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Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection and suggests that, similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonization.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

Background: The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. Results: We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. Conclusions: We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene (lip4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application. [ABSTRACT FROM AUTHOR]

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that CgaDyP1 has high similarity with other fungal DyPs. The recombinant CgaDyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H2O2. CgaDyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. CgaDyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications. [ABSTRACT FROM AUTHOR]

Stress tolerance is a vital attribute for all living beings to cope with environmental adversities. IrrE (also named PprI) from Deinococcus radiodurans enhances resistance to extreme radiation stress by functioning as a global regulator, mediating the transcription of genes involved in deoxyribonucleic acid (DNA) damage response (DDR). The expression of IrrE augmented the resilience of various species to heat, radiation, oxidation, osmotic stresses and inhibitors, encompassing bacterial, fungal, plant, and mammalian cells. Moreover, IrrE was employed in a global regulator engineering strategy to broaden its applications in stress tolerance. The regulatory impacts of heterologously expressed IrrE have been investigated at the molecular and systems level, including the regulation of genes, proteins, modules, or pathways involved in DNA repair, detoxification proteins, protective molecules, native regulators and other aspects. In this review, we discuss the regulatory role and mechanism of IrrE in the antiradiation response of D. radiodurans. Furthermore, the applications and regulatory effects of heterologous expression of IrrE to enhance abiotic stress tolerance are summarized in particular. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Foot-and-mouth disease (FMD) is a great havoc in agri-business-based countries like Bangladesh, for which existing detection system limits the identification and differentiation of serotypes. In this study, an engineered platform was introduced incorporating serotype-specific FMDV VP1 (structural), serotype-independent VP2 (structural) and 3AB (non-structural) proteins for holistic detection. VP1 sequences were engineered combining sequences of BAN/TA/Dh-301/2016 (serotype O), BAN/CH/Sa-304/2016 (serotype A) and BAN/DH/Sa-318/2016 (serotype Asia1). Consensus 3AB sequence was constructed from the selected prevalent viral genomes. Both VP1 and 3AB along with designed VP2 sequences were optimized for codon usage bias, stable mRNA, secondary and tertiary protein structure. Proteins were synthesized in pET-21a (+) plasmid vector followed by transformation of Escherichia coli BL21(DE3) and IPTG-induced- expression. The western blot analysis of engineered proteins showed that purified VP1 prominently bound to anti-VP1 antibodies in vaccinated sera, whereas 3AB and VP2 bound anti-3AB and anti-VP2 antibodies, respectively from infected cattle sera, all previously collected during epidemiological investigation. Furthermore, dot blot hybridization confirmed efficient antibody capture ability of the membrane-immobilized proteins. This holistic diagnostic platform justifies a comprehensive prototype diagnostic kit that would be cost-effective and efficient for serotype specific and non-specific FMDV sero-surveillance. [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Food Industry is the property of Science & Technology of Food Industry Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Terpenoids are known for their diverse structures and broad bioactivities with significant potential in pharmaceutical applications. However, natural products with low yields are usually ignored in traditional chemical analysis. Feature-based molecular networking (FBMN) was developed recently to cluster compounds with similar skeletons, which can highlight trace amounts of unknown compounds. Fusoxypene A is a sesterterpene synthesized by Fusarium oxysporum fusoxypene synthase (FoFS) with a unique 5/6/7/3/5 ring system. In this study, the FoFS-containing biosynthetic gene cluster was identified from F. oxysporum FO14005, and an efficient FBMN-based strategy was established to characterize four new sesterterpenoids, fusoxyordienoid A–D (1–4), based on a small-scale fermentation strategy. A cytochrome P450 monooxygenase, FusB, was found to be involved in the functionalization of fusoxypene A at C-17 and C-24 and responsible for the hydroxylation of fusoxyordienoid A at C-1 and C-8. This study highlights the potential of FBMN as a powerful tool for the discovery and characterization of natural compounds with low abundance. Key points: Combined small-scale fermentation and FBMN for rapid discovery of fusoxyordienoids Characterization of four new fusoxyordienoids with 5/6/7/3/5 ring system Biosynthetic pathway elucidation via tandem expression and substrate feeding [ABSTRACT FROM AUTHOR]

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM. IMPORTANCE Dichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field. [ABSTRACT FROM AUTHOR]

The potato tuber moth, Phthorimaea operculella (Zeller), is a notorious insect pest of potato incurring substantial yield losses in the field as well as in storage. Chemical control is difficult to exercise due to the latent feeding of the caterpillars and their ability to develop resistance against insecticides. One of the essential components of efficient insect-resistant management is using two or more different insecticidal genes in transgenic crops to effectively avoid and delay the resistance development in insect pests. Two constructs, namely DS-1 (cry3A + SN-19 genes) and DS-2 (OCII + SN-19 genes) in pCAMBIA1301 binary vector, were developed and were transformed in potato cultivars (Agria and Lady Olympia) via Agrobacterium-mediated transformation. The molecular analysis confirmed gene integration and expression of the introduced genes in transgenic plants. The insecticidal effects of incorporated genes in transgenic plants were assessed against 1st, 2nd, 3rd, and 4th instar potato tuber moth (PTM) larvae. The transgenic plants endured significantly high mortalities (100%) of larval stages of PTM within 72 h. Our results show that these transgenic potato plants have the potential to control populations of PTM and are also useful tools in managing PTM that would ultimately reduce the dependency on conventional chemical pesticides with potentially less or minimal hazards. These lines can also serve as an excellent source of germplasm for potato breeding program. [ABSTRACT FROM AUTHOR]

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35 °C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater. [ABSTRACT FROM AUTHOR]

The cyclodextrins (CDs) market has increased due to their numerous applications, especially in the food and pharmaceutical industries. The high cost of synthetizing one unique form of CDs, mainly the α- and γ- forms, hinders their use in certain processes. Since CDs are exclusively produced by the enzymatic conversion of starch or starch derivates using cyclodextrin glycosyltransferase (CGTase), enhancing this enzyme production means feasible CD synthesis. Recent discoveries of novel CGTase producers and strategies for optimizing fermentation processes and immobilization techniques have expanded its applications. However, the low yield arising from wild strains remains an obstacle. Studies focused on overexpressing CGTase through heterologous expression and improving their catalytic properties by altering protein structure using protein engineering approaches have been explored to enable upscaling the applications of CGTase. This review surveys recent approaches employed in CGTases production aiming at industrial scale. Overall, the importance of advances in biotechnology for cost reduction in upscale processes and perspectives for future research were highlighted. [Display omitted] • The high demand for cyclodextrins has led to a significant increase in research on CGTases. • Many studies report cultivation optimization to increase CGTase production. • Heterologous expression strategies have been applied to improve CGTase secretion yields. • Molecular engineering approaches have successfully enhanced CGTases catalytic properties. • Immobilization techniques increase enzymatic stability and enable reuse. [ABSTRACT FROM AUTHOR]

Maltotetraose amylase, which catalyzes the hydrolysis of amylaceous polysaccharides into maltooligosaccharides with maltotetraose as the main product, is extensively used in the food industry. However, the lack of efficient expression system for maltotetraose amylase has hampered its production and application. In this study, high-level production of a maltotetraose amylase mutant (referred to as Pp-Mta∆CBM) from Pseudomonas saccharophila was achieved in Pichia pastoris X-33. First, the gene of maltotetraose amylase with the carbohydrate-binding module (CBM) removed was codon-optimized and cloned into the pPICZαA vector, followed by transformation into P. pastoris X-33 for expression. Using the promoter PAOX1 and signal peptide α-factor, high-level production of Pp-Mta∆CBM with minimal extracellular impurity proteins was achieved, resulting in an extracellular activity of 367.9 U/mL after 7 days of cultivation in shake flasks. Next, the expressed Pp-Mta∆CBM was purified and characterized. This recombinant enzyme was glycosylated and has maximum activity at 55 ℃ and pH 7.0. Its Km for soluble starch was 4.1 g/L, and its kcat was 3237.6 s−1. Finally, the Pp-Mta∆CBM was found to produce a maximum maltotetraose yield of 57.1% in the presence of 200 g/L of substrate. The findings presented in this study demonstrate the efficient production of Pp-Mta∆CBM in P. pastoris, providing a new expression system for maltotetraose amylase and laying the foundation for its scale-up production and industrial application. [ABSTRACT FROM AUTHOR]

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species. [ABSTRACT FROM AUTHOR]

Diacetyl is a flavor compound in wine with a low threshold (1–2 mg/L). It is produced during alcoholic fermentation (AF) Saccharomyces and malolactic fermentation (MLF) initiated by lactic acid bacteria (LAB). Whereas, the environment after AF suppresses the normal metabolism of LAB after AF. Researchs have shown the influence on diacetyl mechanisms of the genes ILV2, ILV6, ILV3, ILV5, BDH1, BDH2, and gene aldB from Lactobacillus plantarum in Saccharomyce uvarum WY1. While we found that the diacetyl contents produced by mutants after MLF (Co-fermentation and Seq-fermentation) were significantly improved compared to AF alone. Moreover, the genes mae1 and mae2 from S. pombe, and gene mleS from L. lactis exhibited significant effect on deacidification in our previous study, but the diacetyl of the mutants showed obvious improvement in this study. Thus the effects of association mutation of genes (ILV2, ILV6, ILV3, ILV5, BDH1, BDH2, aldB, mae1, and mleS) on deacidification, diacetyl and other flavors (organic acids, higher alcohols and esters) metabolism in S. uvarum after AF were detected in the study. Among all the mutants, strains V6AmS, V635mS, and V6B12mS showed the most favorable results. Specifically, the L-malic acid contents decreased to 1.26 g/L, 1.18 g/L, and 1.19 g/L, respectively. Concurrently, diacetyl levels were reduced by 52.56%, 61.84%, and 65.31%. The production of n-propanol increased by 18.84%, 20.89%, and 28.12%, whereas isobutanol levels decreased by 37.01%, 42.36%, and 44.04%, and isoamyl alcohol levels decreased by 19.28%, 19.79%, and 16.74%, compared to the parental strain WY1. Additionally, the concentration of lactate ester in the wine increased to 13.162 mg/L, 14.729 mg/L, and 14.236 mg/L, respectively. [ABSTRACT FROM AUTHOR]

The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.

The anti-Clostridium difficile infection (CDI) drug fidaxomicin is a natural polyketide metabolite mainly produced by Micromonosporaceae, such as Actinoplanes deccanensis, Dactylosporangium aurantiacum, and Micromonospora echinospora. In the present study, we employed a stepwise strategy by combining heterologous expression, chassis construction, promoter engineering, activator and transporters overexpression, and optimization of fermentation media for high-level production of fidaxomicin. The maximum yield of 384 mg/L fidaxomicin was achieved with engineered Streptomyces albus D7-VHb in 5 L-tank bioreactor, and it was approximately 15-fold higher than the native strain Actinoplanes deccanensis YP-1 with higher strain stability and growth rate. This study developed an enhanced chassis strain, and for the first time, achieved the heterologous synthesis of fidaxomicin through a combinatorial metabolic engineering strategy.

Abstract Background The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. Results We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. Conclusions We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes.

The aim of this study was to achieve efficient expression of mannanase CtMan26 from Chaetomium thermophilum in Pichia pastoris and to investigate the enzymatic properties of CtMan26 and its potential in the preparation of mannooligosaccharides (MOS). The mannanase CtMan26 contained a total of 448 amino acids and consisted of the family 35 carbohydrate-binding module (CBM35), Linker and the catalytic domain module of the GH26 family. Asp-395 and Leu-396 in the catalytic domain and Leu-100 in the auxiliary module of CBM35 were subjected to multiple hydrophobic forces and could form multiple hydrogen bonds with amino acids in the Linker. Recombinant enzyme CtMan26 was expressed with N-glycosylation modification. CtMan26 showed an apparent molecular mass of about 55 kDa. The optimum temperature of the recombinant enzyme was 55 ℃, and the optimum pH was 5.0. The enzymatic activity was maintained at more than 90% of its original value after being incubated at 50 and 60 ℃ for 1 hour, and it showed good stability at pH ranging from 4.0 to 12.0. The specific activity of CtMan26 was 23.15 U/mg, and its Km and Vmax values were 4.75 mg/mL and 27.17 μmol/(min·mg), respectively, when konjac glucomannan was used as the substrate. MOS prepared with the recombinant enzyme mainly consisted of mannose (14.2 mg/L), mannose disaccharides (20.1 mg/L), mannose trisaccharides (6.1 mg/L) and mannose tetrasaccharides (2.5 mg/L). The MOS exhibited good antioxidant capacity, which scavenged (90.80 ± 0.65)% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, (80.7 ± 1.07)% of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical, (69.10 ± 1.10)% of hydroxyl radical and (76.7 ± 3.14)% of superoxide anion radical at 2.4 mg/mL concentration. Meanwhile, the MOS had obvious prebiotic properties, which could inhibit the growth of Escherichia coli and promote the growth of the beneficial bacteria Pediococcus pentosaceus and Lactobacillu plantarum the effect being more pronounced with increasing concentration and incubation time. In conclusion, the recombinant mannanase CtMan26 has good enzyme properties and therefore has potential application in the preparation of MOS.

Abstract Foot-and-mouth disease (FMD) is a great havoc in agri-business-based countries like Bangladesh, for which existing detection system limits the identification and differentiation of serotypes. In this study, an engineered platform was introduced incorporating serotype-specific FMDV VP1 (structural), serotype-independent VP2 (structural) and 3AB (non-structural) proteins for holistic detection. VP1 sequences were engineered combining sequences of BAN/TA/Dh-301/2016 (serotype O), BAN/CH/Sa-304/2016 (serotype A) and BAN/DH/Sa-318/2016 (serotype Asia1). Consensus 3AB sequence was constructed from the selected prevalent viral genomes. Both VP1 and 3AB along with designed VP2 sequences were optimized for codon usage bias, stable mRNA, secondary and tertiary protein structure. Proteins were synthesized in pET-21a ( +) plasmid vector followed by transformation of Escherichia coli BL21(DE3) and IPTG-induced- expression. The western blot analysis of engineered proteins showed that purified VP1 prominently bound to anti-VP1 antibodies in vaccinated sera, whereas 3AB and VP2 bound anti-3AB and anti-VP2 antibodies, respectively from infected cattle sera, all previously collected during epidemiological investigation. Furthermore, dot blot hybridization confirmed efficient antibody capture ability of the membrane-immobilized proteins. This holistic diagnostic platform justifies a comprehensive prototype diagnostic kit that would be cost-effective and efficient for serotype specific and non-specific FMDV sero-surveillance.

High-yield strains of cold-active lipase are screened to provide production information for the industrial development of lipase. A cold-active lipase-producing strain was isolated from Xing'an larch forest soil in Mohe County using the Rhodamine B plate method. Its morphological, physiological, and biochemical characteristics were identified, and combined with the 16S rDNA gene sequence, the strain was determined to be Bacillus thuringiensis. The lipase gene Lip240 was cloned using the Bacillus thuringiensis genome as a template, and heterologous expression and enzymatic properties of Lip240 were analyzed. The results showed that the recombination lipase Lip240 had an optimal reaction temperature of 30 ℃, and could maintain more than 60% activity when treated at 4~30 ℃ for 6 hours, thus a cold-active lipase. Besides, its optimal pH was 8.0. Additionally, Ca2+, Mn2+, Fe2+, Fe3+, and Cr3+ had a certain enhancing effect on the activity of Lip240. Notably, Lip240 had a good organic solvent tolerance, while SDS could significantly (P

Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.

The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the clean genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

High GC bacteria from the genus Streptomyces harbor expansive secondary metabolism. The expression of biosynthetic proteins and the characterization and identification of biological "parts" for synthetic biology purposes from such pathways are of interest. However, the high GC content of proteins from actinomycetes in addition to the large size and multi-domain architecture of many biosynthetic proteins (such as non-ribosomal peptide synthetases; NRPSs, and polyketide synthases; PKSs often called "megasynthases") often presents issues with full-length translation and folding. Here we evaluate a non-ribosomal peptide synthetase (NRPS) from Streptomyces lavenduale, a multidomain "megasynthase" gene that comes from a high GC (72.5%) genome. While a preliminary step in revealing differences, to our knowledge this presents the first head-to-head comparison of codon-optimized sequences versus a native sequence of proteins of streptomycete origin heterologously expressed in E. coli. We found that any disruption in co-translational folding from codon mismatch that reduces the titer of indigoidine is explainable via the formation of more inclusion bodies as opposed to compromising folding or posttranslational modification in the soluble fraction. This result supports that one could apply any refactoring strategies that improve soluble expression in E. coli without concern that the protein that reaches the soluble fraction is differentially folded.

Abstract The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L−1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8–25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

Background: The important challenge to the biotechnology is to find new effective fusion partners that enable to improve solubility, expression, and optimize the subsequent fine purification of the target protein. Results: The most invariant part of the most immunogenic region of the surface virulence factor A of Streptococcus pneumoniae was selected as a model target protein, while the thermostable chemotaxis polypeptide of W-type from Thermotoga petrophila was used as a fusion partner. The genes encoding fusion variants of these proteins were constructed and cloned into a plasmid vector under the control of the strong bacteriophage T7 transcription regulatory system. Effective Escherichia coli producer strains were obtained, and optimal conditions were chosen for the production of resulting constructs. The optimal pH and temperature ranges of recombinant proteins were determined, and three-dimensional shapes of their molecules were also predicted. Methods of low-stage protein purification were improved. Some of the isolated proteins demonstrated a high level of expression, solubility and purity. Conclusions: Novel chimeric proteins were obtained which had not previously been observed in nature in such domain combinations. It was shown that the biotechnologically valuable characteristics of the hybrid proteins were more marked when the thermal-resistant partner was combined with the N-terminus of pneumococcal protein. The principles of their low-stage purification were performed which does not require any special equipment. That is a basis for significant reduction of prices for diagnostic test-systems components and subunit vaccine production.How to cite: Grishin DV, Sidorov NG, Parfenova OK, et al. Construction, heterological expression and a simple purification of the BP region of the pneumococcal surface protein a fused in different orientations to the chemotaxis adaptor protein CheW from Thermotoga petrophila. Electron J Biotechnol 2024;71. https://doi.org/10.1016/j.ejbt.2024.05.001.

A 61-kb biosynthetic gene cluster (BGC), which is accountable for the biosynthesis of hibarimicin (HBM) B from Microbispora rosea subsp. hibaria TP-A0121, was heterologously expressed in Streptomyces coelicolor M1154, which generated a trace of the target products but accumulated a large amount of shunt products. Based on rational analysis of the relevant secondary metabolism, directed engineering of the biosynthetic pathways resulted in the high production of HBM B, as well as new HBM derivates with improved antitumor activity. These results not only establish a biosynthetic system to effectively synthesize HBMs—a class of the largest and most complex Type-II polyketides, with a unique pseudo-dimeric structure—but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

Trichoderma reesei displays a high capability to produce extracellular proteins and therefore is used as a platform for the expression of heterologous genes. In a previous study, an expression cassette with the constitutive tef1 promoter and the cbh1 terminator compatible with flow cytometry analysis was developed. Independent transformants obtained by a random integration into the genome of a circular plasmid containing the expression cassette showed a wide range of fluorescence levels. Whole genome sequencing was conducted on eight of the transformed strains using two next-generation sequencing (NGS) platforms: Illumina paired-end sequencing and Oxford Nanopore. In all strains, the expression plasmid was inserted at the same position in the genome, i.e., upstream of the tef1 gene, indicating an integration by homologous recombination. The different levels of fluorescence observed correspond to different copy numbers of the plasmid. Overall, the integration of a circular plasmid with the green fluorescence protein (egfp) transgene under the control of tef1 promoter favors multicopy integration and allows over-production of this heterologous protein on glucose. In conclusion, an expression system based on using the tef1 promotor could be one of the building blocks for improving high-value heterologous protein production by increasing the copy number of the encoding genes into the genome of the platform strain. Key points: • Varied eGFP levels from tef1 promoter and cbh1 terminator expression. • Whole genome sequencing on short and long reads platforms reveals various plasmid copy numbers in strains. • Plasmids integrate at the same genomic site by homologous recombination in all strains. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHSPs) act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress, especially heat shock, but the mechanism is not clear. To study the function of sHSPs in Lenzites gibbosa, a common polypore in northern temperate forests that causes spongy white rot of broadleaf trees, under temperature stress, L. gibbosa mycelia were grown at 25 °C for 9 d, treated at 33 °C for 15, 30, 60, and 120 min before sequencing the transcriptomes. From among 32 heat shock protein (HSP) genes found in the screen of the transcriptome data, a highly expressed gene was cloned and named Lghsp17.4. RT-qPCR was used to analyze the expression of the gene Lghsp17.4 under heat shock and dye stress. Both treatments induced higher expression of Lghsp17.4 at the transcriptional level, indicating that Lghsp17.4 might function in the response to heat stress and dye degradation. We previously found that L. gibbosa generally had a heat shock reaction (HSR) during degradation of aromatic compounds, and HSPs were always produced with manganese peroxidases (MnPs) and other lignin-degrading enzymes. Therefore, we measured the activity of MnPs in L. gibbosa after 33 °C heat shock to analyze the relationship between MnPs expression and Lghsp17.4 expression. Heat shocks of 0–30 min increased MnPs activity, and the change in MnPs activity were closely positively correlated with the expression levels of Lghsp17.4 over time, indicating a potential connection and interaction between LgHSP17.4 and MnPs during the HSR in L. gibbosa. Thus, LgHSP17.4 might have a positive regulatory effect on the HSR in L. gibbosa and be a critical component of a stress resistance mechanism. [ABSTRACT FROM AUTHOR]

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L−1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8–25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes. [ABSTRACT FROM AUTHOR]

The experiment aims to develop xylanase XynA and provide a new resource for feeding xylanase. The XynA of Geobacillus stearothermophilus was heterologous expressed in Pichia pastoris, the optimal fermentation conditions, enzymatic properties and antioxidant properties of the hydrolysis product Xylo-oligosaccharide (XOS) were studied. The results showed that the crude XynA with enzyme activity of about 33.7 U/mL could be produced after 96 h induced by 1.0% methanol concentration. The optimum pH value of XynA is 9.0, the optimum temperature is 65 °C, moreover, XynA remains above 80% of the activity at pH value ranging from 5.0 to 10.5 and temperatures between 45 and 75 °C. Thin-layer chromatography (TLC) analysis revealed that the main products of the hydrolysis of bagasse xylan by XynA were xylotriose and xylohexaose. The scavenging rate of DPPH free radical reached 72.04% and the scavenging rate of hydroxylradical reached 99.16% with 1 000 mg/L XOS. The study indicates that XynA exhibits robust activity and stability, efficiently hydrolyzing xylan to produce XOS, thereby facilitating its application in the feed industry. [ABSTRACT FROM AUTHOR]

Actin polymerization is a crucial process in cellular functions, impacting key processes like cell division, development, morphogenesis, motility, and polarity establishment. Abnormalities in actin polymerization can lead to various diseases, including cancer, highlighting its significance in both normal cell function and disease progression. While the amino acid sequences of actin are highly conserved across species, recent studies have revealed differences in their polymerization functions. The animal or plant actin can be used heterologously in yeast cells, but certain types like the rabbit skeletal muscle and maize pollen actin have unique polymerization properties. On the other hand, the yeast actin can interact with the animal or plant actin. This review explores the polymerization characteristics of the animal, plant, and yeast actin, highlighting functional disparities in polymerization both in vivo and in vitro. Additionally, we discuss drugs that target actin polymerization, aiming to offer potential avenues for treating human diseases and advancing our understanding of actin's molecular mechanisms. Further research in this area may uncover the underlying principles of actin diversity and functional evolution, providing valuable insights for related fields. [ABSTRACT FROM AUTHOR]

Background: The important challenge to the biotechnology is to find new effective fusion partners that enable to improve solubility, expression, and optimize the subsequent fine purification of the target protein. Results: The most invariant part of the most immunogenic region of the surface virulence factor A of Streptococcus pneumoniae was selected as a model target protein, while the thermostable chemotaxis polypeptide of W-type from Thermotoga petrophila was used as a fusion partner. The genes encoding fusion variants of these proteins were constructed and cloned into a plasmid vector under the control of the strong bacteriophage T7 transcription regulatory system. Effective Escherichia coli producer strains were obtained, and optimal conditions were chosen for the production of resulting constructs. The optimal pH and temperature ranges of recombinant proteins were determined, and three-dimensional shapes of their molecules were also predicted. Methods of low-stage protein purification were improved. Some of the isolated proteins demonstrated a high level of expression, solubility and purity. Conclusions: Novel chimeric proteins were obtained which had not previously been observed in nature in such domain combinations. It was shown that the biotechnologically valuable characteristics of the hybrid proteins were more marked when the thermal-resistant partner was combined with the Nterminus of pneumococcal protein. The principles of their low-stage purification were performed which does not require any special equipment. That is a basis for significant reduction of prices for diagnostic test-systems components and subunit vaccine production. How to cite: Grishin DV, Sidorov NG, Parfenova OK, et al. Construction, heterological expression and a simple purification of the BP region of the pneumococcal surface protein a fused in different orientations to the chemotaxis adaptor protein CheW from Thermotoga petrophila. Electron J Biotechnol 2024;71. [ABSTRACT FROM AUTHOR]

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application. [ABSTRACT FROM AUTHOR]

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (K m = 0.263 mM, k cat / K m = 562.3 s−1 mM−1 for p -nitrophenyl acetate (p NPA 2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstF P144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstF P144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstF P144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes. [Display omitted] [ABSTRACT FROM AUTHOR]

Glycosaminoglycan (GAG) lyases have been critical in structural and functional studies of GAGs. HCLase_M28, a lyase identified from the genome of Microbacterium sp. M28 was heterologously expressed, enzymatically characterized, and prepared in large-scale fermentation for the production of chondroitin sulfate (CS) oligosaccharides. Results showed that the expression of HCLase_M28 in Escherichia coli BL21 (DE3)-pET24a-HCLase_M28opt1 and Bacillus subtilis W800-pSTOP1622-HCLase_M28opt2 were 108-fold and 25-fold that of wide strain. The optimal lytic reaction of HCLase_M28 happened in 20 mM Tris–HCl (pH 7.2) at 50 °C with a specific activity of 190.9 U/mg toward CS-A. The degrading activity was slightly simulated in presence of 1 mM Ca2+ and Mn2+ while severely inhibited by Hg+, Cu2+, Fe3+, and SDS. TLC and ESI–MS analysis proved HCLase_M28 was an endolytic lyase and degraded CS and hyaluronic acid into unsaturated disaccharides. Through a gradual scale-up of fermentation in 5 L, 100 L, and 1000 L, a highly efficient intracellular expression of HCLase_M28 with an activity of 3.88 × 105 U/L achieved within a 34 h of cultivation. Through ultrafiltration, CS oligosaccharides with DP of 2 to 8 as the main components could be controllably prepared. The successful large-scale fermentation made HCLase_M28 a promising enzyme for industrial production of CS oligosaccharides. [ABSTRACT FROM AUTHOR]

Background: Econazole is a widely used imidazole derivative antifungal for treating skin infections. The molecular targets for its frequent adverse effects of skin irritation symptoms, such as pruritus, burning sensation, and pain, have not been clarified. Transient receptor potential (TRP) channels, non-selective cation channels, are mainly expressed in peripheral sensory neurons and serve as sensors for various irritants. Methods: We investigated the effect of econazole on TRP channel activation by measuring intracellular calcium concentration ([Ca2+]i) through fluorescent ratio imaging in mouse dorsal root ganglion (DRG) neurons isolated from wild-type, TRPA1(−/−) and TRPV1(−/−) mice, as well as in heterologously TRP channel-expressed cells. A cheek injection model was employed to assess econazole-induced itch and pain in vivo. Results: Econazole evoked an increase in [Ca2+]i, which was abolished by the removal of extracellular Ca2+ in mouse DRG neurons. The [Ca2+]i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker. Attenuation of the econazole-induced [Ca2+]i responses was observed in the TRPA1(−/−) mouse DRG neurons but was not significant in the TRPV1(−/−) neurons. Econazole increased the [Ca2+]i in HEK293 cells expressing TRPA1 (TRPA1-HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca2+ mobilization from intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also increased the [Ca2+]i in mouse DRG neurons and TRPA1-HEK, and its nonspecific action was larger than econazole. Fluconazole, a triazole drug failed to activate TRPA1 and TRPV1 in mouse DRG neurons and TRPA1-HEK. Econazole induced itch and pain in wild-type mice, with reduced responses in TRPA1(−/−) mice. Conclusions: These findings suggested that the imidazole derivatives econazole and miconazole may induce skin irritation by activating nociceptive TRPA1 in the sensory neurons. Suppression of TRPA1 activation may mitigate the adverse effects of econazole. [ABSTRACT FROM AUTHOR]

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0–7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Unnatural product (uNP) nonribosomal peptides promise to be a valuable source of pharmacophores for drug discovery. However, the extremely large size and complexity of the nonribosomal peptide synthetase (NRPS) enzymes pose formidable challenges to the production of such uNPs by combinatorial biosynthesis and synthetic biology. Here we report a new NRPS dissection strategy that facilitates the engineering and heterologous production of these NRPSs. This strategy divides NRPSs into "splitting units", each forming an enzyme subunit that contains catalytically independent modules. Functional collaboration between the subunits is then facilitated by artificially duplicating, at the N‐terminus of the downstream subunit, the linker ‐ thiolation domain ‐ linker fragment that is resident at the C‐terminus of the upstream subunit. Using the suggested split site that follows a conserved motif in the linker connecting the adenylation and the thiolation domains allows cognate or chimeric splitting unit pairs to achieve productivities that match, and in many cases surpass those of hybrid chimeric enzymes, and even those of intact NRPSs, upon production in a heterologous chassis. Our strategy provides facile options for the rational engineering of fungal NRPSs and for the combinatorial reprogramming of nonribosomal peptide production. [ABSTRACT FROM AUTHOR]

AbstractNatural products have long served as critical raw materials in chemical and pharmaceutical manufacturing, primarily which can provide superior scaffolds or intermediates for drug discovery and development. Over the last century, natural products have contributed to more than a third of therapeutic drug production. However, traditional methods of producing drugs from natural products have become less efficient and more expensive over the past few decades. The combined utilization of genome mining and synthetic biology based on genome sequencing, bioinformatics tools, big data analytics, genetic engineering, metabolic engineering, and systems biology promises to counter this trend. Here, we reviewed recent (2020–2023) examples of genome mining and synthetic biology used to resolve challenges in the production of natural products, such as less variety, poor efficiency, and low yield. Additionally, the emerging efficient tools, design principles, and building strategies of synthetic biology and its application prospects in NPs synthesis have also been discussed. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CarEs) is an important detoxification enzyme system in phase Ⅰ participating in insecticides resistance. In our previous study, SlCarE054, a CarEs gene from lepidoptera class, was screened out to be upregulated in a pyrethroids and organophosphates resistant population. Its overexpression was verified in two field-collected populations of Spodoptera litura (Lepidoptera: Noctuidae) resistant to pyrethroids and organophosphates by qRT-PCR. Spatiotemporal expression results showed that SlCarE054 was highly expressed in the pupae stage and the digestive tissue midgut. To further explore its role in pyrethroids and organophosphates resistance, its metabolism activity to insecticides was determined by UPLC. Its recombinant protein showed significant metabolism activity to cyhalothrin and fenvalerate, but not to phoxim or chlorpyrifos. The metabolic activity of SlCarE054 to β -cypermethrin showed stereoselectivity, with higher metabolic activity to θ -cypermethrin than the enantiomer α -cypermethrin. The metabolite of β -cypermethrin was identified as 3-phenoxybenzaldehyde. Further modelling and docking analysis indicated that β -cypermethrin, cyhalothrin and fenvalerate could bind with the catalytic triad of the 3D structure of SlCarE054. The interaction of β -cypermethrin with SlCarE054 also showed the lowest binding energy. Our work provides evidence that SlCarE054 play roles in β -cypermethrin resistance in S. litura. [ABSTRACT FROM AUTHOR]

Mammalian odorant receptors (ORs) are crucial for detecting a broad spectrum of odorants, yet their functional expression poses a significant challenge, often requiring Receptor-transporting proteins (RTPs). This study examines mouse Olfr733 and Olfr732, which, despite high homology, show different functional expression profiles in heterologous cell systems. Our research aimed to identify key amino acids impacting Olfr733's functional expression. We discovered that G112FBW3.40 and L148PBW4.49 (Ballesteros–Weinstein numbering in superscript) substitutions in Olfr732 markedly enhance its RTP-independent expression and ligand responsiveness, mirroring Olfr733. These substitutions, particularly Phe112 and Leu148, are crucial for aldehyde recognition and membrane localization in Olfr733, respectively. While Olfr732-type ORs are conserved across species, Olfr733-types, unique to specific rodents, appear to have evolved from Olfr732, with Pro148 enhancing membrane expression and aldehyde sensitivity. Mouse ORs with ProBW4.49 tend to exhibit improved membrane expression compared to their paralogs, especially when co-expressed with RTP1S. This study concludes that the Pro residue in the fourth transmembrane domain significantly contributes to the structural stability of certain olfactory receptors, highlighting the intricate molecular mechanisms underlying OR functionality and evolution. [ABSTRACT FROM AUTHOR]

Extremozymes, enzymes derived from extremophilic organisms, have gained significant attention in industrial applications due to their remarkable resistance to extreme conditions. Their unique properties offer advantages over traditional mesophilic enzymes, making them valuable for applications in biocatalysis, bioremediation, and biotransformations. However, the discovery and downstream processing of them still presents challenges. Native microorganisms that produce extremozymes often exhibit slow growth, resulting in low quantities of specific enzymes. On the other hand, the heterologous expression of extremozymes can be complex, involving challenges such as rare codon usage, formation of secondary mRNA structures, misfolding, aggregation, and inactivation of the enzymes in several cases when they are expressed in conventional mesophilic hosts. In this review, we address the challenges associated to the discovery and production of novel extremozymes. Furthermore, we explore current approaches to overcome these challenges, including use of alternative expression systems through next generation industrial biotechnology (NGIB), cell-free protein synthesis (CFPS), culturomics, new metagenomic approaches, artificial intelligence (AI), among others. • Challenges and opportunities in extremozymes discovery and production. • Strategies for addressing stability problem, misfolding, low yields and codon usage. • Novel opportunities such as NGIB, CFPS, culturomics, AI, among others. [ABSTRACT FROM AUTHOR]