT1 presented high levels of GABA accumulation and significant low-temperature resistance. In summary, we believe that CsGAT1 regulates the ability of tea plants to resist low-temperature stress by changing the concentration of GABA inside and outside the cell. This study provides a theoretical basis for breeding new tea cultivars with strong resistance to low-temperature stress during production.

Biotechnological Production of the Recombinant Two-Component Lantibiotic Lichenicidin in the Bacterial Expression System

Lantibiotics are a family of bacterial antimicrobial peptides synthesized by ribosomes that undergo post-translational modification to form lanthionine (Lan) and methyllanthionine (MeLan) residues. Lantibiotics are considered promising agents for combating antibiotic-resistant bacterial infections. This paper presents a biotechnological method for obtaining two components of the lantibiotic lichenicidin from Bacillus licheniformis B-511 – Lchα and Lchβ. A system has been developed that allows co-expression of the lchA1 or lchA2 genes, encoding the precursors of the α- or β-components, respectively, with the lchM1 or lchM2 genes of the modifying enzymes LchM1 and LchM2 in Escherichia coli cells. The developed system of heterologous expression and purification made it possible to obtain, with high yield, post-translationally modified recombinant Lchβ, completely identical to the natural peptide in structure and biological activity.

Boron confers salt tolerance through facilitating BnaA2.HKT1-mediated root xylem Na+ unloading in rapeseed (Brassica napus L.).

Boron (B) is an important limiting factor for plant growth and yield in saline soils, but the underlying molecular mechanisms remain poorly understood. In this study, we found that appropriate B supply obviously complemented rapeseed (Brassica napus L.) growth under salinity accompanied by higher biomass production and less reactive oxygen species accumulation. Determination of Na+ content in shoots and roots indicated that B significantly repressed root-to-shoot Na+ translocation, and non-invasive micro-tests of root xylem sap demonstrated that B increased xylem Na+ unloading in the roots of rapeseed plants under salinity. Comparative transcriptomic profiling revealed that B strongly upregulated BnaHKT1s expression, especially BnaA2.HKT1, in rapeseed roots exposed to salinity. In situ hybridizations analysis showed that BnaA2.HKT1 was significantly induced in root stelar tissues by high B (HB) under salinity. Green fluorescent protein and yeast heterologous expression showed that BnaA2.HKT1 functioned as a plasma membrane-localized Na+ transporter. Knockout of BnaA2.HKT1 by CRISPR/Cas9 resulted in hypersensitive of rapeseed plants to salinity even under HB condition, with higher shoot Na+ accumulation and lower biomass production. By contrast, overexpression of BnaA2.HKT1 ameliorated salinity-induced growth inhibition under B deficiency and salinity. Overall, our results proposed that B functioned as a positive regulator for the rapeseed growth and seed production under salt stress through facilitating BnaA2.HKT1-mediated root xylem Na+ unloading. This study may also provide an alternative strategy for the improvement of crop growth and development in saline soils.

Calcium-Dependent Lipopeptide Antibiotics against Drug-Resistant Pathogens Discovered via Host-Dependent Heterologous Expression of a Cloned Biosynthetic Gene Cluster.

Historically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium-dependent lipodepsipeptides that are active against drug-resistant Gram-positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new Nϵ-hydroxyarginine-containing cyclic lipodepsipeptides active against drug-resistant Gram-positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that Nϵ-hydroxyarginine is the product of a heme-dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Furthermore, unlike daptomycin, ambocidin A retains potent antimicrobial activity in the presence of lung surfactant, giving it the potential to treat bacterial pneumonia. Our work expands the family of calcium-dependent lipopeptide antibiotics with a new member exhibiting a distinct mechanism of action.

α2δ-2 regulates synaptic GluK1 kainate receptors in Purkinje cells and motor coordination.

Gabapentin and pregabalin are inhibitory ligands of both α2δ-1 and α2δ-2 proteins (also known as subunits of voltage-activated Ca2+ channels) and are commonly prescribed for the treatment of neuropathic pain and epilepsy. However, these drugs can cause gait disorders and ataxia through unknown mechanisms. α2δ-2 and GluK1, a glutamate-gated kainate receptor subtype, are coexpressed in cerebellar Purkinje cells. In this study, we used a heterologous expression system and Purkinje cells to investigate the potential role of α2δ-2 in regulating GluK1-containing kainate receptor activity. Whole-cell patch clamp recordings showed that α2δ-2 coexpression augmented GluK1, but not GluK2, currents in HEK293 cells, and pregabalin abolished this augmentation. Pregabalin lost its inhibitory effect on GluK1 currents in HEK293 cells expressing both GluK1 and the α2δ-2(R282A) mutant. Blocking GluK1-containing receptors with UBP310 substantially reduced the amplitude of excitatory postsynaptic currents at parallel fiber-Purkinje cell synapses in mice. Also, pregabalin markedly attenuated the amplitude of excitatory postsynaptic currents and currents elicited by ATPA, a selective GluK1 receptor agonist, in Purkinje cells in Cacna2d1 knockout mice. Coimmunoprecipitation assays indicated that α2δ-2, but not α2δ-1, formed a protein complex with GluK1 in cerebellar tissues and HEK293 cells through its C terminus. Furthermore, α2δ-2 coexpression potentiated surface expression of GluK1 proteins in HEK293 cells, whereas pregabalin reduced GluK1 proteins in cerebellar synaptosomes. Disrupting α2δ-2-GluK1 interactions using α2δ-2 C-terminus peptide abrogated the potentiating effect of α2δ-2 on GluK1 currents and attenuated the amplitude of GluK1-mediated excitatory postsynaptic currents in Purkinje cells. However, neither pregabalin nor α2δ-2 C-terminus peptide had significant effect on P/Q-type currents in HEK293 cells. Additionally, CRISPR/Cas9-induced conditional knockdown of Cacna2d2 or Grik1 in Purkinje cells, as well as microinjection of α2δ-2 C-terminus peptide or UBP310 into the cerebellum, substantially impaired beam walking and rotarod performance in mice. Our study reveals that α2δ-2 directly interacts with GluK1 independently of its conventional role as a voltage-activated Ca2+ channel subunit. α2δ-2 regulates motor coordination by promoting synaptic expression and activity in GluK1-containing kainate receptors in Purkinje cells.

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model.

The rapid spread of Newcastle disease (ND), driven by extensive commercial exchange in the poultry industry, necessitates urgent preventive measures. Although effective vaccines against the Newcastle disease virus (NDV) have been used since 1940, recent outbreaks and the limitations of current vaccines highlight the need for improved solutions. Advances in synthetic biology, reverse vaccinology, molecular biology, and recombinant DNA technology over the past 20 years have led to the development of recombinant vaccines, which offer enhanced protection and broader immunogenic coverage against NDV. This study aimed to express the immunogenic domains of Hemagglutinin Neuraminidase (HN) and Fusion (F) glycoproteins, linked to the heat-labile enterotoxin B subunit (LTB) bio-adjuvant, to develop an effective and reliable recombinant vaccine for NDV. In this study, the L(HN)2F protein, composed of the LTB bio-adjuvant and the immunogenic regions of the doubled Hemagglutinin Neuraminidase (HN-HN) and Fusion (F) epitope, was expressed in Escherichia coli. Subcutaneous injection was used to evaluate the humoral immune response in mice and the result was compared with B1 vaccine. The induction of strong humoral immune responses proved the strong immunoreactivity of the recombinant protein. The IgG elicited by the recombinant proteins was comparable to that of the commercial B1 vaccine against NDV, indicating its potential as a viable candidate for further development and evaluation as a recombinant vaccine against NDV.

Discovery and Heterologous Expression of Trilenodin, an Antimicrobial Lasso Peptide with a Unique Tri-Isoleucine Motif.

Lasso peptides are an increasingly relevant class of peptide natural products with diverse biological activities, intriguing physical properties, and unique chemical structures. Most characterized lasso peptides have been from Actinobacteria and Proteobacteria, despite bioinformatic analyses suggesting that other bacterial taxa, particularly those from Firmicutes, are rich in biosynthetic gene clusters (BGCs) encoding lasso peptides. Herein, we report the bioinformatic identification of a lasso peptide BGC from Paenibacillus taiwanensis DSM18679 which we termed pats. We used a bioinformatics-guided isolation approach and high-resolution tandem mass spectrometry (HRMS/MS) to isolate and subsequently characterize a new lasso peptide produced from the pats BGC, which we named trilenodin, after the tri-isoleucine motif present in its primary sequence. This tri-isoleucine motif is unique among currently characterized lasso peptides. We confirmed the connection between the pats BGC and trilenodin production by establishing the first Bacillus subtilis 168-based heterologous expression system for expressing Firmicutes lasso peptides. We finally determined that trilenodin exhibits potent antimicrobial activity against B. subtilis and Klebsiella pneumoniae, making trilenodin the first characterized biologically active lasso peptide from Firmicutes. Collectively, we demonstrate that bacteria from Firmicutes can serve as high-potential sources of chemically and biologically diverse lasso peptides.

Heterologous Expression of Type II PKS Gene Cluster Leads to Diversified Angucyclines in Streptomyces albus J1074.

Heterologous expression has emerged as an effective strategy in activating Streptomyces cryptic gene clusters or improving yield. Eight compounds were successfully obtained by heterologous expression of the type II PKS gene cluster spi derived from marine Streptomyces sp. HDN155000 in the chassis host Streptomyces albus J1074. The structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as theoretical NMR calculations and electronic circular dichroism (ECD) calculations. Interestingly, compound WS009 Z (2) contains a rare thiomethyl group, angumycinone T (4) has a novel oxo-bridge formed between C12a and C4, and angumycinone X (3) showed cytotoxicity toward K562 and NCI-H446/EP cell lines.

Systematic discovery of antibacterial and antifungal bacterial toxins.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxin-immunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications.

Assessment of heat tolerance and identification of miRNAs during high-temperature response in grapevine.

With global warming, heat stress has been recognized as a significant factor limiting grapevine development and fruit quality. MicroRNAs (miRNAs) are a class of small non-coding RNAs known to play crucial regulatory roles in stress resistance. Hence, there is an immediate requirement to cultivate and identify grapevine varieties that are resistant to heat and explore miRNA-mediated heat stress defense mechanisms. In this study, we assessed the thermal resistance of 38 grape germplasm resources and identified a series of miRNAs involved in heat stress resistance. The CK (25°C) and HS (45°C) groups of "Shenyue" cuttings of grapes were used as experimental materials for next-generation sequencing and construct libraries of small RNAs. A total of 177 known and 20 novel miRNAs were detected in the libraries. Differential expression analysis identified 65 differentially expressed miRNAs (DEMs) using the DE-Seq procedure. Furthermore, RT-qPCR validation confirmed complementary expression profiles of eight DEMs and their target genes between the HS and CK groups. Heterologous transformation further identified the function of Vvi-miR3633a downregulated under heat stress in Arabidopsis. In the heterologous expression lines, the survival rate was reduced by high temperature treatment indicating the ability of Vvi-miR3633a to regulate heat resistance. Assessing the heat resistance of grape species and the expression patterns of miRNA in response to high temperatures may reveal the molecular processes of heat resistance regulation mediated by miRNA in grapes under heat stress.

Improving Stability of Spiroplasma citri MreB5 Through Purification Optimization and Structural Insights.

MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria Spiroplasma citri, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein. Key features • The protocol is a useful approach to standardize purification of nucleotide-dependent cytoskeletal filaments and other nucleotide-binding proteins. • The mechanistic basis of how different ions could stabilize a protein, and hence improve yield in purification, has been demonstrated. • The change in buffer conditions/salt enabled us to get sufficient yield for biochemical and structural characterization.

Pyrrolizwilline, a Unique Bacterial Alkaloid Assembled by a Nonribosomal Peptide Synthetase and non-Enzymatic Dimerization.

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of heterocyclic specialized metabolites characterized by a core structure comprising a hexahydro-1H-pyrrolizine. PAs are synthesized through two main pathways. In plants, assembly occurs via a homospermidine synthase, and in bacteria, through combined action of a nonribosomal peptide synthetase and a Baeyer-Villiger monooxygenase. While the toxic properties of plant-derived PAs and their prevalence in animal and human foods have been extensively studied, the biological roles and biosynthesis of more complex bacterial PAs are not well understood. Here, we report the identification and characterization of a bacterial biosynthetic gene cluster from Xenorhabdus hominickii, xhpA-G, which is responsible for producing the PA pseudo-dimer pyrrolizwilline. Analysis of X. hominickii promoter exchange mutants together with heterologous expression of xhpA-G in E. coli, revealed a set of pathway intermediates, two of which were chemically synthesized, as well as multiple derivatives. This information was leveraged to propose a detailed biosynthetic pathway to pyrrolizwilline. Furthermore, we have characterized the hydrolase XhpG, the key enzyme in the conversion of the pathway intermediate pyrrolizixenamide to pyrrolizwilline, using X-ray crystallography and small-angle X-ray scattering (SAXS).

Identification and analysis of the key genes for Escherichia coli heterologous protein expression by transcriptomic profiling.

Escherichia coli is a frequently used host for heterologous protein expression, but its expression efficiency is hindered by several limitations, such as formation of inclusion bodies and proteolytic degradation. In this study, we employed high-density fermentation of heterologous protein production in a 5-L bioreactor, resulting in a yield 2.25 times higher than that of the control group. Transcriptional analysis was conducted at three time points after induction for 0h, 4h, and 12h, revealing 420, 301, and 570 upregulated differentially expressed genes, as well as 424, 202, and 525 downregulated genes, respectively. By conducting enrichment analysis, we constructed strains that relieved without iron limitation, exhibiting a 36% increase in biomass and a 32% increase in protein expression. Furthermore, no overflow metabolism of acetic acid was detected during the protein expression process when utilizing chemostat culture, which indicated that the utilization efficiency of glucose was significantly enhanced without iron limitation. This study presents a novel approach to better comprehend the mechanism of high-yield production of heterologous proteins in Escherichia coli.

Modulation of heteromeric glycine receptor function through high concentration clustering.

Ion channels are targeted by many drugs for treating neurological, musculoskeletal, renal and other diseases. These drugs bind to and alter the function of individual channels to achieve desired therapeutic effects. However, many ion channels function in high concentration clusters in their native environment. It is unclear if and how clustering modulates ion channel function. Human heteromeric glycine receptors (GlyRs) are the major inhibitory neurotransmitter receptors in the spinal cord and are active targets for developing chronic pain medications. We show that the α2β heteromeric GlyR assembles with the master postsynaptic scaffolding gephyrin (GPHN) into micron-sized clustered at the plasma membrane after heterologous expression. The inhibitory trans- synaptic adhesion protein neuroligin-2 (NL2) further increases both the cluster sizes and GlyR concentration. The apparent glycine affinity increases monotonically as a function of GlyR concentration but not with cluster size. We also show that ligand re-binding to adjacent GlyRs alters kinetics but not chemical equilibrium. A positively charged N- terminus sequence of the GlyR β subunit was further identified essential for glycine affinity modulation through clustering. Taken together, we propose a mechanism where clustering enhances local electrostatic potential, which in turn concentrates ions and ligands, modulating the function of GlyR. This mechanism is likely universal across ion channel clusters found ubiquitously in biology and provides new perspectives in possible pharmaceutical development.

Boosting Expression of a Specifically Targeted Antimicrobial Peptide K in Pichia pastoris by Employing a 2A Self-Cleaving Peptide-Based Expression System.

Background/Objectives: The current epidemic of drug-resistance bacterial strains is one of the most urgent threats to human health. Antimicrobial peptides (AMPs) are known for their good activity against multidrug resistance bacteria. Specifically targeted AMPs (STAMPs) are a fraction of AMPs that target specific bacteria and maintain the balance of the healthy microbiota of a host. We reported a STAMP Peptide K (former name: peptide 13) for E. coli. The aim of this study was to effectively produce peptide K using methylotrophic yeast Pichia pastoris. Methods: Three inserts (sequence of peptide K (K), two copies of peptide K fused with 2A sequence (KTK), and two copies of peptide K fused with 2A and an extra α mating factor (KTAK)) were designed to investigate the effect of the number of repeats and the trafficking of peptide on the yield. Results: The yield from KTK was the highest-more than two-fold higher compared with K-implying the role of the 2A sequence in heterologous peptide expression apart from the co-translation. Then, the fermentation condition for KTK was optimized. The optimized yield of KTK was 6.67 mg/mL, suggesting the efficiency of the expression system. Selectivity, antibacterial activity, biocompatibility, and the stability of the fermentation product were equivalent to the chemically synthesized peptide. The actional mechanism of the fermentation product included membrane permeabilization and ROS induction. Conclusions: Together, our work provided a new perspective to augment the yield of the antimicrobial peptide in the microbial system, building a technological foundation for their large-scale production and expanding the market application of AMPs.

Integration of mRNA-miRNA Reveals the Possible Role of PyCYCD3 in Increasing Branches Through Bud-Notching in Pear (Pyrus bretschneideri Rehd.).

Bud-notching in pear varieties with weak-branches enhances branch development, hormone distribution, and germination, promoting healthier growth and improving early yield. To examine the regulatory mechanisms of endogenous hormones on lateral bud germination in Pyrus spp. (cv. 'Huangguan') (Pyrus bretschneideri Rehd.), juvenile buds were collected from 2-year-old pear trees. Then, a comprehensive study, including assessments of endogenous hormones, germination and branching rates, RNA-seq analysis, and gene function analysis in these lateral buds was conducted. The results showed that there was no significant difference in germination rate between the control and bud-notching pear trees, but the long branch rate was significantly increased in bud-notching pear trees compared to the control (p < 0.05). After bud-notching, there was a remarkable increase in IAA and BR levels in the pruned section of shoots, specifically by 141% and 93%, respectively. However, the content of ABA in the lateral buds after bud-notching was not significantly different from the control. Based on RNA-seq analysis, a notable proportion of the differentially expressed genes (DEGs) were linked to the plant hormone signal transduction pathway. Notably, the brassinosteroid signaling pathway seemed to have the closest connection with the branching ability of pear with the related genes encoding BRI1 and CYCD3, which showed significant differences between lateral buds. Finally, the heterologous expression of PyCYCD3 has a positive regulatory effect on the increased Arabidopsis growth and branching numbers. Therefore, the PyCYCD3 was identified as an up-regulated gene that is induced via brassinosteroid (BR) and could act as a conduit, transforming bud-notching cues into proliferative signals, thereby governing lateral branching mechanisms in pear trees.

Trehalulose: Exploring its benefits, biosynthesis, and enhanced production techniques

The increasing concern over sugar-related health issues has sparked research interest in seeking alternatives to sucrose. Trehalulose, a beneficial structural isomer of sucrose, is a non-cariogenic sugar with a low glycemic and insulinemic index. Besides its potential as a sugar substitute, trehalulose exhibits high antioxidant properties, making it attractive for various industrial applications. Despite its numerous advantages and potential application in various sectors, the industrial adoption of trehalulose has yet to be established due to lack of studies on its characteristics and practical uses. This review aims to provide a comprehensive overview of the properties of trehalulose, emphasizing its health benefits. The industrial prospects of trehalulose as sweetener and reducing agent, particularly in food and beverages pharmaceutical, and cosmeceutical sectors, are explored. Additionally, the review delves into the sources of trehalulose and the diverse organisms capable of producing trehalulose. The biosynthesis of this sugar primarily involves an enzyme-mediated process. Thus, these enzymes' properties, mechanisms, and the heterologous expression of genes associated with trehalulose production are explored. The strategies discussed in this review can be improved and applied to establish trehalulose bio-factories for efficient synthesis of trehalulose in the future. With further research and development, trehalulose holds promise as a valuable component across various industries.